WO2009100955A1 - Antisense rna targeting cxcr4 - Google Patents

Abstract

MiR-146a and antagomirs thereof are useful in controlling expression of CXCR4 and megakaryopoiesis, tumor metastasis, HTV entry and/or Rheumatoid Arthritis.

Description

Antisense RNA Targeting CXCR4

The present invention relates to the use of antisense RNA.

MicroRNAs (miRs) are a class of regulatory, single-stranded RNAs of about 22 nucleotides (Bartel, 2004; He and Harmon, 2004; Ambros, 2004). MiRs repress protein expression at post-transcriptional level, mostly through base pairing to the 3' untranslated region (UTR) of the target mRNA, thus leading to its reduced translation and/or degradation (Valencia- Sanchez et al., 2006). MiRs control basic biological functions, such as cell proliferation and differentiation (Zhao et al., 2005; Naguibneva et al., 2006; Chen et al., 2006). Furthermore, in diverse types of cancer miRs may function as "oncomirs" (Esquela-Kerscher and Slack, 2006). Despite these advances, only few targets for the >500 known mammalian miRs have been validated so far. Particularly, relatively little is known on the role of miRs in hematopoietic differentiation (Chen et al., 2004; Fazi et al., 2005; Felli et al., 2005; Fontana et al., 2007).

The promyelocytic leukemia zinc finger protein, PLZF, is a transcription factor involved in the regulation of development (Kelly and Daniel, 2006), hematopoietic proliferation and differentiation (Ball et al., 1999; Labbaye et al., 2002), leukemogenesis (Melnick and Licht, 1999; Parrado et al., 2000) and tumorigenesis (Felicetti et al., 2004). However, the molecular mechanisms through which PLZF exerts its regulatory effects on hematopoiesis are still unknown,

The chemokine receptor 4, CXCR4, and its ligand SDF-I, are key molecules in the process of homing/mobilization of normal hematopoietic and leukemic cells (Peled et al., 1999; Gazitt, 2004; Lapidot et al., 2005; Ratajczak et al., 2006), particularly at stem cells level (Kucia et al., 2005; Burger and Kipps 2006). SDF-I acts as a mobilization or retention signal in hematopoiesis (Cottier-Fox et al., 2003; Ruiz de Almodovar et al., 2006), while CXCR4 regulation by cytokines is an important step in myeloid cell mobilization (Kim et al., 2006). CXCR4 acts as a transporter for internalization and secretion of SDF-I across the bone marrow-blood barrier (Dar et al., 2005). Inhibition of CXCR4 blocks thrombopoiesis in vivo (Avecilla et al., 2004), indicating that the integrity of the SDF-1/CXCR4 pathway is indispensable for megakaryocyte proliferation and differentiation/maturation (Pang et al., 2005). Nevertheless, the mechanism controlling CXCR4 expression in angiogenesis and hematopoiesis is not fully understood.

Surprisingly, we have discovered that miR-146a targets CXCR4 mRNA. Accordingly, by up-modulating miR146a, CXCR4 can be inhibited. Conversely, by down-modulating miR- 146a, CXCR4 inhibition can be lifted and upregulation of CXCR4 stimulates megakaryopoiesis.

Analysis of the sequence of the CXCR4 gene promoter did not reveal any functional PLZF binding site, suggesting that CXCR4 is not a direct target gene of PLZF. However, we discovered a discrepancy between stable CXCR4 mRNA levels and increased protein levels in K562-PLZF cells. Since miR-146 is sharply downmodulated in K562-PLZF cells, we formed a hypothesis that CXCR4 mRNA may be targeted by miR-146a. To confirm this, we used a range of different bioinformatic algorithms. However, only a single algorithm supported this hypothesis. Despite this setback, we conducted further experiments and surprisingly found that miR-146a does in fact target CXCR4 mRNA in vivo.

Furthermore, we have also surprisingly found that that PLZF represses miR-146a transcription. In hematopoietic progenitor cell (HPC) culture undergoing unilineage megakaryocytic (Mk) differentiation and maturation, the increasing expression of PLZF inhibits miR-146a expression, leading to enhanced CXCR4 protein levels. Functional studies showed that miR-146a or PLZF siRNA transfection similarly downmodulate CXCR4 translation and impair proliferation, differentiation and maturation of Mk cells, indicating that the PLZF/miR-146a/CXCR4 cascade controls normal megakaryopoiesis.

Thus, in a first aspect, the present invention provides use of antisense RNA specific for all or part of the 3' untranslated region of CXCR4 protein mRNA in therapy.

Up-modulating miR-146a (down-modulation of CXCR4) may be achieved, for instance, by administering or increasing expression of miR-146a, for instance administering it directly or in the form of a vector with a coding sequence for miR-146a preferably under the control of a suitable promoter, or by reducing antagomir (anti-miR 146a) levels. It may also preferably be achieved by decreasing the levels or expression of PLZF, for instance by increasing the levels of inhibitory antisense RNA targeted to PLZF. Down-modulation of CXCR4 leads to reduced megakaryopoiesis.

Conversely, down-modulating miR-146a (up-modulation of CXCR4) may be achieved, for instance, by increasing antagomir (anti- miR-146a) levels, increasing PLZF levels, for instance using a vector with a coding sequence for anti-miR-146a and/or PLZF, preferably under the control of a suitable promoter. It may also preferably be achieved by reducing the levels of inhibitory antisense RNA targeted to PLZF. Up-modulation of CXCR4 leads to increased megakaryopoiesis.

Preferably, therapy is treatment of cancer, preferably tumour metastasis. Preferably, therapy is treatment of HIV, most preferably by prevention of HIV entry into host cells. It is also preferred that the therapy is treatment of Rheumatoid Arthritis. Preferably, the therapy is treatment of other CXCR4-dependent tumours or disease conditions, such as leukaemia and solid tumours.

It is also preferred that the therapy is treatment of the therapy is the modulation of Megakaryopoiesis. By using antisense RNA, such as miR-146a, Megakaryopoiesis can be inhibited. Conversely, use of sense RNA, such as an antagomir can stimulate Megakaryopoiesis.

Preferably, the therapy is via CXCR4 receptor down-modulation or inhibition, particularly by posttranscriptional control of CXCR4 by miR-146a.

It is preferred that the antisense RNA is a micro RNA. Preferably, the antisense RNA has at least 60% homology with a selected region of the 3' untranslated region of CXCR4 protein mRNA. Preferably, the antisense RNA is between about 12 bases and 45 bases in length.

The antisense RNA is preferably a sequence having the same sequence as mature miR- 146a, which is preferably the RNA sequence of SEQ E) NO 1 (mature miR-146a 5'- ugagaacugaauuccauggguu-3 ' ) . Where a sequence capable of inhibiting miR-146a is required (anti- miR-146a, antagomirs), its sequence is preferably the RNA sequence of SEQ ID NO 2 (anti-sense miR-146a 5'- aacccatggaattcagttctca-3 ' ) .

The nucleotide sequence of human CXCR4 (SEQ ID NOs. 3 and 4) and PLZF (SEQ ID NO. 5) are given below:

SEQ ID NO.3 (CXCR4)

1 5'-UUUUCt tccctctagt gggcggggca gaggagttag ccaagatgtg actttgaaac

61 cctcagcgtc tcagtgccct tttgttctaa acaaagaatt ttgtaattgg ttctaccaaa

121 gaaggatata atgaagtcac tatgggaaaa gatggggagg agagttgtag gattctacat

181 taattctctt gtgcccttag cccactactt cagaatttcc tgaagaaagc aagcctgaat

241 tggtttttta aattgcttta aaaatttttt ttaactgggt taatgcttgc tgaattggaa

301 gtgaatgtcc attcctttgc ctcttttgca gatatacact tcagataact acaccgagga

361 aatgggctca ggggactatg actccatgaa ggaaccctgt ttccgtgaag aaaatgctaa

421 tttcaataaa atcttcctgc ccaccatcta ctccatcatc ttcttaactg gcattgtggg

481 caatggattg gtcatcctgg tcatgggtta ccagaagaaa ctgagaagca tgacggacaa

541 gtacaggctg cacctgtcag tggccgacct cctctttgtc atcacgcttc ccttctgggc

601 agttgatgcc gtggcaaact ggtactttgg gaacttccta tgcaaggcag tccatgtcat

661 ctacacagtc aacctctaca gcagtgtcct catcctggcc ttcatcagtc tggaccgcta

721 cctggccatc gtccacgcca ccaacagtca gaggccaagg aagctgttgg ctgaaaaggt

781 ggtctatgtt ggcgtctgga tccctgccct cctgctgact attcccgact tcatctttgc

841 caacgtcagt gaggcagatg acagatatat ctgtgaccgc ttctacccca atgacttgtg

901 ggtggttgtg ttccagtttc agcacatcat ggttggcctt atcctgcctg gtattgtcat

961 cctgtcctgc tattgcatta tcatctccaa gctgtcacac tccaagggcc accagaagcg

1021 caaggccctc aagaccacag tcatcctcat cctggctttc ttcgcctgtt ggctgcctta

1081 ctacattggg atcagcatcg actccttcat cctcctggaa atcatcaagc aagggtgtga

1141 gtttgagaac actgtgcaca agtggatttc catcaccgag gccctagctt tcttccactg

1201 ttgtctgaac cccatcctct atgctttcct tggagccaaa tttaaaacct ctgcccagca

1261 cgcactcacc tctgtgagca gagggtccag cctcaagatc ctctccaaag gaaagcgagg

1321 tggacattca tctgtttcca ctgagtctga gtcttcaagt tttcactcca gctaacacag

1381 atgtaaaaga ctttttttta tacgataaat aacttttttt taagttacac atttttcaga

1441 tataaaagac tgaccaatat tgtacagttt ttattgcttg ttggattttt gtcttgtgtt

1501 tctttagttt ttgtgaagtt taattgactt atttatataa attttttttg tttcatattg

1561 atgtgtgtct aggcaggacc tgtggccaag ttcttagttg ctgtatgtct cgtggtagga

1621 ctgtagaaaa gggaactgaa cattccagag cgtgtagtga atcacgtaaa gctagaaatg

1681 atccccagct gtttatgcat agataatctc tccattcccg tggaacgttt ttcctgttct

1741 taagacgtga ttttgctgta gaagatggca cttataacca aagcccaaag tggtatagaa

1801 atgctggttt ttcagttttc aggagtgggt tgatttcagc acctacagtg tacagtcttg

1861 tattaagttg ttaataaaag tacatgttaa acttaaaaaa aaaaaaaaaa aa-3 '

The preferred coding sequence: 305 ..1375 is shown in bold and underlined above and in the Sequence Listing.

A preferred nucleotide sequence encoding PLZF is shown in SEQ ID NO. 5. The 3' untranslated (UTR) region of human CXCR4 protein mRNA is provided as SEQ ID

NO. 6 :

1376 5'-cacag

1381 atgtaaaaga ctttttttta tacgataaat aacttttttt taagttacac atttttcaga

1441 tataaaagac tgaccaatat tgtacagttt ttattgcttg ttggattttt gtcttgtgtt

1501 tctttagttt ttgtgaagtt taattgactt atttatataa attttttttg tttcatattg

1561 atgtgtgtct aggcaggacc tgtggccaag ttcttagttg ctgtatgtct cgtggtagga

1621 ctgtagaaaa gggaactgaa cattccagag cgtgtagtga atcacgtaaa gctagaaatg

1681 atccccagct gtttatgcat agataatctc tccattcccg tggaacgttt ttcctgttct

1741 taagacgtga ttttgctgta gaagatggca cttataacca aagcccaaag tggtatagaa

1801 atgctggttt ttcagttttc aggagtgggt tgatttcagc acctacagtg tacagtcttg

1861 tattaagttg ttaataaaag tacatgttaa acttaaaaaa aaaaaaaaaa aa-3'

The antisense RNA is preferably specific for all or part of the 3' UTR of CXCR4 protein mRNA. Preferably, it is specific for the full length 3' UTR of CXCR4 mRNA. Preferably, it is specific for at least one, and preferably both, of the following putative miR-146a binding sites: "Site 2" (from 1575 to 1596 bp); and "Site 3" (from 1800 to 1821 bp) as described in Didiano and Hobert, 2006, as shown above in bold and underlined text and also shown in Figure 3c. The binding sites for the present polynucleotide on CXCR4 are preferably one or more of those shown in Figure 3 c, especially the highlighted portions thereof and above.

Preferred PLZF binding site sequences shown in Figures 3e and 3g are provided in SEQ ID NOs 7 and 8, where GTAC in the first sequence is mutated to CGGC.

Also provided is an inhibitor or suppressor (antagomir) of miR-146a. Preferably, this is a sense RNA. The invention provides for the use of this inhibitor or suppressor in therapy, preferably in mediation, and preferably stimulation, of megakaryopoiesis.

The sense RNA preferably hybridises to miR-146a under highly stringent conditions, such as 6 x SSC. Similarly, the invention also provides the use of PLZF to increase CXCR4 expression or reduce miR 146a-mediated inhibition of CXCR4, in therapy, preferably in mediation, and preferably stimulation, of Megakaryopoiesis.

Also provided is a vector comprising the present antisense or antagomir RNA, or DNA encoding said RNA. Preferably, the vector encodes or comprises the mature form of the RNA, where the RNA is a micro RNA. The invention also provides a vector comprising nucleotides encoding PLZF.

The invention also provides a method of treating Cancer, HIV and/or Rheumatoid Arthritis, for example, or other CXCR4-dependent tumours or disease conditions, comprising administering to a patient an antisense RNA, preferably miR- 146a, and/or antisense RNA targeted to PLZF, to thereby inhibit CXCR4 expression, as discussed above.

Antisense RNA may be specific for any part of the 3' UTR of CXCR4 protein mRNA, and it will be appreciated that the 3' UTR may vary slightly from individual to individual.

The invention also provides the use of PLZF, or inhibitors thereof, to modulate CXCR4 expression, for instance via miR- 146a, and for treatment of conditions associated with CXCR4. In particular, we have also found that siRNA targeted to PLZF, thereby inhibiting or downmodulating PLZF expression, also has a similar effect to upmodualting miR- 146a, i.e. inhibition of CXCR4, as well as Mk cell proliferation and differentiation/maturation.

Provides is a method of inhibiting megakaryopoiesis or treating cancer, HIV and/or Rheumatoid Arthritis, or other CXCR4-dependent tumours or disease conditions, comprising administering to a patient an antisense RNA, preferably miR- 146a, and/or antisense RNA targeted to PLZF, to thereby inhibit CXCR4 expression.

Alternatively, also provided is a method of stimulating megakaryopoiesis comprising administering to a patient an antagomir of miR- 146a and/or PLZF or nucleotides encoding it, to thereby increase CXCR4 expression.

In addition, as noted above, miR need not be 100% faithful to the target, sense sequence. Indeed, where they are 100% faithful, this can lead to cleavage of the target mRNA through the formation of dsRNA. While the formation of dsRNA and cleavage of CXCR4 protein mRNA is included within the scope of the present invention, it is not a requirement that the antisense RNA be 100% faithful to the target sequence, provided that the antisense RNA is capable of binding the target 3' UTR to inhibit or prevent translation.

Thus, it will be appreciated that the antisense RNA of the present invention need only exhibit as little as 60% or less homology with the target region of the 3' UTR. More preferably, the antisense RNA exhibits greater homology than 60%, such as between 70 and 95%, and more preferably between 80 and 95%, such as around 90% homology. Homology of up to and including 100%, such as between 95 and 100%, is also provided. Suitable methods for assessment of such homology include the BLAST program.

The antisense RNA of the present invention may be as long as the 3' UTR, or even longer. However, it is generally preferred that the antisense RNA is no longer than 50 bases, and it may be a short as 10 bases, for example. More preferably, the antisense RNA of the present invention is between about 12 bases and 45 bases in length, and is more preferably between about 15 and 35 bases in length.

The preferred miR is miR- 146a, according to SEQ ID NO. 1. Its mature length is of 22 bases. Thus, a particularly preferred length is between 20 and 25 bases, and especially 22.

The area of the 3' UTR to be targeted may be any that prevents or inhibits translation of the ORF, when associated with an antisense RNA of the invention. The particularly preferred regions are those targeted by miR- 146a, and targeting either of these regions with antisense RNA substantially reduces translation of CXCR4 protein.

Regions of the 3' UTR that it is preferred to target include the central region of the 3' UTR and regions between the central region and the ORF. Such regions which are proximal to the ORF are particularly preferred. Other CXCR4 mRNA sequences, such as the coding region for instance, may also be targeted. It is preferred that the antisense RNA of the present invention is a short interfering RNA or a micro RNA.

The present invention further provides mutants and variants of miR- 146a. In this respect, a mutant may comprise at least one of a deletion, insertion, inversion or substitution, always provided that the resulting miR is capable of interacting with the 3' UTR to inhibit or prevent translation of the associated coding sequence. Enhanced homology with the 3 ' UTR is preferred. A variant will generally be a naturally occurring mutant, and will normally comprise one or more substitutions. Particularly preferred stretches of the microRNA of the present invention correspond to the so-called "seed" sequences.

It will be appreciated that reference to any sequence encompasses mutants and variants thereof, caused by substitutions, insertions or deletions, having levels of sequence homology (preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%, and most preferably at least 99.5% sequence homology), or corresponding sequences capable to hybridising to the reference sequence under highly stringent conditions (preferably 6x SSC).

The antisense RNAs of the present invention may be provided in any suitable form to the target site. In this respect, the target site may be in vivo, ex vivo, or in vitro, for example, and the only requirement of the antisense RNA is that it interacts with the target 3' UTR sufficiently to be able to inhibit or prevent translation of the CXCR4 ORF.

The antisense RNA may be provided directly, or a target cell may be transformed with a vector encoding the antisense RNA directly, or a precursor therefor. Suitable precursors will be those that are processed to provide a mature miR, although it is not necessary that such precursors be transcribed as long primary transcripts, for example.

Where the antisense RNA is provided directly, then this may be provided in a stabilised form such as is available from Dharmacon (www.dharmacon.com. Boulder, CO, USA).

A large number of microRNAs are known from WO 2005/013901, the patent specification of which alone is over 400 pages. However, no specific function is provided therefor.

Thus, although microRNAs are known, we are the first to establish that naturally-occurring RNA sequences, in particular miR- 146a, or inhibitors thereof, are in fact capable of modulating the expression of CXCR4 protein.

Insofar as miR- 146a is known, and any stabilised versions thereof, such as provided by Dharmacon are known, then the present invention does not extend to these compounds per se. However, the present invention extends to these and all other antisense RNAs provided by the present invention, for use in therapy and other processes. More particularly, the present invention provides the use of antisense RNA specific for all or part of the 3' untranslated region of CXCR4 protein mRNA in therapy.

The nature of the therapy is any that is affected by expression of CXCR4 protein. In particular, antisense RNAs of the present invention may be used in the treatment of CXCR4-dependent tumours, as well as other CXCR4-dependent, such as HIV and RA.

Solid, non-diffuse tumours may be targeted by direct injection of the tumour with a transforming vector, such as lenti virus, or adenovirus. If desired, the virus or vector may be labelled, such as with FITC (fluorescein isothiocyanate), in order to be able to monitor success of transformation.

Thus, it is also preferred that the present invention is used in the modulation of Megakaryopoiesis.

For the treatment of a more diffuse condition, then systemic administration may be appropriate, and antisense RNA may be administered by injection in a suitable vehicle, for example.

Levels of antisense RNA to be administered will be readily determined by the skilled physician, but may vary from about 1 μg/kg up to several hundred micrograms per kilogram.

The present invention further provides miR-146a inhibitors, and their use in therapy. These are referred to as "sense inhibitors" in that they are complementary, at least in part, to the antisense miRNA of the present invention.

Also provided is the use of a sense or antisense polynucleotide according to present invention in the manufacture of a medicament for the treatment or prophylaxis of the conditions specified herein.

Suitable inhibitors for miR-146a include antibodies and sense RNA sequences capable of interacting with these miRs. Such sense RNAs may correspond directly to the concomitant portion of the 3' UTR of CXCR4 mRNA, but there is no requirement that they do so. Indeed, as miRs frequently do not correspond entirely to the 3 ' UTR that they target, while the existence of dsRNA often leads to destruction of the target RNA, then it is a preferred embodiment that the inhibitor of miR-146a is entirely homologous for the corresponding length of miR-146a. The length of the inhibitor need not be as long as miR-146a, provided that it interacts sufficiently at least to prevent either of these miRs interacting with the 3' UTR or CXCR4 mRNA, when so bound.

Conditions treatable by miR-146a inhibitors include those associated with downmodulation of CXCR4, thereby requiring upregulation of CXCR4.

Preferred methods of delivery of the antisense miRNA or sense inhibitors may be by any gene therapy method known in the art, as will be readily apparent to the skilled person. Such methods include the so-called "gene-gun" method or delivery within viral capsids, particularly adenoviral or lentiviral capsids encapsulating or enclosing said polynucleotides, preferably under the control of a suitable promoter.

Preferred means of administration by injection include intravenous, intramuscular, for instance. However, it will also be appreciated that the polynucleotides of the present invention can be administered by other methods such as transdermally or per orally, provided that they are suitably formulated.

Also provided is a "test kit" capable of testing the level of expression of the CXCR4 protein such that the physician or patient can determine whether or not levels of the CXCR4 protein should be increased or decreased by the sense or antisense sequences of the present invention.

The present invention also encompasses a polynucleotide sequence, particularly a DNA sequence, which encodes the microRNAs of the present invention, operably linked to a suitable first promoter so that the MicroRNAs can be transcribed in vivo. Similarly, the present invention also provides a polynucleotide, particularly DNA, providing polynucleotides encoding the sense microRNA inhibitors of the present invention, also operably linked to a suitable second promoter for in vivo expression of said sense microRNA inhibitors.

hi particular, it is also preferred that the first and second promoters mentioned above can be controlled by a third element, such that the level of expression of the antisense microRNA and the level of expression of the sense microRNA inhibitors can be controlled in a coordinated manner. In this regard, it is preferred that a feedback mechanism is also included for establishing this level of control. Chimeric molecules are also provided, consisting of a polynucleotide according to the present invention, i.e. the antisense MicroRNAs or the sense microRNA inhibitors, linked to a second element. The second element may be a further polynucleotide sequence or may be a protein sequence, such as part or all of an antibody. Alternatively, the second element may have the function or a marker so that the location of microRNAs can be followed.

Thus, miR-146a and antagomirs thereof are useful in controlling or mediating expression of CXCR4 and controlling or mediating Megakaryopoiesis.

Here, we have shown that miR-146a masters Mk differentiation by targeting the CXCR4 receptor. This is surprising because miR-146a was only previously known in carcinogenesis (He et al., 2005) and in the regulation of the innate immune response (Taganov et al., 2006). However, our results now indicate that miR-146a plays a pivotal role in the control of CXCR4 expression in Mk differentiation. Although bioinformatic algorithms suggest that several miRs putatively target CXCR4 (see Results), their absence or specific expression profile in Mk cell lines suggests that they do not control CXCR4 protein levels in Megakaryopoiesis. In contrast, we demonstrated that miR-146a interacts directly with in the 3' UTR CXCR4 mRNA region and that CXCR4 is a specific target of miR-146a in the Mk lineage. Thus, our study represents the first report showing a role for a specific miR in the regulation of CXCR4 expression.

We identified a multistep regulatory mechanism in normal Megakaryopoiesis, including the transcription factor PLZF, miR- 146a and CXCR4, i.e., the receptor for stromal cell-derived factor- 1. In leukemic cell lines PLZF overexpression downmodulated miR- 146a and upregulated CXCR4 protein, whereas PLZF knock-down induced opposite effects.

In vitro assays indicated that: (a) miR-146a targets the CXCR4 mRNA, impeding its translation; (b) PLZF interacts with the miR- 146a promoter, inhibiting its transcriptional activity. In unilineage Megakaryopoietic (Mk) culture of cord blood CD34⁺ progenitors, PLZF expression was upregulated, whereas miR- 146a expression declined and CXCR4 protein level inversely increased. In functional experiments miR- 146a transfection impaired proliferation and differentiation/ maturation of Mk cells, as well as Mk colony formation. Altogether, our data indicate that normal Megakaryopoiesis is controlled by a novel cascade pathway, whereby an increased PLZF level suppresses transcription of miR-146a, hence activating CXCR4 translation.

The CXCR4 receptor is involved in the process of cancer metastasis (Wang et al. 2006), mediates HIV entry into CD4+ lymphocytes (Littman DR, 1998) and plays a major role in the pathogenesis of rheumatoid arthritis (Nanki et al. 2000). Particularly, the interaction between SDF-Ia and CXCR4 has a pivotal role in the directional migration of hematopoietic and epithelial cancer cells during the metastatic process (Burger and Kipps, 2006): therefore, small CXCR4 antagonist molecules have been developed for potential use in metastasis therapy (Tamamura and Fujii, 2005). In view of the capacity of anti-miR antisense oligonucleotides ("antagomirs") to exert effective in vivo actions (Krutzfeldt et al 2005; Care et al., 2007), our results indicate that these anti-miR- 146a molecules may represent a key tool for molecular therapy in the above clinical settings.

The invention will now be described in relation to the Figures and accompanying Examples. The Figures show:

Figure 1. PLZF expression induces miR-146a downmodulation and CXCR4 protein upmodulation in K562-PLZF cells

(a) Western blot analysis of PLZF protein expression in K562 cells infected with control (LXSN) or PLZF vectors. Actin is the internal control. One representative experiment (left panel) and PLZF protein expression ± SEM values from 3 independent clones of K562- PLZF cells (right panel) are shown, (b) Northern blot analysis of miR-146a expression in K562-LXSN and K562-PLZF cells. tRNA is shown for normalization. One representative experiment (left panel) and the ratio of miR-146a/ tRNA expression ± SEM values from 3 independent clones of K562-PLZF cells (right panel) are shown. ***P< 0.001 when compared to LXSN. (c) Real time PCR analysis of CXCR4 mRNA expression in K562- LXSN and K562-PLZF cells. The error bars represent the mean ± s.d. (n = 3). (d) Flow cytometry analysis of CXCR4 protein expression, performed on permeabilized K562-LXSN and -PLZF cells stained with PE-conjugated anti-CXCR4 mAb (CXCR4 PE, clear bar) or isotype control (shaded). The Geometric Mean Fluorescence Intensity Ratio (MFIR) of one representative experiment (middle panel) and mean CXCR4 expression ± SEM values from 3 independent clones (right panel) are shown. **P< 0.01 when compared to LXSN. (d) Left panel: Growth curve of K562-LXSN and K562-PLZF cultures treated with the Mk inducer, PDB, compared to untreated cells (C). Mean ± SEM values from 3 independent experiments is shown, *P< 0.05 when compared to LXSN Cont.+/- PDB; Right panel: FACS analysis of Mk markers CD61, CD41 and CD9 antigens expression at sequential days in cultures from cells treated with PDB or untreated (C).

Regulation loop among PLZF, miR-146a and CXCR4 protein expression in leukemic cells: (e) Left panel: Real time PCR analysis of PLZF mRNA expression level; right panels: Western blot analysis of PLZF protein level, in PLZF-siRNAs transfected HEL cells (siR) as compared to control- siRN As transfected HEL cells (C). (f) Northern blot analysis of miR-146a expression in PLZF-siRNA transfected HEL cells (siR) as compared to control siRNA-transfected HEL cells (C). tRNA was used for normalization, (g) Left panel: Real time PCR analysis of CXCR4 mRNA expression in C and siR HEL samples. The error bars represent the mean ± s.d. (n - 3); right panels: FACS analysis of CXCR4 protein level in siR-compared to C-HEL samples. A representative experiment out of three (left panel) and mean ± SEM of 3 independent experiments (right panel) are shown.

Figure 2. Regulation loop among PLZF, miR-146a and CXCR4 protein expression in leukemic cells

(a) Real time PCR analysis of PLZF mRNA expression level in PLZF siRNAs-transfected HEL cells (siR) as compared to control siRNAs-transfected HEL cells (C).

(b) Northern blot analysis of miR-146a expression in PLZF siRNA-transfected HEL cells (siR) as compared to control siRNA-transfected HEL cells (C). tRNA was used for normalization.

(c) Real time PCR analysis of CXCR4 mRNA expression in C and siR HEL samples.

(d) FACS analysis of CXCR4 protein level in siR-compared to C-HEL samples. A representative experiment out of four is shown.

Figure 3. PLZF, miR-146a and CXCR4 expression in purified HPCs induced to megakaryocyte (Mk) differentiation

(a) Immunofluorescence analysis of PLZF protein expression (α-PLZF) at the indicated sequential days in HPCs grown in liquid-phase unilineage Mk cultures. (-): no primary antibody negative control. The upper panels show the phase contrast microscopy fields.

(b) RT-PCR analysis of pre-miR-146a expression; and

(c) real time PCR analysis of miR-146a expression, performed at the indicated sequential days, in HPCs grown in liquid-phase unilineage Mk cultures. GAPDH is presented for normalization. (d) Real time PCR analysis of CXCR4 mRNA expression performed in HPCs during Mk differentiation. A, B, C and D show one representative experiment out of three.

(e) Flow cytometry analysis of CXCR4 protein expression in fixed and permeabilized HPCs during liquid-phase unilineage Mk cultures, stained with a PE-conjugated anti-CXCR4 mAb (shaded) or isotype control (clear). The Geometric Mean Fluorescence Intensity Ratio (MFIR) of one representative experiment is indicated.

Figure 4. mik-146a directly interacts with CXCR4 mRNA-3'UTR

(a) Flow cytometry analysis of CXCR4 protein expression in miR-146a transfected Jurkat cells. The transfected increasing amounts of miR-146a are indicated. CD3 expression was evaluated as a negative control.

(b) Real time PCR of CXCR4 mRNA in the same cells. One representative experiment out of three is shown.

(c) Sequence of the three putative miR-146a binding sites (in red) in the 3' UTR-CXCR4 mRNA region.

(d) Luciferase targeting assay on lysates from Phoem^'x cells co-transfected with miR-146a or non targeting RNA and psiCHECK 2 empty vector (E), or with the same vector containing the full length 3' UTR of CXCR4 mRNA (FL) or fragments of the CXCR4 3'UTR containing Site 2 (S2), Site 3 (S3), Sites 2 and 3 (S2/3), a mutated S2 site (Mut2) or a mutated S3 site (Mut3). Values represent the percentage of the luciferase activity obtained when co-transfecting the nontargeting oligonucleotide. Mean ± SEM values from three separate experiments. ** P< 0.01 where compared to control group.

Figure 5. PLZF binds and represses miR-146a promoter

(a) Scheme of the miR-146a promoter on chromosome 5 showing the PLZF binding site in the region upstream the pre-miR-146a. The arrows indicate the position of the primers used for the ChIP experiments.

(b) Chromatin immunoprecipitation from lysates of HEL cells with an anti-PLZF mAb (α- PLZF), an unrelated antibody (α-c-abl) or no antibody (no-Ab). Immunoprecipitated chromatin was analysed by PCR amplification of the miR-146a promoter region surrounding the PLZF binding site (Prom 146a) or a region that does not contain PLZF binding sites (Control Prom) or a GAPDH coding region, to detect non relevant DNA sequences. A sample representing 0.02% of total chromatin (input) was included in the PCR analysis. (c) Promoter activity assay of the pre-miR-146a upstream DNA sequence (Prom 146a) cloned in a promoterless luciferase reporter vector (pGL3Basic) and transfected into Phoenix cells in the presence (clear bars) or absence (black) of a PLZF expression vector. Vector indicates the empty reporter vector.

(d) Promoter activity assay of the pre-miR-146a upstream DNA sequence (Prom 146a), as in C, or the same region with a mutated PLZF binding site (shown on the right) (Mut. Prom 146a), cloned in a reporter vector containing a minimal promoter (pGL3Prom) and transfected with or without a PLZF expression vector.

The data in c and d are reported as percentage of the value obtained with the promoter constructs in the absence of the PLZF vector. Mean values of three independents experiments.

Figure 6. Ectopic miR-146a expression in Mk cells downmodulates CXCR4 protein expression and impairs cell growth and Mk differentiation-maturation

(a) Growth curve of Mk culture transfected with rm^'R-146a (miR-146a), or a control oligonucleotide (Cont. oligo), compared to untreated Mk cells (Control Mk). Mean ± SEM values from 3 independent experiments. **P< 0.01 when compared with control oligonucleotide transfected cells.

(b) Left panel: real time PCR analysis of CXCR4 mRNA expression in unilineage Mk culture transfected on day 1 (arrow) with miR-146a oligonucleotide (miR-146a), or non targeting oligonucleotide (Cont. oligo), compared to control Mk culture (Control Mk). Right panel: CXCR4 protein expression, evaluated at sequential days by flow cytometry in Mk culture in the miR-146a, Cont. oligo and Control Mk groups. The cells were permeabilized and stained with a PE-conjugated anti-CXCR4 mAb (shaded bar) or isotype control (clear). The Geometric Mean Fluorescence Intensity Ratio (MFER) of one representative experiment is indicated.

(c) Morphological analysis of the differentiation and maturation of Mk cells transfected with miR-146a (miR-146a), or control oligonucleotide (Cont. oligo) or untreated (Control Mk)₅ collected at day 14 and stained with May-Grϋnwald-Giemsa for evaluation of cells featuring polylobated nuclei {Left panel). Mean ± SEM values from 3 independent experiments. **P< 0.01 when compared with Cont. oligo transfected cells.

(d) FACS analysis of CD61 and CD41 antigens expression at sequential days in Mk cultures from cells transfected with miR-146a (miR-146a), or control oligonucleotide (Cont. oligo) or untreated (Control Mk). (e) Megakaryocyte (upper panel), erythroid and granulo-monocytic (left and right lower panel respectively) colony-forming capacity of HPCs transfected with miR-146a (miR-

146a), or control oligonucleotide (Cont. oligo) or untreated HPCs cells (C). Mean ± SEM values from 3 independent experiments. **P< 0.01 when compared with Cont. oligo transfected cells.

Figure 7. Inhibition of PLZF expression by siRNA treatment impairs megakaryopoiesis

(a) Growth curve of Mk culture cells transfected with siRNA targeting PLZF mRNA (PLZF-siRNA) or non targeting control siRNA (C-siRNA), as compared to untreated Mk cells (Control Mk). Mean ± SEM values from 3 independent experiments.

(b) Real time PCR analysis of PLZF mRNA expression {Left panel) (mean ± SEM values from 3 independent experiments) and RT-PCR analysis of pre-miR-146a expression {Right panel) (results from a representative experiment out of three are presented) in unilineage Mk culture transfected on day 1 (arrow) with PLZF-siRNA, as compared to control C- siRNA Mk culture.

(c) Flow cytometry analysis of CXCR4 protein expression, at sequential days in Mk culture in the PLZF-siRNA, C-siRNA and Control Mk groups. The Geometric Mean Fluorescence Intensity Ratio (MFIR) indicates mean ± SEM values from 3 independent experiments. **P< 0.01 when compared with C-siRNA transfected cells.

(d) Morphological analysis of the differentiation and maturation of Mk cells transfected with PLZF-siRNA (PLZF), or control C-siRNA (C) or untreated (Cont. Mk), collected at day 14 and stained with May-Grunwald-Giemsa for evaluation of cells featuring polylobated nuclei {Left panel). Mean ± SEM values from 3 independent experiments. **P< 0.01 when compared with control C-siRNA transfected cells.

(e) FACS analysis of CD61 and CD9 antigens expression at sequential days in Mk cultures from cells transfected with PLZF-siRNA, or control C-siRNA or untreated (Control MK).

(f) Megakaryocyte (upper panel), erythroid and granulo-monocytic (left and right lower panel respectively) colonies generated by HPCs transfected with PLZF-siRNA (PLZF) or control C-siRNA (C), or by untreated HPCs (Cont.) (C). Mean ± SEM values from 3 independent experiments. **P< 0.01 when compared with control C-siRNA transfected cells.

Figure 8: MicroRNA expression profiling in leukemic cell lines Northern Blot analysis of human microRNAs expression in leukemic cell lines: K562-PLZF compared to K562-LXSN cells, HEL and Jurkat (JURK). MiRs expressed in the cell lines are shown (except for miR-9). MiR-Ib, 7b, 9, 122a, 124, 125a, 129, 133, 139*, 148a, 153, 183, 184, 191, 200b, 205, 217, 221, 222, 224*, 281-1, 281-2, 381*, 410* were not expressed in these cell lines. tRNA was used for RNA normalization. Representative results from three independent experiments are shown, * Putative miRs targeting CXCR4 mRNA (www.TargetScan.org^').

Figs 9-11 are described in more detail in the Examples below.

EXAMPLES

EXPERIMENTAL PROCEDURES

Cell lines. K562, HEL, Jurkat and Phoenix cells were cultured using standard methods. Transduced K562-LXSN and three selected clones 1, 2, 3 of K562-PLZF cells

were prepared as described³².

PDB treatment of K562 cells. Cells were incubated for 4 days in the presence of PDB (Phorbol Dibutyrate, Sigma St Louis, USA) as described in³⁵ and analyzed for Mk membrane markers (CD9, CD41, CD61) expression by Flow cytometry analysis, as

described below.

Unilineage and clonogenic Mk culture. Collection of CB, isolation of CD34⁺ cells, unilineage Mk culture and morphology analysis were performed as described³⁵. For clonogenicity assays, after miR-146a or nontargeting oligonucleotides transfection, 1x10³ HPCs were plated in methylcellulose medium containing a saturating concentration of TPO³⁵. HPC colonies were scored on days 10-14. Multilineage cultures of CD34⁺ HPCs

were performed as previously described³⁷. Western blotting

Western blot analysis to evaluate PLZF protein expression was performed using the anti-PLZF mAb (Oncogene Research Products, Boston, MA)³².

MiR expression analysis

Northern blotting of 25 μg of total RNA and miRs expression analysis were performed as described¹¹.

Quantitative real-time (qRT)-PCR analysis of miR-146a was performed by TaqMan technology, using the kit ABI PRISM 7700 DNA Sequence Detection System specific for miR-146a reverse transcription and PCR analysis (Applied Biosystems, Foster City, CA) according to the manufacturer's procedure.

Reverse transcription (RT)-PCR analysis for pre-miR-146a was performed using random primers-RT kit (Invitrogen), according to the manufacturer's procedure. PCR analysis was performed with the following pre-miR-146a primers: forward - SEQ ID NO. 9; Reverse - SEQ ID NO. 10. PCR products were analyzed by Southern blot using a miR-146a antisense ³²P-labelled probe: SEQ ID NO. 11. Normalization was performed using GAPDH RT-PCR analysis³². qRT-PCR analysis for GAPDH, PLZF (ZNF 145), CXCR4 was performed according to standard procedures³². Commercial ready-to-use primers/probe mixes were used (Assays on Demand Products, Applied Biosystems, Foster City, CA).

Flow cytometry analysis

To evaluate total CXCR4 protein expression, cells were fixed and permeabilized (Permi-Fix, BD/Pharmingen) before incubation with either PE-conjugated anti-CXCR4 mAb (clone 1245, BD/Pharmingen) or a control PE-conjugated mAb of the same isotype. The cells were analyzed using the FACSCAN flow cytometer and the Cell Quest software (BD) for acquisition and analysis. The reported mean fluorescence intensity (MFIR) indicates the ratio between the geometric mean fluorescence values observed in CXCR4 labeled cells and cells labeled with a negative control, gated on FSC/ SSC dot plot to select viable cells. The same procedure was followed for CD3 detection, using a PE-conjugated anti-CD3 mAb (clone UCHTl, BD/Pharmingen).

Flow cytometric analysis of K562-LXSN and K562-PLZF cells untreated or treated with PDB, and Mk cultures, were performed as described³⁵.

Morphological and immunofluorescence analysis

For morphological analysis, F£PCs were harvested on different days of Mk unilineage culture, transferred to glass slides by cytospin centrifugation and stained with May- Grϋnwald-Giemsa.

For immunofluorescence labelling, cells were fixed on glass slides in absolute methanol at room temperature for 5 min, permeabilized in absolute acetone at -20° C for 2 minutes and washed in PBS. The slides were then incubated 30 min. with the anti-PLZF mAb (1 : 100 in PBS/BSA, final dilution 10 μg/ml), washed in PBS and incubated for 30 min with FITC-labeled F(ab⁷)₂ fragments of affinity goat purified α-Mouse IgG 1 :40 (Dakopatts, Copenhagen, Denmark). After extensive wash in PBS, slides were mounted with antifade glycerol mounting medium (Moulecular Probes, Eugene, OR, USA) and analyzed under an inverted Olympus FV500 equipped for confocal microscopy.

ChIP assay

3x10⁶ HEL cells were crosslinked by addition to the culture medium of formaldehyde at 1% final concentration and incubation for 10 min at 37°C. After sonication, ChIP assay kit (Upstate USA, Charlottesville, VA) was used according to the manufacturer's procedure and protein-DNA complexes were immunoprecipitated overnight with the anti-PLZF mAb, an anti c-abl mAb (Oncogene Research Products, Boston, MA) or protein-A sepharose only. A genomic fragment of 128 bp containing the PLZF binding site in the pre-miR-146a upstream region (Prom 146a) {Ensembl Genomic Gene ID ENSGOOOOO 199083) was amplified by PCR using primers flanking the PLZF site: forward SEQ ID NO. 12; reverse SEQ ID NO. 13. PCR products were Southern blotted and hybridized with the internal probe ³²P-labeled oligonucleotide SEQ ID NO. 14. As control, a flanking 140 bp genomic region, not containing any PLZF site, was amplified by PCR and analyzed using the following primers: sense SEQ ID NO. 15; antisense SEQ ID NO. 16 and internal probe SEQ ID NO. 17. Non relevant cellular DNA sequences were detected by amplification of a GAPDH coding region using primers and PCR conditions as described³².

Promoter assay

A 475 bp DNA fragment of Prom 146a was PCR-amplified from genomic DNA using the primers forward SEQ ID NO. 18 and reverse SEQ ID NO. 19, and cloned upstream to the luciferase gene into the pGL3Basic (pGL3Basic/Prom 146a) and pGL3 Promoter (pGL3 Prom/Prom 146a) vectors (Promega). By mutagenesis of the PLZF site into the pGL3 Prom/Prom 146a vector using, according manufacturer's instructions, the QuickChange Site-Directed mutagenesis kit (Stratagene), we prepared the PLZF mutated Prom 146a vector (pGL3Prom/Mut. Prom 146a). The pcDNA3/PLZF (PLZF) was previously reported³¹. All vectors were checked by automated sequencing.

In luciferase assay experiments, Phoenix cells were transfected using Lipofectamine 2000 (Invitrogen), with a Renilla luciferase vector (50 ng), together with luciferase vectors described above (0,8 ug) and, where indicated, with the pcDNA3/PLZF. Luciferase activity was measured 48 hr post-transfection with the Dual Luciferase Reporter System (Promega, Madison, WI, USA) according to the manufacturer's instructions, by using Microlite TLXl (Dynatech Laboratoires, Chantilly, CA) and then normalized for Renilla Luciferase activity. Data are presented as mean values, obtained from at least three independents experiments. Silencing of PLZF or CXCR4 expression by siRNAs

Two double-stranded small interfering PLZF-siRNA sequences (NM_001018011), or CXCR4-siRNAs sequences (NM_003467) and the control C-siRNA sequence with no homology to the human genome were purchased from Dharmacon, Lafayette, CO. Cells were co-transfected with 90 nM PLZF-siRNA or CXCR4-siRNAs or C-siRNA and FITC- conjugated nontargeting oligonucleotide using Lipofectamine according to the manufacturer's instruction. After 48 hrs, cells were harvested and analysed for PLZF, miR- 146a and CXCR4 mRNA and protein expression as described above.

Transfection of miR-146a oligonucleotide

Stability-enhanced miR-146a oligonucleotide (miR-146a), control nontargeting RNA oligonucleotide (Cont. oligo), antagomir-146a (α-miR-146a), unrelated antagomir-133a (Cont. α-miR) and FITC-conjugated nontargeting oligonucleotide were purchased from Dharmacon. Cell lines were transfected using Lipofectamine and collected for analysis 48 hrs after transfection. HPCs were transfected at day 0 or 1 of Mk cultures and collected at sequential times through Mk differentiation/maturation.

Luciferase target assay

Plasmids and constructs We used the psiCHECK 2- 3'UTR vector (Promega, Madison, WI USA) to clone, downstream to the Renilla luciferase gene, the full length 3' UTR of CXCR4 mRNA (FL wild type, wt vector) (NM_001008540, from position 1450 to 1862), which includes 3 putative miR-146a binding sites (Site 1 : from 1452 to 1473 bp; Site 2: from 1575 to 1596 bp; Site 3: from 1800 to 1821 bp). In the same plasmid we cloned CXCR4 mRNA fragments: Sl (1450 to 1573 bp, including Site 1), S2 (1554 to 1775 bp, including Site 2), S3 (1756 to 1862 bp, including Site 3), S2/3 (1554 to 1862 bp, including both Sites 2 and Site 3) and Cont. (1621 to 1750 bp) a fragment without putative miR-146a binding site to use as a control. By mutagenesis (QuickChange Site-Directed mutagenesis kit ,Stratagene) of the Site

1, Site 2 and/or Site 3 in the reporter vectors FL wt and S2, S3, we prepared the constructs FL-Mutl, FL-Mut2, FL-Mut3, FL-Mut2/3 and MutS2, MutS3 respectively. We used the following primers: for the mutation of Site 1, SEQ ID NO. 20 5'- GACCAATATTGTAGCGGCCGCA TTGCTTGTTGGATTT-3 ' (from 1452 to 1489); Site

2, SEQ ID NO. 21 5'-GCAGGACCTGTGGCCAGCGGCCGCGTTGCTGTATGTCTCG- 3' (from 1573 to 1612); Site 3, SEQ ID NO. 22 5'- ATAGAAATGCTGGTTTGCGGCCGC7OGGAGTGGG TTGATTTC-3' (from 1795 to 1837).

Luciferase assay. Phoenix cells were cotransfected with 75 ng of psiCHECK-2 reporter vectors and 60 pmol of either the nontargeting RNA oligonucleotide or stability- enhanced miR-146a oligonucleotide, using Lipofectamine. After 48 hrs, cells were lysed with Passive Lysis Buffer (Promega, Madison, WI USA), and their Renilla luciferase activity was measured by using the Femto-master FB 12 (Zylux, Oak Ridge, TN). The relative reporter activity was obtained by normalization to the psiCHECK-2- 3' UTR/nontargeting oligonucleotide, cotransfection.

Transfection of miR-146a and CXCR4-3'UTR mutated expression vectors Plasmids constructs. We used the pcDNA 3.1 expression vector (Promega, Madison, WI USA) to subclone into Hind III /Eco RI sites, the full length CXCR4 coding sequence and its 3'UTR region (CXCR4-3'UTR wild type, CXCR4-wt vector). The oligonucleotides used for PCR amplification were: CXCR4-forward SEQ ID NO. 23 and 3'UTR-reverse SEQ ID NO. 24. By mutagenesis (QuickChange Site-Directed mutagenesis kit ,Stratagene) of the 3' UTR region of CXCR4-wt vector into both Sites 2 and 3, we prepared the construct CXCR4-3'UTR/S2/S3 mutated (CXCR4-Mut2/3) . We also subcloned pre-miR-146a in pcDNA 3.1 vector (miR-146a). All vectors were checked by automated sequencing. The transfection of CXCR4-wt, CXCR4-Mut2/3 or miR-146a were performed in K562-LXSN and overexpression tested by FACS analysis and Northern blot analysis respectively (data not shown).

Transfection assays. K562 cells were cotransfected with 100 ng of pcDNA 3.1 empty vector, CXCR4-wt or CXCR4-Mut.2/3 vector and miR-146a vector, using Lipofectamine. After 48 hrs, cells were treated with PDB and analyzed 2/4 days later for CXCR4 and Mk markers expression, as compared to untreated cells.

RESULTS

PLZF, miR-146a and CXCR4 protein expression correlate in hematopoietic cell lines

In previous studies we transduced K562 cells with PLZF (K562-PLZF cells) and characterized them, as compared to control cells transduced with empty vector (K562- LXSN)³² (Fig.la). In three clones of K562-PLZF (clones 1, 2, 3)³² the miRNA expression profile was evaluated by Northern blot (Fig 8). Among several miRNAs of interest, we found that miR-146a expression is consistently downmodulated in K562-PLZF cells (Fig. Ib, PLZF), compared to control ones (Fig. Ib, LXSN and Fig. 8), suggesting that miR-146a may be a target of PLZF. In K562-PLZF cells, CXCR4 mRNA expression is not significantly modulated, whereas CXCR4 protein level is upregulated, as indicated respectively by quantitative Real Time (qRT-) PCR (Fig. Ic, left panel) and FACS evaluation (Fig. Ic, middle and right panels). Analysis of the sequence of the CXCR4 gene promoter did not reveal any functional PLZF binding site, suggesting that CXCR4 is not a direct target gene of PLZF. On the other hand, the discrepancy between CXCR4 mRNA and protein levels in K562-PLZF cells suggested that CXCR4 mRNA may be targeted by miR- 146a: this hypothesis was supported by a single bioinformatic algorithm (https://microrna.sanger.ac.uk/).

Importantly, the algorithm www.TargetScan.org suggested that CXCR4 mRNA is a putative target of other miRNAs, including miR-P, -93, -224, -381, -410. However, miR-P, - 224, -381, -410 were not expressed in hematopoietic cell lines; in the case of miR-95, we found no correlation with the levels of CXCR4 protein expression (Fig. 8 and legend). miR- 23b is not consistently regulated in different K562-PLZF clones (Fig.8), does not target CXCR4 3'UTR and does not modify the expression of CXCR4 protein when overexpressed in Jurkat cells (Fig 9).

To further investigate the regulatory network involving PLZF and miR-146a in the control of CXCR4 expression, we used the PLZF positive cell line HEL to downmodulate PLZF expression by small interfering RNAs (siRNA), as shown by qRT-PCR (Fig 10a left panel) and Western blot analysis (Fig 10a middle and right panels), and examined the effects on miR-146a, and CXCR4 expression at mRNA and protein level. PLZF siRNA (siR), but not control siRNA (C), promoted miR-146a expression (Fig. 10b), while inducing CXCR4 protein downmodulation (Fig. 10c, middle and right panels), without any significant change of CXCR4 mRNA level (Fig.10, left panel).

Overall, our results showed an inverse correlation between PLZF and miR-146a level, as well as between miR-146a and CXCR4 protein expression in both K562 -PLZF cells and PLZF siRNA-treated HEL cells.

K562 cells induced to Mk differentiation: a model system to investigate the role of the PLZF/miR-146a/CXCR4 receptor pathway in megakaryopoiesis.

We then analyzed the growth and Mk differentiation of K562-PLZF cells, as compared to K562-LXSN control³³. Their proliferation rate was slightly lower than in control cells (Fig Ie, left panel). In three clones of K562-PLZF cells the expression of the Mk differentiation antigens CD61, CD41 and CD9 was increased, as compared to the control cells (right panel). Upon Mk differentiation induced by phorbol dibutyrate (PDB)³⁴, PLZF expression was enhanced (Fig. Id, left panel), while the level of pre-miR-146a was reduced (middle panel) and that of CXCR4 protein was increased (right panel). PDB treatment impaired cell proliferation (Fig Ie, left panel), while inducing the expression of CD41, CD61, CD9 antigens at significantly higher levels in K562-PLZF than in control cells (right panel). Altogether, these data indicate that PLZF potentiates the Mk differentiation of K562 cells and is coupled with diminished miR-146a and enhanced CXCR4 protein expression. We hence postulate that these cells represent a useful model system for evaluation of the role of the PLZF/miR-146a/CXCR4 complex in Mk differentiation.

PLZF, miR-146a and CXCR4 expression in unilineage Mk culture

Our observations in K562 cells prompted us to evaluate the expression of PLZF, miR- 146a, CXCR4 mRNA and protein, at sequential days, in human cord blood (CB) CD34⁺ HPCs induced to unilineage differentiation and maturation through the Mk pathway in serum-free liquid suspension culture³⁵.

In agreement with the effects of PDB in K562 cells, in these Mk cultures, PLZF protein expression is gradually upregulated starting from day 1-3, as shown by anti-PLZF immunofluorescence and Western blot analysis (Fig. 2a). In parallel, we analyzed pre-miR- 146a (pre-miR-146a) and miR-146a expression by RT-PCR (Fig. 2b, left panel) and qRT- PCR (Fig. 2b, right panel) analysis respectively. The data showed a high level of pre-miR- 146a and miR-146a expression in quiescent CD34⁺ cells (day 0), followed by a gradual, marked downmodulation in the day 1-3 period, which persisted thereafter. Thus, we observed an inverse correlation between PLZF protein and miR-146a expression levels during Mk differentiation/maturation (Fig. 2d).

In the same Mk cultures, the CXCR4 mRNA level, analysed by RT-PCR, showed a relatively mild fluctuation: specifically, it was elevated at day 0 and 1, lower at day 3-8 and then elevated again at day 10 (Fig. 2c, left panel). These fluctuations are seemingly mediated by transcriptional regulatory mechanisms. Conversely, FACS analysis showed that total CXCR4 protein expression was markedly upregulated, peaking at day 3 and sustainedly enhanced through day 10 (Fig. 2c, right panel). The rise of CXCR4 protein, in presence of a decreased mRNA, suggests that the sharp decline of miR-146a effectively unblocks CXCR4 mRNA translation.

Altogether, these data indicate that during Mk differentiation, (i) miR-146a level was inversely related to PLZF expression (Fig. 2d); (ii) miR-146a expression was inversely related to CXCR4 protein level (Fig. 2e). The results hence suggest a transcriptional control by PLZF on miR-146a expression, coupled with a posttranscriptional control of miR-146a on CXCR4 protein level.

CXCR4 is a direct target of miR-146a

To verify whether CXCR4 is a direct target of miR-146a, we over-expressed miR- 146a in the Jurkat cell line, since it is negative for miR-146a expression (Fig. 8). We cotransfected the cells with: (a) a nontargeting scrambled oligonucleotide or miR-146a and (b) a FITC-conjugated non-targeting oligonucleotide used as a tracer for FACS analysis. 80% of the cells were FITC-positive (data not shown). FACS evaluation of CXCR4 protein expression revealed that CXCR4 protein was downmodulated by miR-146a in a dose- dependent way (Fig. 3a, right panel), without any significant modulation of CXCR4 mRNA expression (Fig. 3b). Conversely, CXCR4 level was unchanged in cells transfected with the nontargeting oligonucleotide (Fig. 3a, left panel). The inhibition of CXCR4 protein expression was time-dependent, with the lowest level at 48 h post-transfection (not shown). The specificity of these effects was confirmed by FACS analysis of the CD3 molecule, which was not modulated by the nontargeting oligonucleotide or miR-146a transfection (Fig. 3a). To demonstrate the direct interaction between miR-146a and CXCR4 mRNA, we inserted downstream ofa Renilla luciferase reporter cDNA the full length 3' UTR region of CXCR4 mRNA (FL wt), including three sequences complementary to miR-146a (Fig. 3c, Site 1, 2, 3), as well as the FL region containing a mutagenized Sl, S2, S3 or S2/S3 binding sites (vectors FL-Mutl, FL-Mut2, FL-Mut3, FL-Mut2/3, respectively). We also inserted in the reporter vector, shorter fragments containing the single putative binding site 1, 2, 3, or 2 and 3 (vectors Sl, S2, S3, S2/3, respectively) or their mutants (MutS2, MutS3) (Fig. 3d). Each vector was transfected in the miR-146a negative Phoenix cell line, together with miR- 146a or a control non-targeting oligonucleotide (Cont. oligo). The luciferase activity of the FL wt, FL-Mutl, FL-Mut2, FL-Mut3, and also of the S2, S3 or S2/3 reporter vectors was markedly diminished after miR-146a cotransfection (Fig. 3d, miR-146a), while not affected by the non-targeting oligonucleotide (Fig. 3d, Cont. oligo). We did not notice any modulation of luciferase activity with the Sl reporter vector (Fig. 3d, Fragments/miR-146a) excluding the Site 1 as a miR-146a binding site. Importantly, we did not detect any modulation of luciferase activity after miR-146a cotransfection with the FL-Mut2/3, MutS2 or MutS3 reporter vector, containing a mutagenized S2/S3, S2 or S3 binding site respectively (Fig. 3d). The results hence indicated that miR-146a interferes with CXCR4 mRNA translation via direct interaction with the 3' UTR-FL (FL-wt), specifically via miR- 146a binding of S2 and S3 sites.

PLZF is a transcriptional repressor of miR-146a expression

To investigate the potential interaction between PLZF and miR-146a expression, we analyzed the genomic region upstream the pre-miR-146a on chromosome 5 (ENSGOOOOO 199083). At position (-137; -117) we found the sequence SEQ ID NO. 25 5'- TAGACCTGGZ4 CT AGGAAGC A-3' (Fig. 3e), related to the previously described PLZF DNA-binding consensus³⁶. We evaluated whether PLZF was recruited at the pre-miR-146a DNA upstream region (Fig. 3e) by chromatin immunoprecipitation (ChIP) experiments performed on the PLZF- expressing HEL cell line. Protein-DNA complexes were immunoprecipitated with either the anti-PLZF mAb, or no antibody, or the unrelated anti-c-abl mAb used as negative control (Fig. 3f, α-PLZF, no-Ab, α-c-abl respectively). The immunoprecipitates were then amplified by PCR using: (i) primers flanking the putative PLZF DNA-binding site in the Prom 146a region (Fig. 3f, Prom 146a); (ii) primers recognizing a region upstream to Prom 146a sequence, which does not contain any identifiable PLZF site (Fig. 3f, Control Prom). Our ChIP assays indicated that in HEL cells PLZF is associated to the region surrounding the putative PLZF binding site into the Prom 146a genomic region.

We then cloned this region in a luciferase reporter construct (pGL3Basic) and investigated its ability to promote transcription. The pGL3Basic/Proml46a was able to enhance by 30-fold the luciferase activity over pGL3Basic level, whereas PLZF expression was able to repress 40% of this effect (Fig. 3g, left panel). To further demonstrate that PLZF acts as a repressor on the newly identified PLZF-binding site described above, we cloned the region containing this site (Prom 146a) or a mutagenized version (Mut. Prom 146a) into a reporter vector containing an artificial minimal promoter (pGL3 Promoter) and performed luciferase assays. PLZF was able to repress the luciferase activity detected for the Prom 146a vector, but not the activity of the Mut. Prom 146a vector (Fig. 3g, right panel).

Together our data identified a new PLZF DNA-binding site in Prom 146a and indicated PLZF as a direct transcriptional repressor of miR-146a.

Enforced expression of miR-146a in unilineage Mk cultures blocks CXCR4 protein expression and impairs cell proliferation and differentiation/maturation

We further evaluated whether ectopic expression of miR-146a affects the proliferation, differentiation and maturation of Mk cultures. CD34⁺ cells, seeded in unilineage Mk liquid suspension cultures, were co-transfected on day 1 with: (a) miR-146a or a nontargeting scrambled oligonucleotide and (b) a FITC- conjugated non-targeting oligonucleotide used as a tracer for FACS analysis. Transfection efficiency was 70% (data not shown). We sorted the FITC-positive cells by FACS and seeded them in unilineage Mk liquid suspension culture or in semisolid Mk medium for clonogenic analysis³⁵.

In Mk liquid culture, we analyzed at sequential times miR-146a transfected cells (miR-146a), as compared to nontargeting oligonucleotide transfected (Cont. oligo) and untransfected cells (Control Mk) (Fig. 4a). Analysis of CXCR4 mRNA expression by qRT- PCR (Fig. 4b, left panel) and CXCR4 protein level by FACS evaluation (Fig. 4b, right panel) indicated that miR-146a enforced expression induced protein downmodulation throughout megakaryopoiesis, without significantly affecting mRNA expression, indicating that CXCR4 is a target of miR-146a in the Mk lineage.

Notably, miR-146a-transfection induced a marked decrease in proliferation rate (Fig. 4a) and a marked reduction of cells with polylobated nuclei, suggesting a reduced Mk maturation (Fig. 4c). This was confirmed by analysis of CD61 and CD41 (Fig. 4d) and CD9 (not shown) antigens, whose expression was clearly lower in miR-146a-transfected cells than in control ones.

By clonogenic Mk³⁵ and multilineage³⁷ progenitor assay, we observed that miR-146a overexpression reduced the number of Mk colonies to ~ 30% of control value (Fig. 4e, upper panel), whereas it did not affect the number of BFU-E and CFU-GM colonies (lower panels), thus indicating that the miR-146a effect is Mk-specific.

Mk cultures were also transfected with siRNAs that specifically blocked PLZF expression (PLZF-siRNA) or nontargeting control siRNA (C-siRNA), together with FITC- conjugated double-stranded RNA. FITC positive cells were then sorted and cultured. Silencing of PLZF mRNA (Fig.5b, right panel) i) enhanced pre-miR-146a expression (Fig. 5b, left panel), ii) decreased CXCR4 protein level as evaluated by FACS (Fig. 5c), iii) decreased cell proliferation (Fig. 5a), and iv) impaired Mk differentiation/maturation, as shown by morphology (Fig. 5d), reduced expression of the surface antigens CD61, CD9 (Fig. 5e), CD41 and CD42b (not shown), as well as by clonogenic Mk and multilineage progenitor assay (Fig. 5f). These results are equivalent to those obtained when miR-146a, was transfected in Mk cultures (Fig. 4), confirming that the effects of PLZF on megakaryopoiesis are mediated by miR-146a.

Altogether, our results indicated that a sustained overexpression of miR-146a impaired Mk proliferation and induced retardation of Mk differentiation/maturation. This was associated with a decrease of CXCR4 expression at protein but not mRNA level, showing that normal Mk proliferation and differentiation/maturation is promoted by the PLZF mediated down-modulation of miR-146a, which causes an increased CXCR4 protein expression.

MiR-146a suppression in unilineage Mk cultures increases CXCR4 protein expression and stimulates cell proliferation and differentiation/maturation

We then evaluated whether the early suppression of miR-146a activity affects cell proliferation, differentiation and maturation in Mk cultures.

CD34⁺ cells, seeded in unilineage Mk liquid cultures, were co-transfected after 10 hrs with: (i) antagomiR-146a oligonucleotide (α-miR-146a) or control unrelated antagomir- 133a (Cont. α-miR) and (ii) a FITC-coηjugated non-targeting oligonucleotide used as a tracer for FACS analysis. We sorted the FITC-positive cells by FACS and seeded them again in unilineage Mk liquid culture. We next analyzed at sequential times antagomir-146a transfected cells (α-miR- 146a), as compared to control (Cont. α-miR) and untransfected cells (Control Mk) (Fig. 11). Antagomir-146a transfection induced a significant CXCR4 protein upmodulation throughout megakaryopoiesis (Fig. l ib right panel) without any significant change of CXCR4 mRNA level (left panel): this was coupled with a modest increase in proliferation rate (Fig. Ha) and an accelerated Mk differentiation and maturation, as indicated by the increase of cells with polylobated nuclei (Fig. l ie), as well as the rise of CD61 and CD41 expression (Fig. l id). Actually, the accelerated block of miR-146a produced a 2-3-day earlier upregulation of CXCR4 and a correspondingly earlier onset of Mk differentiation. Altogether, the results observed by transfection of antagomir- 146a in Mk cells mirror those observed upon miR-146a enforced expression (Fig. 4).

Knockdown of CXCR4 in unilineage Mk cultures impairs cell proliferation and differentiation/maturation

We then blocked CXCR4 expression in Mk cultures by siRNA. Cells were cotransfected with siRNA targeting CXCR4 mRNA (CXCR4-siRNA) or nontargeting control siRNA (C-siRNA), and FITC-conjugated double-stranded RNA. FITC positive cells were then sorted and cultured. Silencing of CXCR4 mRNA (Fig. 6b) decreased CXCR4 protein level as evaluated by FACS (Fig. 6c), decreased cell proliferation (Fig. 6a) and impaired Mk differentiation/maturation, as shown by morphology (Fig. 6d) and reduced expression of the surface antigens CD61, CD9 (Fig. 6e), CD41 and CD42b (not shown), as well as by clonogenic Mk and multilineage progenitor assay (Fig. 6f).

These results are equivalent to those obtained when miR-146a or PLZF-siRNA, were transfected in Mk cultures (Fig. 4 and 5) and confirm that the crucial effects of miR-146a on megakaryopoiesis are mediated by CXCR4.

Rescue of miR-146a expression blocks CXCR4 translation and impairs PDB- induced Mk differentiation of K562-PLZF cells

To further strengthen the link between PLZF, miR-146a and CXCR4 expression, we transfected miR-146a in K562-PLZF and K562-LXSN cells, used as a model of inducible Mk differentiation³⁴ (Fig. 1). After co-transfection with: (a) a nontargeting scrambled oligonucleotide (C-oligo) or miR-146a and (b) a FITC-conjugated non-targeting oligonucleotide used as a tracer for FACS analysis, 85% of the cells were FITC-positive (data not shown). FACS evaluation of CXCR4 protein expression in miR-146a transfected cells revealed that CXCR4 protein was downmodulated by miR-146a (Fig. 7a). We then analyzed the expression of Mk markers in miR-146a transfected cells after four days of PDB treatment (Fig. 7b,c): i) Mk membrane markers are clearly induced in K562-PLZF cells, as compared to LXSN cells; ii) miR-146a transfection abrogated the increase of the Mk markers CD9, CD41, CD61 expression induced by PDB; (iii) notably, in miR-146a- trasfected K562-PLZF cells the expression level of these markers was downmodulated to that observed in K562-LXSN cells. Therefore, the rescue of miR-146a expression blocks CXCR4 translation and impairs Mk-like differentiation in K562-PLZF cells, thus confirming that the effects of PLZF are exerted through the miR-146a/CXCR4 pathway.

Expression of a CXCR4 mutated in the 3' UTR rescues the inhibitory effect induced by miR-146a on Mk differentiation of K562 cells

To obtain critical evidence linking the effects of miR-146a on Mk differentiation to its activity on CXCR4 translation, we investigated the rescue of these effects upon expression of a mutated CXCR4 not targeted by miR-146a. Specifically, K562 cells induced to Mk differentiation by PDB treatment were co-transfected with both miR-146a and expression vectors for the full length CXCR4 cDNA including the 3'UTR region (wt), or a CXCR4 cDNA deleted of the entire 3'UTR, (del) or a full length CXCR4 cDNA carrying mutations of the miR-146a binding sites 2 and 3 in the 3'UTR (mut). We next measured the expression of CXCR4 and Mk differentiation markers. The results indicate that the deletion or mutation of the CXCR4 3' UTR almost abolished the effects of miR-146a on CXCR4 expression (Fig 7 d) and restored the induction of the Mk differentiation markers CD41 and CD61 (Fig 7 e, f). Thus, the inhibitory effects of miR-146a on Mk differentiation of K562 cells are rescued by CXCR4, indicating that these effects are mostly exerted through the control of CXCR4 translation. DISCUSSION

We have identified a molecular cascade controlling normal megakaryopoiesis. This regulatory pathway involves: (a) enhanced expression of PLZF, which inhibits miR-146a transcription; (b) miR-146a downmodulation, which causes an increased translation of the target CXCR4 mRNA. In this regulatory loop miR-146a acts as an effector of the PLZF transcription factor, mediating its control activity on CXCR4 protein level and megakaryopoiesis .

MiRNAs are posttranscriptional regulators of gene expression, which play a key role in basic cell functions under normal and abnormal conditions³'³⁸. Although their role in the control of normal⁹ and leukemic³⁹ hematopoiesis has been recognized, relatively little is known so far on the specific pathways involved, particularly in megakaryopoiesis. We have previously shown that miR-221 and -222 are critical regulators of erythropoiesis through targeting of the kit receptor¹¹. In addition, enhanced expression of miR-223 repressing translation of the NFI-A transcription factor is necessary for granulocytic differentiation¹⁰. Recently, we discovered a control network in monocytopoiesis: this pathway comprises miR-17-5p/20a/106a, which target the transcription factor AML-I, activating in turn the expression of the M-CSF receptor¹². Here we show that miR-146a masters Mk differentiation by targeting the CXCR4 receptor. MiR- 146a is involved in carcinogenesis⁴⁰ and in the regulation of the innate immune response⁴¹. Our results now indicate that miR- 146a plays a pivotal role in the control of CXCR4 expression in Mk differentiation.

Although informatic algorithms suggest that several miRNAs might putatively target CXCR4 (see Results), their absence or specific expression profile in Mk cell lines suggests that they do not control CXCR4 protein levels in megakaryopoiesis. In contrast, we demonstrated that miR-146a interacts directly with two "seed" regions in the 3' UTR

CXCR4 mRNA region, thereby inhibiting CXCR4 translation. The CXCR4 receptor is upmodulated through all steps of the Mk differentiation pathway, from CFU-Mk through platelets (ref. 42 and our results). In normal megakaryopoiesis, chemokines, cytokines and adhesive interactions contribute to differentiation control by regulating both proliferation and the spatial relationships among precursor cells within the bone marrow microenvironment³⁰'³¹'⁴². Specifically previous reports³⁰'³¹, confirmed by the hereby presented data, show that inhibition of CXCR4 blocks normal megakaryopoiesis and thrombopoiesis, indicating a major role for CXCR4 in these processes.

Our functional studies demonstrate that the upregulation of CXCR4 protein expression, triggered by the downmodulation of miR-146a, is required for Mk progenitor proliferation, as well as for Mk precursor differentiation and maturation. This conclusion is directly supported by the fact that enforced expression of miR-146a in HPCs markedly inhibits Mk colony formation, as well as initial and more advanced megakaryopoiesis in liquid suspension culture. This effect is specific for the Mk lineage, since we did not observe any inhibition of erythropoiesis or granulo-monocytopoiesis in culture assays. An opposite effect is induced by antagomir-146a transfection. In fact, the early treatment of the cells with antagomir-146a accelerated by ~ 2-3 days the upregulation of CXCR4 and produced a similar acceleration of Mk differentiation/maturation. Remarkably, the siRNA- mediated inhibition of CXCR4 protein expression in Mk culture mimics the effects of miR- 146a overexpression, suggesting that CXCR4 is the major mediator of the effects of miR- 146a on megakaryopoiesis. Rescue experiments with CXCR4, mutated in the 3'UTR at the level of the miR-146a target sequences or deleted of the 3' UTR, indicate that CXCR4 is the major or only mediator of the miR-146a effect on the Mk lineage. Altogether, we postulate that the miR-146a/CXCR4 pathway is a major controller of both early and late phases of megakaryopoiesis, triggering the regulated expression of the Mk-specific gene program. This program, particularly in its late stages, may well involve the modulation of other miRNAs ⁴³.

The molecular pathway acting through the miR-146a/CXCR4 complex is controlled by the PLZF suppressor. This transcription factor inhibits miR-146a transcription by interacting directly with a newly discovered PLZF binding site within the miR-146a promoter region. ChIP experiments, luciferase-based promoter assays and PLZF silencing studies coherently indicate that the progressive increase of PLZF expression during megakaryopoiesis represses miR-146a transcription, thereby unblocking CXCR4 protein expression and stimulating megakaryopoiesis. Here again, the effects of PLZF silencing on Mk differentiation mimic those observed upon either miR-146a overexpression or CXCR4 silencing. Furthermore, rescue experiments indicate that the PLZF action on megakaryopoiesis is essentially mediated by miR-146a.

Altogether, these studies indicate that a three-step suppressor pathway including PLZF, miR-146a and CXCR4 plays a key role in the control of megakaryopoiesis.

The PLZF/miR-146a/CXCR4 action may be in part mediated by regulation of the interactions of Mk precursor cells with other cells and, in vivo, with the extracellular matrix, hi line with this hypothesis, PLZF down-regulates the adhesion molecule VLA-4 involved in the mobilization of hematopoietic and leukemic cells³², while CXCR4 is an essential regulator of hematopoietic stem cells traffic in stem cells niches²² and a mediator of myeloid cells homing/mobilization .

The CXCR4 receptor is involved in the process of cancer metastasis⁴⁴, mediates HIV entry into CD4+ lymphocytes⁴⁵ and plays a major role in the pathogenesis of rheumatoid arthritis⁴⁶. Therefore, the discovery of inhibitors of the SDF1/CXCR4 pathway is an important objective in molecular pharmacology⁴⁷. In view of the capacity of miRNAs and anti-miRNAs oligonucleotides to exert effective in vivo actions⁴⁸'⁴⁹, our results indicate that miR-146a may represent a key tool for the development of molecular therapy in these

clinical settings.

The identification of the PLZF/miR-146a/CXCR4 cascade pathway defines a new model for the miRNA-mediated regulation of megakaryopoiesis, although we cannot

exclude that PLZF and miR-146a may exert additional, minor actions through other targets. This model suggests that miRNAs act as intermediate effectors through which transcription factors exert their control on differentiation and proliferation of precursor cells. MiRNA- based regulatory mechanisms have been described in erythropoiesis ^l, granulopoiesis and monocytopoiesis¹². All these control pathways target a key hematopoietic receptor:

specifically, c-kit in erythropoiesis, CXCR4 in megakaryopoiesis, G-CSF receptor in granulopoiesis (our unpublished results) and M-CSF receptor in monocytopoiesis. The receptor is either a direct miRNA target, as in erythropoiesis and megakaryocytopoiesis, or is regulated by a miRNA-controlled transcription factor, as in granulocytopoiesis and monocytopoiesis. Altogether, we postulate that the cascade model depicted here may represent a general mechanism in the regulation of hematopoiesis, linking lineage(s)- specific transcription factors, miRNAs and key functional mRNA targets, particularly

growth factor/chemokine receptors.

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Claims

Claims

1. Use of antisense RNA specific for all or part of the 3' untranslated region of CXCR4 protein mRNA in therapy.

2. Use according to claim 1 for the treatment of cancer.

3. Use according to claim 2 for the treatment of tumour metastasis, leukaemia and/or solid tumours.

4. Use according to claim 1 for the treatment of HIV.

5. Use according to claim 4 for prevention of HIV entry into host cells.

6. Use according to claim 1 for the treatment of Rheumatoid Arthritis.

7. Use according to claim 1 for the inhibition of megakaryopoiesis.

8. Use according to any preceding claim via CXCR4 receptor down-modulation or inhibition, especially by posttranscriptional control of CXCR4 by miR-146a.

9. Use according to any preceding claim, wherein the antisense RNA is a micro RNA having has at least 60% homology with a selected region of the 3' untranslated region of CXCR4 protein mRNA.

10. Use according to claim 9, wherein the micro RNA has the sequence of SEQ ED NO 1.

11. Use according to any preceding claim, wherein the 3 ' untranslated (UTR) region of human CXCR4 protein mRNA is that provided in SEQ ID NO. 5.

12. Use of an inhibitor or suppressor (antagomir) of miR-146a in therapy.

13. Use according to claim 12, for the stimulation of Megakaryopoiesis.

14. Use according to claim 12 or 13, wherein the antagomir has the sequence of SEQ ID NO 2.

15. Use of PLZF to increase CXCR4 expression or reduce miR 146a-mediated inhibition of CXCR4, in therapy.

16. Use of antisense RNA specific for the mRNA sequence of PLZF to reduce CXCR4 expression or increase miR 146a-mediated inhibition of CXCR4, in therapy.

17. A vector comprising the antisense or antagomir RNA, according to any of claims 1-14 or 16, or DNA encoding said RNA.

18. A vector comprising nucleotides encoding PLZF.

19. A method of inhibiting megakaryopoiesis or treating cancer, HIV and/or Rheumatoid Arthritis, or other CXCR4-dependent tumours or disease conditions, comprising administering to a patient an antisense RNA, preferably miR- 146a, and/or antisense RNA targeted to PLZF, to thereby inhibit CXCR4 expression.

20. A method of stimulating megakaryopoiesis comprising administering to a patient an antagomir of miR- 146a and/or PLZF or nucleotides encoding it, to thereby increase CXCR4 expression.