KR101374585B1 - The shRNA downregulating HSP27 for treatment of tumor - Google Patents

Abstract

The present invention relates to shRNAs that inhibit HSP27 expression.
According to the present invention, it is possible to provide an anti-tumor composition using shRNA that inhibits HSP27 expression.

Description

The shRNA downregulating HSP27 for treatment of tumor}

The present invention relates to shRNAs that inhibit HSP27 expression and antitumor compositions comprising the same.

HSP27 (heat shock protein 27) is a phosphorylated protein that forms oligomers as large as 800 kD. It has anti-apoptotic properties and molecular chaperones, and it can protect against heat shock and oxidative stress. It is one of the small HSPs that protect cells. Small HSPs (referring to HSPs in the range of 15-42 kD in the size of the monomer) act as chaperone molecules when the oligomers are large, inhibiting irreversible aggregation of the released proteins and cooperating with HSP70 and ATP To become folded proteins that are active again. In addition, mammalian sHSPs are phosphorylated very rapidly through mitogen-activated protein kinase (MAPK) -activated protein kinase 2, a substrate of p38, against a variety of extracellular stresses. In the case of HSP27, the phosphorylation induced decreases the oligomer size from 70 kD to 250 kD, reducing the saffron function and increasing resistance to oxidative stress. Low molecular heat shock proteins are known to form about 800 kDa oligomeric proteins, with about 32 subunits gathering. Low molecular heat shock proteins are commonly characterized by having an α-crystallin region at the C-terminal region. Among low molecular heat shock proteins, HSP27 and αBcrystallin (αBC) are known to play a role in molecular chaperone, which is involved in protein folding under thermal shock conditions to prevent protein aggregation and maintain structure stability. . It has been reported that low molecular heat shock proteins are closely related to major neurological diseases such as Parkinson's disease, Alzheimer's disease and multiple sclerosis.

In addition, HSP27 is overexpressed in many malignant tumor cells such as prostate cancer, breast cancer, gastric cancer, ovarian cancer and bladder cancer, and it is functionally related to tumor formation and treatment resistance of cancer by directly interfering with drug resistance and cell death. . HSP27 can be an attractive therapeutic target because suppressing it affects a wide variety of pathways involved in the survival and resistance of cancer cells. When Src, a non-receptor tyrosine kinase, is activated, the transcription factor STAT3 is activated. STAT3 is involved in cell proliferation, angiogenesis, inhibition of apoptosis, migration and invasion of cancer cells, and metastasis, as well as the production of STAT3 to stimulate tumor cells to produce elements involved in immunosuppression of immune cells. Activated STAT3 increases the gene expression of HSP27. In contrast, inhibiting HSP27 reduces the level of STAT3 and decreases its downstream molecule, c-fos. Therefore, it is expected that effectively inhibiting HSP27 will act on tumor formation, metastasis of formed tumors and overcoming immune suppression.

As a previous study related to HSP27, Non-Patent Document 1 discloses that the DNA type OGX-427 (5'-GGGACGCGGCGCTCGGTCAT-3 'and siRNA form (5'-GUCUCAUCGGAUUUUGCAGC-3') increases cell death and reduces cell growth. It has been reported to increase drug sensitivity to Paclitaxel.

Non-Patent Document 2 is synthesized in the form of HSP27 siRNA duplex (sense: 5'- UGAGAGACUGCCGCCAAGUAA-3 '; antisense: 5'-UUACUUGGCGGCAGUCUCAUU-3') and transfected together with lipofectamine, head and neck squamous cell carcinoma Although metastasis is reduced in head and neck squamous cancer cells, transfection of siRNA is possible in vitro, but is easily degraded in vivo and is problematic in terms of delivery. impossible.

HSP27 knockdown using nucleotide-based therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer cells (Mol Cancer Ther 6: 299-308, 2007 by Kamada et al) Silencing heat shock protein 27 (HSP27) decreases metastatic behavior of human head and neck squamous cell cancer cells in vitro (Mol Pharm. 2010 August 2; 7 (4): 1283-1290 by Zhu et al)

Accordingly, the present inventors have made efforts to solve the above problems, and as a result, by selecting a target that effectively induces the silencing of HSP27 to produce a shRNA, and loaded it into an adenovirus siRNA by a conventional non-viral agent The present invention has been completed by drastically improving the transmission ability of the.

Therefore, an object of the present invention is to provide a shRNA that suppresses HSP27 expression by using a nucleotide sequence represented by SEQ ID NO: 1 as a target sequence.

The present invention also has another object to provide an antitumor composition comprising the shRNA as an active ingredient.

The present invention also has another object to provide a recombinant expression vector expressing the shRNA.

Another object of the present invention is to provide an antitumor composition comprising the recombinant expression vector as an active ingredient.

The present invention also has another object to provide an adenovirus into which the recombinant expression vector is introduced.

The present invention as a means for solving the above problems,

Using the nucleotide sequence shown in SEQ ID NO: 1 or 2 as the target sequence, shRNA that inhibits HSP27 expression is provided.

As another means for solving the above problems,

There is provided an antitumor composition comprising the shRNA as an active ingredient.

The present invention, as another means for solving the above problems,

A recombinant expression vector for shRNA expression is provided.

The present invention, as another means for solving the above problems,

And an antitumor composition comprising the recombinant expression vector as an active ingredient.

The present invention, as another means for solving the above problems,

And provides the adenovirus into which the recombinant expression vector is introduced.

The present invention produced a novel shRNA that inhibits HSP27 expression, and significantly increased specificity, delivery capacity and expression suppression ability compared to the prior art by increasing the infection rate using adenovirus as a gene carrier.

The shRNA that inhibits HSP27 expression according to the present invention exhibits excellent antitumor effect by reducing tumor survival, metastasis, and immunosuppression.

The anti-cancer effect was confirmed by mounting the shRNA of the present invention on adenovirus and directly administering it to the tumor. Unlike the lentivirus, the adenovirus does not integrate into the chromosome and extracts the effect only when cancer tissue is present. There is an advantage. In particular, most cancer cells can be applied to all cancers by imparting targeting to adenoviruses having excellent delivery efficiency.

1 shows a pSP72 / ΔE3 / si-negative vector, which is an E3 shuttle vector.
2 is a schematic diagram of a process of homologous recombination with dl324, an adenovirus backbone, after subcloning HSP27 shRNA into a shuttle vector.
Figure 3 illustrates the virus generation by homologous recombination of the shuttle vector and the backbone containing the gene of interest to be introduced.
Figure 4 shows the results of PCR, E1, E3 region of adenovirus for the selection of homologously recombined colonies in bacteria that are actually easy to recombine.
5 shows screening of the final recombinant colonies with HindIII digestion pattern (C: dl324-IX, 1: Sample 6 of FIG. 4, 2: Sample 7 of FIG. 4).
Figure 6 shows the result of the appearance of fragment DNA after PacI cleavage to confirm the transfection of homologous recombination adenovirus genomic DNA [C: dl324-IX, 1: Sample 6, 2: Figure 4 of Figure 4 Sample of 7].
Figure 7 shows the sequencing results to confirm that the shRNA gene of the desired HSP27 entered correctly.
Figure 8 confirms the ability to inhibit HSP27 expression by adenovirus expressing human HSP27 shRNA of the present invention by real-time PCR.
9 confirms the result of decreasing cell viability and increasing apoptosis effect when adenovirus expressing HSP27 shRNA was reduced to decrease the amount of HSP27 expression in cancer cells.

RNA interference (RNAi) is a natural mechanism that selectively inhibits the expression of target genes. The mediator of sequence specific mRNA degradation is a small interfering RNA of 19-23 nucleotides produced by cleavage of ribonuclease III from longer ds RNA. The cytoplasmic RNA-induced silencing complex (RISC) binds to siRNA and directs the degradation of mRNA containing a sequence complementary to one of the siRNAs. The application of RNA interference in mammals has the effect of therapeutic gene silencing. Despite the advantages of siRNAs, siRNAs have limitations in clinical applications in that they must be prepared in vitro and knockdown genes are typically delivered by transient transfection for 6-10 days. The shRNA (small-hairpin RNA) expression system of the present invention can solve the aforementioned disadvantages.

A shRNA is a molecule of about 20 bases or more having a double-stranded structure in a molecule and having a structure similar to a hairpin, by partially including a circular base sequence in single-stranded RNA.

The present invention relates to a shRNA that inhibits HSP27 expression, characterized in that the following sequence is used as a target sequence.

Human target sequence: 5'-GACGAGCATGGCTACATCTCCCGGT-3 '[SEQ ID NO: 1]

In the present invention, the shRNA that inhibits HSP27 expression has a sequence complementary to a portion of the HSP27 gene, and may degrade mRNA or inhibit translation of the HSP27 gene. When the complementarity is 80-90%, translation of mRNA can be inhibited, and when it is 100%, mRNA can be degraded.

Therefore, in the present invention, the shRNA that inhibits HSP27 expression preferably includes a nucleotide sequence having 100% homology to the complementary sequence for the 385-409 nucleotides of the human mRNA.

In one embodiment, the mouse shRNA consists of the nucleotide sequence shown in SEQ ID NO: 3 and its complementary nucleotide sequence, and the human shRNA may consist of the nucleotide sequence shown in SEQ ID NO: 2 and its complementary nucleotide sequence. Each base sequence and its complementary base sequence may be linked palindrome by a loop region of 4 to 10 bp to form a hairpin structure.

Specific examples of shRNAs of the present invention may include the following sequences:

ShRNA for human target sequence of SEQ ID NO: 5'-gatcc gacgagcatggctacatctcccggt tctc accgggagatgtagccatgctcgtc tttttt a -3 '[SEQ ID NO: 2].

As a substance for inhibiting the expression of HSP27 by RNAi, shRNA (short hairpin RNA) composed of a short hairpin structure having a protrusion at the 3 'end can also be used.

The substance which inhibits the expression of HSP27 by RNAi may be artificially chemically synthesized, and the DNA of the hairpin structure in which the DNA sequences of the sense strand and the antisense strand are reversely connected by T7 RNA polymerase is used in laboratory conditions (in vitro). RNA may also be synthesized and produced. When synthesized under laboratory conditions, antisense and sense RNA can be synthesized from template DNA using T7 RNA polymerase and T7 promoter. After annealing these in laboratory conditions, introducing them into cells causes RNAi to inhibit the expression of HSP27. Introduction into a cell can be performed, for example by the calcium phosphate method or the method using various transfection reagents (for example, oligofectamine, lipofectamine, lipofection, etc.).

As a substance which inhibits the expression of HSP27 by RNAi, an expression vector containing shRNA or DNA may be used, or a cell containing the expression vector may be used. The type of the expression vector or cell is not particularly limited, but an expression vector or cell already used as a medicament is preferable.

In the present invention, shRNA having a nucleotide sequence represented by SEQ ID NO: 1 as a target sequence can be used.

Accordingly, the present invention includes a recombinant expression vector for shRNA expression.

The recombinant vector of the present invention can be constructed by a recombinant DNA method known in the art.

Adenoviruses, retroviruses, lentiviruses, adeno-associated viruses and the like are useful viruses (or viral vectors) useful for delivering shRNA in the present invention, and adenoviruses are preferred for reasons of temporary induction of expression as in tumors.

In order to introduce the shRNA into adenovirus, the following DNA can be prepared based on the shRNA sequence.

≪ DNA for human target sequence >

Top Strand: 5'-gatcc gacgagcatggctacatctcccggt tctc accgggagatgtagccatgctcgtc tttttt a-3 '[SEQ ID NO: 3]

Bottom strand: 5'-agctt aaaa gacgagcatggctacatctcccggt gaga accgggagatgtagccatgctcgtc g-3 '[SEQ ID NO: 4]

In addition, non-viral vectors useful for transferring shRNA in the present invention include all vectors commonly used in gene therapy except for the viral vectors described above, and examples thereof include various plasmids and liposomes that can be expressed in eukaryotic cells .

Meanwhile, in the present invention, shRNAs that inhibit HSP27 expression are preferably operably linked to at least a promoter in order to be properly transcribed in the delivered cells. The promoter may be any promoter capable of functioning in eukaryotic cells, but it is particularly preferable that the U6 promoter is advantageous for producing small size RNA as RNA polymerase III. For efficient transcription of shRNAs that inhibit HSP27 expression, additional regulatory sequences including leader sequences, polyadenylation sequences, promoters, enhancers, upstream activation sequences, signal peptide sequences, and transcription terminators may be added as needed. It may also include.

The term "operably linked" as used herein means that the binding between nucleic acid sequences is functionally related. When any nucleic acid sequence is operably linked, any nucleic acid sequence is located so as to be functionally related to another nucleic acid sequence. In the present invention, it is said that when any transcriptional control sequence affects the transcription of an shRNA, the transcriptional control sequence is operably linked to the shRNA.

In addition, the present invention is a shRNA that inhibits the HSP27 expression of SEQ ID NO: 1, the top strand represented by SEQ ID NO: 3 and the bottom strand (bottom strand) represented by SEQ ID NO: 4 or expression thereof It relates to an anti-tumor composition comprising a recombinant expression vector as an active ingredient.

The route of administration of the antitumor composition of the present invention is not particularly limited and may be selected from oral or parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, Topical administration to a patient, skin administration, etc.). Suitable forms for oral administration may be in the form of solids or liquids, and suitable forms for parenteral administration may be in the form of injections, drops, suppositories, external preparations, eye drops, nasal drops, and the like. The antitumor composition of the present invention may contain a pharmaceutically acceptable additive, if necessary, depending on the form of the preparation. Specific examples of the pharmaceutically acceptable additives include additives such as excipients, binders, disintegrators, lubricants, antioxidants, preservatives, stabilizers, isotonizing agents, colorants, mildewers, diluents, emulsifiers, suspending agents, , Extenders, buffers, delivery carriers, carriers, excipients and / or pharmaceutical adjuvants.

The antitumor composition of the present invention in the form of a solid preparation for oral use can be prepared, for example, by adding an excipient to an active ingredient and, if necessary, adding a formulation additive such as a binder, a disintegrant, a lubricant, a colorant or a mating agent After that, it can be prepared as tablets, granules, powders and capsules according to a conventional method. The antitumor composition of the present invention in the form of a liquid preparation for oral use can be prepared by adding one or more additives for formulation such as a mating agent, a stabilizer, or a preservative to the active ingredient, , Elicitation practice, and the like.

As the solvent used for prescribing the antitumor composition of the present invention as a liquid preparation, any of water and non-aqueous solvents may be used. Liquid preparations can be prepared by methods well known in the art. For example, the injection may be prepared by dissolving the injectable solution in a solvent such as physiological saline, a buffer such as PBS, sterilized water, etc., filtering sterilized with a filter paper or the like, and then filling the sterilized container (for example, ampule). The injectable preparation may contain a conventional pharmaceutical carrier if necessary.

Also, an administration method using a non-invasive catheter may be used. Carriers usable in the present invention include neutral, buffered physiological saline, or physiological saline containing serum albumin.

Regarding gene delivery such as a shRNA expression vector that inhibits HSP27 expression, the method is not particularly limited as long as the shRNA or shRNA expression vector that inhibits HSP27 expression is expressed in the cell to be applied. For example, a viral vector or a liposome It is possible to use gene transduction using. Examples of the viral vector include animal viruses such as retroviruses, vaccinia viruses, adenoviruses, and sindividual semicircular viruses.

Substances that inhibit HSP27 expression by RNAi may be injected directly into cells.

The effective ingredient of the antitumor composition of the present invention is used in a therapeutically effective amount, and the dose of the composition may vary depending on the purpose of use, the degree of addiction of the disease, the age, body weight, sex, history of the patient, And the like can be determined by those skilled in the art. For example, about 1x10 < ¹⁰ > particles to 1x10 < ¹² > particles per kg of adult as an active ingredient. The frequency of administration of the antitumor composition of the present invention may be, for example, once per day to several months.

Since the shRNA of the present invention inhibits HSP27 expression, the pharmaceutical composition of the present invention can be used for various diseases or diseases associated with tumors, such as brain cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, esophageal cancer, It can be used for the treatment of pancreatic cancer, bladder cancer, prostate cancer, colon cancer, colon cancer and cervical cancer. As used herein, the term “treatment” means (i) prevention of tumor cell formation; (Ii) inhibition of a disease or condition associated with the tumor following removal of the tumor cells; And (iii) alleviation of a disease or disorder associated with a tumor following removal of tumor cells. Thus, the term “therapeutically effective amount” herein means an amount sufficient to achieve the above pharmacological effect.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited by the following examples.

Example 1: shRNA Preparation-Target Selection Effectively Inducing Silencing of HSP27

In order to induce the silencing of HSP27, the present invention provides a shRNA based on sense 25 mer / antisense 25 mer (with a 4-base loop in the middle) and introduces it into a shuttle vector for expression in adenovirus. Homologous recombination virus was produced.

In order to verify the specificity of shRNA HSP27, a shuttle vector having a scrambled shRNA was also prepared. The specificity and the ability to inhibit the expression were greatly improved as compared with the conventional method.

To this end, siRNA having an effect of inhibiting at least 80% of human HSP27 mRNA at least 10 nM of shRNA of HSP27 was obtained through real-time PCR.

To this end, human siRNA was prepared using the HeLa cell line, which is a cervical cancer cell, and then transfected and examined to decrease after 24 hours.

The experimental method is as follows.

Real-time RT-PCR transfected several types of candidate shRNA 10nM to 30% B16F10 and incubated for 24 hours, screening five shRNAs against human HSP27 through validation, 82.68% silencing in target shRNAs (silencing) effect was confirmed.

For real-time RT-PCR, the forward primer was 5'-CTACATCTCCCGGTGCTTCA-3 '[SEQ ID NO: 5] and the reverse primer was 5'-GGATGGTGATCTCGTTGGAC-3' [SEQ ID NO: 6], and the reaction conditions were performed as follows. .

Step 1: Reverse transcription (42 ° C for 5 min, 95 ° C for 10 sec)

Step 2: PCR reaction (95 ° C for 5 sec, 60 ° C for 20 sec) 50 cycles,

Step 3: The separation was carried out (60 ° C-> 95 ° C).

As a result of validation experiment, the following target was selected based on Table 1 among five target sequence candidates.

Human target sequence: 5′-GACGAGCATGGCTACATCTCCCGGT-3 ′ [SEQ ID NO: 1]: HSP27 # 3 in Table 1 below

 Note: ct: cycle threshold, the number of cycles required to reach saturation, the smaller the amount of original mRNA.

Δct: ct for HSP27 minus ct for actin,

ΔΔct: Δct of the HSP27 shRNA treated sample minus HSP27 Δct of the control.

2-ΔΔct: A negative index ΔΔct value of 2.

Expression inhibition rate is expressed as a percentage of 2-ΔΔct

ShRNAs with 25/25 +4 loops were synthesized against the target sequence and their inhibitory effect on the target sequence was confirmed by real-time PCR.

ShRNA with human target sequence (SEQ ID NO: 1): 5'-gatcc gacgagcatggctacatctcccggt tctc accgggagatgtagccatgctcgtc tttttt a -3 '[SEQ ID NO: 2]

In order to express the base sequence of SEQ ID NO: 2 selected by the real-time PCR described above in adenovirus, BamHI and HindIII base sites were inserted at both ends, and a loop having four bases of tctc was formed in the middle. . That is, the basic structure of human shRNA is composed of 5'-25 mer-loop (4 mer) -25mer-3 '.

Based on this, the following 2 strands of DNA strand were introduced into the adenovirus for induction of shRNA production.

Top Strand: 5'-gatcc gacgagcatggctacatctcccggt tctc accgggagatgtagccatgctcgtc tttttt a-3 '[SEQ ID NO: 3]

Bottom strand: 5'-agctt aaaa gacgagcatggctacatctcccggt gaga accgggagatgtagccatgctcgtc g-3 '[SEQ ID NO: 4]

Example 2: Construction of non-replicating adenovirus vectors expressing shRNA against a target sequence

The oligonucleotide consisting of bases with BamH I and Hind III restriction enzyme sequences at both ends is placed with the most effective inhibitory shRNA sequence identified by real-time RT-PCR between the sense and antisense sequences tctc or tctctc. Oligonucleotides complementary to the nucleotides were synthesized and annealed, respectively, and then adenovirus E3L (26591-28588) and the pSP72 / ΔE3 / si-negative vector (Fig. 2, pSP72 cloning vector (Promega)) were used as E3 shuttle vectors. The plasmid into which E3R (30504-31057) was inserted was inserted into the gamlencc psilencer 2.1-U6 hygro from Ambion's psilencer 2.1-U6 hygro + ggatcc actaccgttg ttataggtgt tcaagagaca cctataacaacggtagtttt ttggaaaagctt-HindSP72 △ in the form of -EcoRI-U6 promoter + -BamHI-nonsense shRNA / si negative (scrambled)] the first Bam HI and H ind ⅲ after treatment, to the above copolymer Bam HI and Hind ⅲ inserts the HSP27 shRNA exposed to each end pSP72 △ E3-sh-human HSP27 [ Column No. 7 was produced in the [2]. Negative control adenoviruses with BamHI and HindIII at both ends and scrambled sequences (actaccgttgttataggtgt) and loop (ttcaagaga) were prepared.

For homologous recombination, dl324-IX was cut with enzyme Spe I and the pSP72ΔE3-U6-sh-human Hsp27 shuttle vector containing human shHSP27 (sequence: 5'-gatcc gacgagcatggctacatctcccggt tctc accgggagatgtagccatgctcgtc tttttt a-3 ') was restricted. Cleavage with the enzyme Xmn I. In order to proceed with homologous recombination, plasmids cut with each enzyme were transformed into E. coli BJ5183, a competent cell for homologous recombination at a ratio of dl324-IX: pSP72ΔE3-U6-shHsp27 = 200ng: 500ng.

(When a shRNA is produced in a replicable adenovirus, a non-replicating adenovirus was produced because the inhibitory effect by the shRNA and the cell lysis effect are mixed and it is difficult to clearly identify only the inhibitory effect).

The method of confirming whether the homologous recombination is properly is to discriminate by PCR of the E1, E3 parts [Fig. 3]. shHsp27 (shRNA-human Hsp27) was constructed in the E3 shuttle vector, and since it was homologous to the E3 part, the E1 part should be the same as the band of dl324-IX, and the E3 part was the band of pSP72ΔE3-U6-sh-human Hsp27. Must be equal to Therefore, it can be assumed that the sample 2, 6, 7, 11 in Figure 3 can proceed to the next step. DNA fractions were extracted after inoculation of the fractions separated by colony PCR. In E. coli BJ5183, even after extracting DNA, the concentration is very low, and the E. coli DH5α was transformed again and the pattern was confirmed again.

After inoculation, the DNA was extracted and the patterns of Hind III and Pac I were confirmed. Only positive clones (# 2, 6, 7, 11) were selected by E1 and E3 site PCR of adenoviruses [FIG. 3], and the final recombinant was selected by Hind III digestion pattern as shown in FIG. .

Samples presumed to be Hind III homologous recombination (in this case, only 4 samples are used instead of 4 samples. The reason for this is that when Hind III cutting is performed on BJ5183, all 4 samples have a positive pattern or # 2 and # 11 faintly. Electrophoresis only in # 6 and # 7, where # 1 is a sample 6 when PCR and # 2 is a sample 7 when PCR), and the band is dl324- which is a control. Unlike IX, it can be seen that two more bands are formed [FIG. 5]. In addition, when cutting with Pac I, one band appears as in the control group dl324-IX. This inserts the viral gene into the plasmid so that the viral gene dl324-IX can grow when transformed into bacterial cells in a medium containing ampicillin. The plasmid has only one band when cut with Pac I because all of the sites linked to the viral gene are Pac I sites. This confirms that the homologous recombination.

The homologous recombinant plasmid DNA was obtained as described above, treated with Hind Ⅲ restriction enzyme to confirm the change of DNA pattern, and finally sequenced to confirm homologous recombination, and the identified plasmids were digested with Pac I. The non-replicating adenovirus expressing shRNA HSP27 was produced by transformation. The adenovirus was grown in a 293 cell line, concentrated to a CsCl gradient, and the titer of the virus was determined by limiting dilution or plaque assay.

The final virus titer was 6.32 × 10 ⁹ PFU / ml by limiting dilution titration.

Example  3: Confirmation of Effects on Cancer Cells shRNA  Expressed Adenovirus  To inhibit HSP27 expression

1) Check by real-time RT-PCR

HSP27 Inhibition of expression was confirmed by infection with human prostate cancer cells DU-145 with 1 to 100 moi 2 days later, the cells were lysed with Trizol and continuously treated with chloroform, isopropanol, ethanol and the like. HSP27 after harvesting RNA The degree of mRNA expression inhibition was confirmed by real-time PCR.

Real-time PCR primers for human HSP27 inhibition were used as forward primer: 5'-CTACATCTCCCGGTGCTTCA-3 '[SEQ ID NO: 8] and reverse primer: 5'-GGATGGTGATCTCGTTGGAC-3' [SEQ ID NO: 9], AB powerSYBR Green RNA- 0.2 μl RT enzyme mix (125X), 12.5 μl RT-PCR Mix (2x), 0.5 μl Forward Primer (100 pM), 0.5 μl reverse Primer (100 pM), RNA (10ng / μl) using a to-Ct 1step kit 5 μl, 6.3 μl of Nuclease-free water was added to make the total volume 25 μl, and the reaction conditions are shown in Table 2 below.

Step Temp (캜) Duration Cycles RT step 48 30min Hold Enzyme Activation 95 10 min Hold Denature 95 15 sec 40 Anneal / Extend 60 1 min

As a result of confirming shRNA of human HSP27, the inhibition of HSP27 expression of about 80% was observed in 50 moi of adenovirus, such as about 66% of silencing effect in 1 moi of adenovirus [Table 3, FIG. 8]. .

division Expression inhibition rate (%) NC 0 moi 0 1 moi 0 10 moi 0 50 moi 0 100 moi 0 hHSP27
shRNA 0 moi 0 1 moi 65.7 10 moi 75.7 50 moi 78.8 100 moi 87.1

NC: Adenovirus with Scrambled shRNA sequence

2) Effect of HSP27 Expression Inhibition on Apoptosis and Cell Viability

To determine whether decreased expression of HSP27 in cancer cells increases the sensitivity to anticancer agents, PARP was reduced by apoptosis of adenovirus expressing HSP27 shRNA and combined with TRAIL and curcumin. It was confirmed that the division was also increased to increase apoptosis [FIG. 9].

The specific experimental procedure is as follows. As a control, adenovirus expressing nonsense shRNA and adenovirus expressing shRNA HSP27 are infected with cancer cells (DU-145 prostate cancer cells) at 10 moi, and after 48 hours, TRAIL, a cytokine that specifically kills cancer cells, and natural After pretreatment with curcumin 50 mM, an extract-derived chemosensitizer for 1 hour, and TRAIL (200 ng / ml) for 4 hours, the levels of Src or phosphorylated Akt phosphorylated with apoptosis markers PARP or cell viability markers were measured. Measured by Western blot.

The results of this study indicate that the truncated fragments of PARP increased and the phosphorylated Src and Akt decreased over time when HSP27 decreased, indicating increased cell death and decreased viability.

Attach an electronic file to a sequence list

Claims (9)

A shRNA that inhibits HSP27 expression represented by SEQ ID NO: 2 as a target sequence represented by SEQ ID NO: 1 as a target sequence.
delete An antitumor composition comprising the shRNA of claim 1 as an active ingredient.
A recombinant expression vector expressing the shRNA of claim 1.
5. The method of claim 4,
A recombinant expression vector comprising a DNA comprising a top strand represented by SEQ ID NO: 3 and a bottom strand represented by SEQ ID NO: 4.
5. The method of claim 4,
A recombinant expression vector obtained by recombining DNA into a vector containing a U6 promoter.
5. The method of claim 4,
A recombinant expression vector of pSP72ΔE3-U6-shHSP27 represented by SEQ ID NO: 7.
An antitumor composition comprising the recombinant expression vector of claim 4 as an active ingredient.
Adenovirus introduced with a recombinant expression vector of claim 4.

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