CN116270710A - Application of circRBM33 as target in diagnosis and treatment of prostate cancer - Google Patents
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- CN116270710A CN116270710A CN202310124261.3A CN202310124261A CN116270710A CN 116270710 A CN116270710 A CN 116270710A CN 202310124261 A CN202310124261 A CN 202310124261A CN 116270710 A CN116270710 A CN 116270710A
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Abstract
The invention relates to application of circRBM33 serving as a target spot in diagnosis and treatment of prostate cancer. The invention fully reveals the function of the circRBM33 in cancers, especially prostate cancer cells and the target genes regulated and controlled by the circRBM33, clarifies the new mechanism of regulating and controlling the prostate cancer by the circRBM33, and finds a key target for inhibiting the prostate cancer. The invention carries out intensive research on the function of the circRBM33 in the prostate cancer progression process, and defines the influence of the circRBM33 on the proliferation and migration capacity of prostate cancer cells and the influence of the circRBM33 on androgen receptor pathway inhibitor (ARSI) treatment. The invention makes a brand-new elucidation on the progress mechanism of the prostate cancer, provides a brand-new thought for later-stage related drug development and clinical diagnosis and treatment, and has great social significance and market prospect.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to application of circRBM33 serving as a target spot in diagnosis and treatment of prostate cancer.
Background
Prostate cancer (PCa) is second in incidence among global male malignancies and fifth in mortality. Androgen-blocking therapy (Androgen deprivation therapy, ADT), which reduces androgen levels by surgery or drugs, is currently the cornerstone for the treatment of advanced prostate cancer. However, all prostate cancers inevitably develop castration-resistant prostate cancer (castration resistant prostate can cer, CRPC) around 18 months after ADT treatment due to aberrant reactivation of the androgen receptor signaling pathway. Fortunately, a range of androgen receptor signaling pathway inhibitors (AR signaling inhibitor s, ARSIs) including androgen synthesis inhibitors (Abiraterone et al) and AR antagonists (Enzalutamide, darlutamide, aplutamide et al) have been applied to CRPC treatment, significantly extending the survival of patients. However, even so, CRPC will eventually have reduced therapeutic sensitivity to ARSIs, gradually developing resistance, due to AR splice variants or alternative activation.
N6-methyladenosine (m 6A) is one of the most common modifications in mammalian mRNA. It is a reversible epigenetic regulation, comprising three groups of catalytic enzymes, methyltransferase, demethylase and m6A recognition protein, respectively. METTL3 is the core catalytic unit of the methyltransferase complex, which has been reported to regulate proliferation, metastasis, angiogenesis and metabolism in a variety of cancers. FTO was the first demonstrated m6A demethylase, and was initially demonstrated to play an important role in leukemia aggressiveness, and subsequently demonstrated to play a different role in a variety of solid cancers. In addition, the m6A recognition protein has wide influence on the stability, degradation, translation and other processes of the corresponding binding RNA, for example, YTH DF1 can bind to the m6A modification site of EIF3C mRNA and increase the translation thereof, thereby promoting the occurrence and metastasis of ovarian cancer.
In addition to modifying mRNA, m6A is also found in many non-coding RNA types, such as circular RNA (circRNA). The circRNA is a post-spliced non-coding RNA that was previously thought to be the product of misprocessing. However, so far, more and more studies have shown that circRNA is involved in the occurrence and pathogenesis of various diseases by different means, such as miRNA sponge, interacting with proteins and even translating new peptide fragments. It is reported that the m6A modification not only contributes to the synthesis and nuclear transport of the circular RNA, but also promotes the interaction of the circular RNA with proteins and even confers the circular RNA translation ability. Conversely, cir cRNA can also regulate m6A modifications, such as the interaction of circ0008399 with WTAP, promote assembly of the m6A complex, and enhance cisplatin resistance of bladder cancer. Thus, the interaction between the m6A modification and the circRNA is complex, and thus, there is an urgent need to find new therapeutic perspectives, screen for circRNA closely related to prostate cancer, and make intensive studies on its mechanism of action.
Disclosure of Invention
The invention aims to solve the technical problems that the molecular mechanism of cancer, especially prostate cancer proliferation and metastasis is not clear enough and an effective treatment means is lacking in the prior art, so that the research on the progress mechanism of the prostate cancer is carried out, and a novel prevention, treatment and prognosis evaluation means is provided for the prostate cancer.
In order to solve the technical problems, the invention is realized by the following technical scheme.
In a first aspect, the invention provides the use of a circRBM33 inhibitor for the preparation of a product for the prophylaxis and/or treatment of prostate cancer.
Preferably, the circRBM33 inhibitor is selected from shRNA designed based on the circRBM33 gene.
Preferably, the shRNA designed based on the circRBM33 gene is selected from one or more of sh-C1 and sh-C2, wherein the sh-C1 sequence is as follows: ccggGATGAATTTACAATGA TGActcgagTCATCATTGTAAATTCATCtttttg, sh-C2 sequence: ccggGAATTT ACAATGATGACTTctcgagAAGTCATCATTGTAAATTCtttttg.
In a second aspect, the invention provides the use of an agent for detecting the expression level of circRBM33 in the manufacture of a product for use in the assisted diagnosis and/or prognosis evaluation of prostate cancer.
Preferably, the reagent for detecting the expression level of the circRBM33 includes a primer pair for detecting the expression level of the circRBM33 gene.
Preferably, the forward primer sequences of the primer pairs are: GAATTGTATACTCAA GAGTACC, the reverse primer sequence is: CTGGTCAAAGTCATCATTGTA.
It is to be understood that, unless otherwise specified, in the context of the present invention, the primers and/or primer pairs refer to PCR primers used to synthesize the cDNA strand of the circRBM33 gene in PCR, thereby detecting the RNA expression level of the circRBM33 gene. In addition to the primers and/or primer pairs listed in the present invention, it is fully within the ability of one skilled in the art to design corresponding primers and/or primer pairs based on the gene sequence of circRBM33 using means conventional in the art, including but not limited to molecular biology, and to screen the designed primers and/or primer pairs by means of conventional experimentation, provided that specific detection of the expression level of circRBM33 is achieved.
In a third aspect, the invention provides the use of a circRBM33 inhibitor in the manufacture of a medicament for promoting sensitivity of Androgen Receptor Signaling Inhibitors (ARSIs) to cancer treatment.
Preferably, the cancer is selected from prostate cancer.
Preferably, the androgen receptor signaling pathway inhibitor is selected from one or more of Enzalutamide Lu An (Enzalutamide), dariluamide (dariluamide), apatamine (apalutamide), abiraterone (Abiraterone).
Preferably, the circRBM33 inhibitor is selected from shRNA designed based on the circRBM33 gene.
Preferably, the shRNA designed based on the circRBM33 gene is selected from one or more of sh-C1 and sh-C2, wherein the sh-C1 sequence is as follows: ccggGATGAATTTACAATGA TGActcgagTCATCATTGTAAATTCATCtttttg, sh-C2 sequence: ccggGAATTT ACAATGATGACTTctcgagAAGTCATCATTGTAAATTCtttttg.
In a fourth aspect, the invention provides a pharmaceutical composition for the prevention and/or treatment of prostate cancer comprising a circRBM33 inhibitor and an FMR1 inhibitor.
Preferably, the circRBM33 inhibitor is selected from shRNA designed based on the circRBM33 gene; the FMR1 inhibitor is selected from siRNA designed based on FMR1 gene.
Preferably, the shRNA designed based on the circRBM33 gene is selected from one or more of sh-C1 and sh-C2, wherein the sh-C1 sequence is as follows: ccggGATGAATTTACAATGA TGActcgagTCATCATTGTAAATTCATCtttttg, sh-C2 sequence: ccggGAATTT ACAATGATGACTTctcgagAAGTCATCATTGTAAATTCtttttg; the siRNA designed based on the FMR1 gene is selected from one or more of si-1 and si-2, wherein the si-1 sequence is CCAGAAGACUUACGGCAAATT, and the si-2 sequence is GCAUCAAAUGCUUCUGAAATT.
In a fifth aspect, the present invention provides the use of the above pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of prostate cancer.
The inventors of the present invention found through studies that circRBM33 was significantly elevated in prostate cancer cells, and that the elevation was associated with poor prognosis for prostate cancer patients; prostate cancer patients with higher expression of circRBM33 have no biochemical recurrence (Biochemical recurrence-free, BCR-free) significantly shorter time than patients with higher expression of circRBM 33; meanwhile, after the cir cRBM33 is inhibited, the proliferation and migration of the prostate cancer cells can be obviously inhibited in vitro and in vivo. Clinical sample detection shows that the expression level of the circRBM33 is positively correlated with the Gleason score, and the high expression of the circRBM33 indicates that the prostate cancer patient has poorer survival without BCR. In addition, the present invention also demonstrates that the expression level of cirrbm 33 has a significant effect on the therapeutic effect of ARSI, and that when the intracellular cirrbm 33 level is reduced by inhibiting the expression of cirrbm 33, the sensitivity of PCa to ARSIs treatment can be effectively increased.
As a new way of epigenetic modification, RNA m6A is widely present in various types of RNAs, and a number of studies report that m6A modification is involved in biogenesis and various functions of circRNA, for example METTL3 mediates m6A modification of circIGF2BP3 to circularize it, and the like. Here, the present invention focused on the relationship between m6A modifications and circrnas and studied their functional role in prostate cancer progression, confirming that circRBM33 is methylated by MET TL3 and then interacts with FMR1 in m6A fashion. Whereas FMR1 has no effect on the stability of the circRNA, but rather forms a binary complex with circRBM33 to regulate downstream target molecules. Numerous studies have shown that FMR1 protein deficiency is closely related to fragile X syndrome (including mental retardation, behavioral abnormalities, etc.). Currently, there is increasing evidence that FMR1 plays an important role in tumorigenesis and metastasis, and the results of the present invention consistently indicate that silencing FMR1 also inhibits the growth and migration of prostate cancer cells; clinical sample testing also suggests that high expression of FM R1 predicts a shorter progression free survival for prostate cancer patients. The research of the invention shows that the protein expression level of FMR1 is positively correlated with the expression level of circRBM33, the circRBM33 interacts with FMR1 in an m6A mode to form a binary complex, and then the binary complex is combined with PDHA1mRNA to enhance the stability and increase the translation output, thereby enhancing mitochondrial respiration and promoting PCa growth and transfer; and by inhibiting the circRBM33 and/or FMR1, the progression of the prostate cancer can be effectively inhibited.
Compared with the prior art, the invention has the following technical effects:
(1) The invention carries out intensive research on the pathogenesis and development mechanism of the prostate cancer, and discovers that the expression level of the circRBM33 is a factor highly related to the prostate cancer; furthermore, the expression level index of the circRBM33 in the subject is obtained by detecting the circRBM33 in the subject, so that the probability of the prostate cancer of the subject can be effectively and reasonably predicted, namely, the expression level of the circRBM33 can be used as a biomarker for clinically assisting in diagnosing the prostate cancer diseases, and when the expression level of the prostate cancer is obviously increased, the condition that the subject is a prostate cancer patient or a high-risk group suffering from the prostate cancer can be clarified, and the irreversible health damage to the patient caused by rapid development and deterioration of the diseases can be effectively prevented.
(2) By detecting the expression level of the circRBM33 in the prostate cancer patient, the reasonable prediction of the tumor differentiation degree in the patient can be judged, so that a personalized treatment scheme is provided to improve the clinical treatment effect; the prognosis of the patient can be reasonably evaluated, and a reasonable and effective guiding effect is provided for treatment and rehabilitation. Meanwhile, by inhibiting the expression of the circRBM33, the sensitivity of the prostate cancer to ARSIs treatment can be effectively improved.
(3) The invention has the advantages that the expression of the circRBM33 is inhibited to generate obvious inhibition effect on the prostate cancer cells, and simultaneously, the circRBM33 is interacted with FMR1 in an m6A mode to form a binary compound, so that the progress of the prostate cancer is influenced; by inhibiting cir cRBM33 and/or FMR1, progression of prostate cancer will be effectively inhibited. The invention provides a new drug treatment target for human to attack the prostate cancer, provides a new direction for the control of the prostate cancer for searching a novel tumor marker or a new treatment strategy related to the diagnosis of the prostate cancer, is beneficial to the subsequent drug research and development, clinical treatment and the like, and has important scientific significance.
Drawings
FIG. 1 is a schematic representation of the results of Merrip experiments with circRBM33 in 2 prostate cancer cell lines.
FIG. 2 is a schematic diagram showing the results of the convergent and convergent primer amplification experiments performed in 4 prostate cancer cell lines.
FIG. 3 is a schematic representation of the results of RNase R-tolerance experiments performed on 4 prostate cancer cell lines.
FIG. 4 is a schematic representation of the results of the colistin D assay in 2 prostate cancer cell lines.
FIG. 5 is a graph showing the results of KM analysis of the expression level of circRBM33 in a prostate cancer patient.
FIG. 6 is a graph showing results of the expression level of circRBM33 in various cell lines.
FIG. 7 is a schematic representation of the results of over-expression or inhibition of intracellular circrRBM 33.
FIG. 8 is a graph showing the effect of circRBM33 inhibition on proliferation of prostate cancer cells.
FIG. 9 is a graph showing the effect of inhibition of circRBM33 on prostate cancer cell clone formation.
FIG. 10 is a graph showing the effect of circRBM33 inhibition on prostate cancer cell migration.
FIG. 11 is a graph showing the effect of circRBM33 over-expression on proliferation of prostate cancer cells.
FIG. 12 is a graph showing the effect of circRBM33 overexpression on prostate cancer cell clone formation.
FIG. 13 is a graph showing the effect of circRBM33 over-expression on prostate cancer cell migration.
FIG. 14 is a graph showing the effect of circRBM33 on mouse subcutaneous tumor growth.
FIG. 15 is a graph showing the effect of inhibition of circRBM33 on ARSIs therapeutic activity.
FIG. 16 is a graph showing the effect of inhibition of circRBM33 in combination with Enzalutamide on growth of subcutaneous tumors in mice.
FIG. 17 is a graph showing the effect of circRBM33 inhibition in combination with Darolutamide on mouse subcutaneous tumor growth.
FIG. 18 is a graphical representation of FISH sublocalization results of circRBM33 in 2 prostate cancer cell lines.
FIG. 19 is a schematic representation of the results of expression of circRBM33 in the nucleus and cytoplasm of 2 prostate cancer cell lines.
FIG. 20 is a schematic representation of the results of a circRBM33 and FMR1 co-localization experiment.
FIG. 21 is a graphical representation of the results of the Churp analysis of the circRBM33 and FMR 1.
FIG. 22 is a graph showing the results of RIP analysis of FMR1 in prostate cancer cells.
FIG. 23 is a graph showing the role of METTL3 in m6A modification of circRBM33 by MeRIP analysis.
FIG. 24 is a graph showing that the circRBM33 interacts with FMR1 in an m6A dependent manner as demonstrated by FMR1-RIP experiments.
FIG. 25 is a graph showing the WB results of FMR1 inhibition by siRNA.
FIG. 26 is a graph showing the effect of FMR1 inhibition on proliferation of prostate cancer cells.
FIG. 27 is a graph showing the effect of FMR1 inhibition on prostate cancer cell clone formation.
FIG. 28 is a graph showing the effect of FMR1 inhibition on prostate cancer cell migration.
FIG. 29 is a graph showing the results of DFS analysis for prostate cancer patients with different FMR1 expression levels.
FIG. 30 is a graph showing results of the expression levels of circRBM33 in prostate cancer tissues with different Gleason scores.
FIG. 31 is a graphical representation of results of FMR1 immunohistochemical analysis of prostate cancer tissue at different Gleason scores.
FIG. 32 is a graph showing the results of the difference in FMR1 scores in prostate cancer tissues at different levels of circRBM33 expression.
FIG. 33 is a schematic diagram showing the results of verifying correlation between circRBM33 and FMR1 expression using chi-square test.
FIG. 34 is a graph showing the effect of FMR1 knockdown on PDHA1 expression in circRBM33 overexpressing cells.
FIG. 35 is a graph showing the effect of circRBM33 and FMR1 on proliferation of prostate cancer cells.
FIG. 36 is a graph showing the effect of circRBM33 and FMR1 on prostate cancer cell clone formation.
FIG. 37 is a graph showing the effect of circRBM33 and FMR1 on prostate cancer cell migration.
Detailed Description
In order to make the objects, technical solutions and effects of the present invention more clear and clear, the present invention will be described in further detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Prostate cancer cell lines including PC-3, C4-2, DU145, 22Rv1, etc., as listed in the context of the present invention were purchased from BeNa Culture Collection, where C4-2 and 22Rv1 were cultured in RPMI-1640 medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin, without specific explanation; PC-3 and DU145 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. RWPE-1 was purchased from iCell Biosci ence and cultured in keratinocyte free serum medium (K-SFM) containing 0.05mg/mL bovine pituitary extract and 5ng/mL human recombinant EGF. All cell lines were identified by short tandem repeat analysis by the chinese collection of typical cultures (martial arts) and verified for the presence of mycoplasma contamination using PCR detection kit (biothorive Sci, shanghai) while being stored in liquid nitrogen and used for subsequent experiments. All of the reagents used in the present invention are commercially available. For the use of clinical specimens, the application of the clinical specimens and patients are signed with informed consent, and related procedures and methods meet the medical ethics requirements and the quality management standards of clinical trials of medicines and are approved by the ethics committee of Zhujiang hospitals at the university of south medical science. The experimental methods used in the invention, such as bioinformatics analysis, nucleic acid extraction, transcriptome sequencing, primer design, tumor cell culture, PCR, lentiviral vector construction, cell transfection, westernblot, molecular cloning, small molecule interference technology, immunohistochemistry, immunofluorescence staining, cell proliferation experiments, cell migration experiments, cell clone formation experiments, fluorescence in situ hybridization experiments, methylated RNA immunoprecipitation experiments, RIP experiments and the like are all conventional methods and technologies in the field, and animal experiments are all approved by the animal protection Committee of Zhujiang hospitals of southern medical university.
Representative results of selection from the biological experimental replicates are presented in the context figures, and data are presented as mean±sd and mean±sem as specified in the figures. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 9.0 or SPSS 20.0 software. And comparing the average value difference of two or more groups by adopting conventional medical statistical methods such as t-test, chi-square test, rank sum test, analysis of variance and the like. The difference was considered significant with p < 0.05.
Example 1 screening for targets associated with prostate cancer
In order to determine the m6A related types of circR NA which can be used for prostate cancer treatment and prognosis evaluation, firstly, the expression matrix of all the circRNAs in the tissues of a prostate cancer patient is obtained, and the circRNAs with high abundance characteristics (FPKM < 0.5) are screened. Subsequently, circular RNAs associated with biochemical recurrence of prostate cancer were screened out of the circRNA data containing follow-up data, for a total of 1382 standard-meeting circrnas; next, methylated RNA co-immunoprecipitation sequencing (methylated RNA immunoprecip itation sequencing, meRIP) experiments were performed with prostate cancer cells to study m 6A-related circRNA profile, as follows:
(1) MeRIP assays were performed using a methylated RNA immunoprecipitation kit (BersinBio, bes, 5203) and an RNA immunoprecipitation kit (BersinBio, bes, 5101), in which total RNA was isolated using Trizol and was isolated to about 300bp using an ultrasonic cell disruptor.
(2) The RNA fragment was co-immunoprecipitated with anti-N6-methyladenosine antibody in a vertical rotator at 4℃for 4h, incubated with protein A/G beads for 1h, and proteinase K eluted at 55℃for 45min to give m6a modified RNA.
(3) The collected cells were lysed with lysis buffer on ice and DNA was removed with DNase. Subsequently, immunoprecipitation with anti-m6A antibody at 4℃for 4h, incubation with protein A/G beads for 1h, elution with proteinase K at 55℃for 45min, and finally detection of the resulting RNA samples using transcriptome second generation sequencing techniques.
In total 355 m 6A-related circRNAs were obtained by the MeRIP-seq described above; finally, the circRNA associated with m6A, namely circRBM33 (hsa_circ_ 0001771), which has prognostic evaluation value, was obtained by Venn diagram. The gene encoding the circRBM33 is located on chromosome 7, and the transcript sequence comprises 4 exons (i.e., exons 2, 3, 4, 5) and is reverse spliced from exons 2 and 5. Two of the motifs were found to be potential targets by motif analysis of the MeRIP-seq data to define the possible m6A binding sites.
Subsequently, an m6A-RNA co-immunoprecipitation (MeRIP) experiment was performed to determine whether the circRBM33 could be modified by m6A, and the results are shown in fig. 1. The results showed that in 2 prostate cancer cells (22 Rv1, C4-2), the circRBM33 fragment appeared in both the m6a antibody lane and the Input lane, but not the IgG lane, indicating that circRBM33 was able to be modified by m6 a. To further verify the above results, PCR amplification was performed using convergent and divergent primers in cDNA and gDNA samples of 4 prostate cancer cell lines. As a result, it was found that, of the 4 prostate cancer cell lines, the circRBM33 was amplified only in cDNA, but not in gDNA (see FIG. 2). Next, by performing an RNase R tolerance experiment to compare the stability of the circumrbm 33 and the linear RBM33 (linear RBM 33), the following steps are performed:
(1) 4 prostate cancer cell lines were treated with 2mg/mL actinomycin D (Sigma, USA) at 0, 6, 12 and 24 hour isocratic time points, respectively.
(2) Collecting total RNA of the corresponding cell sample; RNase R (2U/. Mu.g RNA) was added thereto and cleaved at 37℃for 30min.
(3) The expression level of the above genes was detected by qPCR.
The results showed that the circRBM33 was more stable than the linear RBM33 after RNase R treatment (see FIG. 3). On the other hand, the actinomycin D assay also showed that the stability of the circRBM33 was better than that of the linear RBM33 (see FIG. 4), and the results were consistent with the above.
After defining the basic properties of circRBM33, it is necessary to determine whether it can play a critical prognostic evaluation role in prostate cancer. In this regard, survival prognosis analysis was performed on the expression of circRBM33 using the Kaplan-Meier (KM) method in a public prostate cancer patient cohort, with PCa patients divided into two subgroups according to the median of the expression level of circRBM 33: namely, the cir cRBM33 high expression group and the circRBM33 low expression group, and the results are shown in FIG. 5. The results show that therefore, the survival rate of BCR-Free in the circRBM33 high expression group is significantly worse than in the patients in the circRB M33 low expression group (p < 0.05). Through studies of the relationship between the expression level of circRBM33 and some of the clinical pathological features of prostate cancer, it was found that the expression level of circRBM33 was proportional to the Gleason score and the tumor progression to some extent (not shown in the figure). From the above study, it is clear that circRBM33 can be modified by m6A and can be used as a key index for assessing the prognosis of prostate cancer BCR.
EXAMPLE 2 study of expression of circRBM33 in prostate cancer cells
To determine the potential role of circRBM33 in PCa in vitro and in vivo, the expression levels of circRBM33 in four prostate cell lines were first examined by qPCR experiments. The detection results are shown in FIG. 6. The results show that circRBM33 is low expressed in normal prostate tissue cells, whereas it is significantly high expressed in prostate cancer cells (< 0.01, p < 0.0001). Whereas 22Rv1 and DU145 cells exhibited very significant high expression levels of circRBM33 in the four prostate cancer cell lines, the expression levels were relatively low in C4-2 and PC-3 cells. In this regard, shRNA was designed based on the circRBM33 sequence, shC (sequence: ccggGATGAATTTACAATGATGActcgagTCATCATTGTAAATTCATCtttttg) and shC2 (sequence: ccggGAATTTACAATGATGACTTctcgagAAGTCATCATTGTA AATTCtttttg), respectively, transfected into 22Rv1 and DU145 cells, respectively, to silence expression of circRBM33, using sh-NC (empty vector) as a negative control; the circRBM33 was also overexpressed in C4-2 and PC-3 cells using lentiviral vectors, with ciR5 as a negative control. The results are shown in FIG. 7, and the results show that sh-C1 and sh-C2 can successfully inhibit the expression of circRBM33 and reduce the intracellular level of circRBM 33; the expression level of the circRBM33 in the C4-2 and PC-3 cells after lentivirus transfection is obviously improved. It was also found that at the transcriptional level, lentiviral transfection had only a regulatory effect on the expression level of circRBM33, without any effect on linear RBM33 (not shown).
Example 3 Effect of circRBM33 on prostate cancer behavior and function in vitro and in vivo
From the foregoing examples, it is expected that the circRBM33 may have a promoting effect on the progression of prostate cancer, i.e., the circRBM33 plays a role as a pro-cancerous factor in the progression of prostate cancer disease. To verify this conclusion, it was confirmed by a series of in vitro experiments.
By designing shRNA targeting circRBM33, its effect on prostate cancer cell proliferation was studied as follows:
(1) shRNA targeting circRBM33 (sh-C1 and sh-C2) were transfected into 22Rv1 and DU145 cells, respectively;
(2) When the cells grow to the logarithmic phase, pancreatin is digested and counted, the proper cell density is selected according to the doubling time of various cells, and the cells are inoculated into a 96-well plate (3 repeats);
(3) Cultures were carried out in an incubator at 37℃and at CCK-8 for 24h, 48h, 72h and 96h, respectively: the medium containing CCK-8 was added at a ratio of medium (10. Mu.L: 90. Mu.L), and the plates were incubated in an incubator for 2 hours, and the absorbance at 450nm was measured to evaluate the proliferation status of the cells.
The results are shown in FIG. 8. The results show that after the shRNA is utilized to silence the circRBM33, the proliferation capacity of the prostate cancer cells is obviously reduced, the growth of the prostate cancer cells can be obviously inhibited, the proliferation activity of the prostate cancer cells is obviously reduced, and the difference has statistical significance (p is less than 0.01).
Subsequently, the effect of shRNA targeting circRBM33 on prostate cancer cell clone formation was studied as follows:
(1) shRNA targeting circRBM33 (sh-C1 and sh-C2) were transfected into 22Rv1 and DU145 cells, respectively;
(2) When the cells grow to the logarithmic phase, the pancreatin is digested and counted, the proper cell density is selected according to the multiplication time of various cells, and the cells are inoculated into a six-hole plate containing 2mL of a pre-temperature culture solution with the temperature of 37 ℃ and gently swayed back and forth and left and right to ensure that the cells are uniformly dispersed, and the cells are placed at the temperature of 37 ℃ and contain 5 percent CO 2 Is cultured in a cell culture box;
(3) When macroscopic clones appear in the culture dish, the culture is stopped, the supernatant is discarded, the culture dish is carefully immersed and washed 2 times with PBS, 1mL of methanol containing 0.5% crystal violet is added to each well, and the culture dish is dyed for 30min; discarding the methanol and washing the residual methanol with water; cell clones can be observed; the number of cells was > 50, counted as an effective clone, as observed under a microscope.
The detection results are shown in FIG. 9. The results show that, compared with the blank vector sh-NC group, after the shRNA is used for silencing the circRBM33, the clonogenic capacity of the prostate cancer cells is significantly reduced, the clonogenic capacity of the prostate cancer cells can be significantly inhibited, and the difference has statistical significance (p < 0.01, p < 0.001).
Further, by targeting shRNA of circRBM33, its effect on prostate cancer cell migration was studied as follows:
(1) When the cells grow to the logarithmic phase, the pancreatin is digested and counted, the proper cell density is selected according to the multiplication time of various cells, and the cells are diluted by a serum-free culture medium to prepare a cell suspension;
(2) Adding 200 mu L of cell suspension into the upper chamber, adding 500 mu L of culture medium containing 10% FBS into the lower chamber, and culturing in a 37 ℃ incubator;
(3) Taking out after 24/48 hours, sucking out redundant liquid in the upper chamber, washing twice with PBS, lightly rotating a cotton stick in the upper chamber, sucking up water and wiping off cells on the inner side of the membrane;
(4) Adding 0.5% crystal violet dye solution into the upper chamber, dyeing for 20min, recovering the dye solution, slowly buffering to remove the dye solution with PBS, lightly rotating with cotton stick in the upper chamber again, and drying by suction;
(5) A slide was placed on the overhead microscope, the cell chamber was inverted on the slide, photographed under a 100-fold field of view, and the membrane was counted up, down, left, right, and middle.
The detection results are shown in FIG. 10. The results show that, both for 22Rv1 cells and DU145 cells, the expression level of cirrbm 33 in the cells was significantly inhibited by shRNA compared to the blank vector sh-NC group, and the differences were statistically significant (< 0.01, < 0.001).
Subsequently, PC-3 and C4-2 cells were transfected by the lentiviral transfection method of example 2, and the above cell proliferation, clone formation and cell migration experiments were repeated, and the results are shown in FIGS. 11-13, respectively. The results show that the expression level of circRBM33 in PC-3 and C4-2 was significantly increased after lentiviral transfection, whereas overexpression of circRBM33 significantly increased the proliferation activity, clonogenic level and migration capacity of prostate cancer (< 0.01, p < 0.001). From the above results, it is clear that the circRBM33 has a significant effect on the functional activity of the prostate cancer cells, and can effectively inhibit proliferation, cloning and metastasis of the prostate cancer by inhibiting the expression of the circRBM33, and can significantly promote proliferation, cloning and metastasis of the prostate cancer when the circRBM33 is overexpressed. From this, it is known that cir cRBM33 is a key target closely related to the progression of prostate cancer, and by intervening in this, it can effectively inhibit the progression of prostate cancer and other processes.
To study the effect of circRBM33 on prostate cancer in vivo, 4 week old BALB/c-nu mice were selected for in vivo experiments, with the following specific steps:
first, tumor volumes were measured every 3 days after day 7, and by the end of day 28, all mice were sacrificed for dissection and tumor measurements.
(1) The day before the experiment, the Matrigel which has been packaged is put into a refrigerator at 4 ℃ for overnight from-20 ℃ in advance, so that the Matrigel is melted from a solid state to a liquid state.
(2) Taking 20 BALB/c-nu mice of 4 weeks old, randomly dividing into 4 groups, marking as groups 1-4, and subcutaneously injecting the circlRBM 33 up-regulated or circlRBM 33 silenced prostate cancer cells mixed with Matrigel matrix into the back respectively; wherein, group 1 mice subcutaneously inject PC-3 cells over-expressed by circR BM33, group 2 mice subcutaneously inject PC-3 containing 3_cir5 as a negative control, group 3 mice subcutaneously inject 22Rv1 cells inhibited by circRBM33 expression, and group 4 mice subcutaneously inject 22Rv1 cells containing sh-NC as a negative control.
(3) Mice were observed daily for growth and mental status, tumor size was measured for each group of mice every 3 days after 7 days of injection, and tumor volume was calculated using the following formula: volume (mm) 3 )＝Length(mm)×Width 2 (mm 2 )/2。
(4) Mice were sacrificed on day 28, and after the mice were sacrificed by excessive anesthesia, the tumors of each group of mice were stripped off and photographed and the volume was measured.
The experimental results are shown in FIG. 14. The results show that overexpression of circRBM33 significantly increases tumor volume and growth rate, while inhibition of circRBM33 expression effectively impedes tumor progression.
Prior studies have found that castration resistant prostate cancer cells have a greater capacity for mitochondrial metabolism, resulting in increased oxidative phosphorylation, making them susceptible to treatment by mitochondrial metabolism. Given the indirect effect of circRBM33 on mitochondrial respiration, it was investigated whether silencing cir cRBM33 would affect PCa cell sensitivity to ARSIs by assessing. To evaluate the response of PC a cells to ARSIs, two ARSIs were used in cell culture, namely Enzalu amide and dareutamide (see fig. 15). With increasing administration concentration, the growth of the prostate cancer cells tends to decrease, and in contrast, in the cells with the suppressed expression of the circRBM33, the cell growth is more significantly suppressed, i.e., the circRBM33 can effectively enhance the therapeutic effect of ARS I on prostate cancer.
Subsequently, xenograft tumors were induced subcutaneously by subcutaneously injecting circRBM33 expression-inhibiting cells (sh-C1) and negative control cells (sh-NC) into BALB/C-nu mice, and after tumor formation, the random mice were divided into two groups, and each group was subjected to gastric administration with Enzalutamide and DMSO, and the mice were periodically monitored for status and tumor volumes. Mice were sacrificed one month later, tumor growth curves were drawn for the mice, and weights and volumes of the mice were measured. The experimental results are shown in fig. 16, and the results show that the expression of the circRBM33 is down-regulated to effectively inhibit the growth of tumors in mice in accordance with the previous experiments, namely, compared with a negative control group; meanwhile, the inhibition of the cir cRBM33 expression can more effectively increase the anti-tumor activity of Enzalutamide. The above experiment was repeated using daroutamide and similar results were obtained (see fig. 17), thus demonstrating that the therapeutic effect of ARSIs can be improved by inhibition of circRBM 33.
EXAMPLE 4 mechanism of action study of circRBM33
In order to search how the circRBM33 affects prostate cancer, intensive studies have been made on its mechanism of action. First, the subdocalization of cirrbm 33 in cells was detected by FISH (Fluorescence in Situ Hybrid ization, fluorescent in situ hybridization), wherein Cy 3-labeled cirrbm 33 and 18S probe (cytoplasmic indicator) were synthesized by GenePharm, using the kit of RiboTM fluorescent in situ hybridization kit (RIBOBI O, C10910), the specific experimental procedure was as follows:
(1) Prostate cancer cells (PC-3, C4-2) were fixed with 4% paraformaldehyde and then pre-perforated with 1% Triton.
(2) Prehybridization buffer was incubated at room temperature for 30min and hybridization buffer mixed with 20. Mu.M probe at 37℃overnight.
(3) Cells were washed sequentially with 4 XSSC, 0.1% Tween-20, 2 XSSC, and 1 XSSC buffer, and stained with DAPI-containing anti-fluorescence quencher (P0131).
The results are shown in FIG. 18. The results show that circRBM33 is primarily localized in the cytoplasm of prostate cancer. Furthermore, the results of the nucleoplasmic isolation extraction experiments are consistent with the FISH results described above, with the circRBM33 being predominantly localized in the cytoplasm (see FIG. 19).
Subsequent bioinformatics analysis by catapid and circum-intellect found that FMR1 (also known as FMRP (Fragile X Mental Retardation Prote in)) may potentially interact with circumrbm 33. For this, FISH experiments were used to co-localize the circRBM33 and FMR1, specifically as follows:
(1) Prostate cancer cells (PC-3, C4-2) were fixed with 4% paraformaldehyde and then pre-perforated with 1% Triton.
(2) Prehybridization buffer was incubated at room temperature for 30min and hybridization buffer mixed with 20. Mu.M probe at 37℃overnight.
(3) Cells were washed sequentially with 4 XSSC, 0.1% Tween-20, 2 XSSC and 1 XSSC buffer, blocked with 1% Bovine Serum Albumin (BSA) solution at room temperature for 30 minutes, and then incubated overnight at 4℃with anti-FMR1 primary antibody.
(4) Cells were incubated with Coralite 488-labeled secondary antibody (proteontech, SA 00013), DAPI stained, and co-localized observations were performed using a confocal imaging system (ZEISS, LSM 900). The results show that there is indeed a possible interaction of circRBM33 with FMR1 (see fig. 20).
To verify whether FMR1 is one of the molecules that interacts with the circRBM33, a probe was designed for the circRBM33 for the ChIRP assay (BersinBio, bes 5104) to lock the molecule that interacts with the circRBM33 as follows:
(1) The cells were collected, crosslinked with 4% formaldehyde solution, and neutralized with glycine at room temperature.
(2) Cells were lysed with lysis buffer containing protease and RNase inhibitor.
(3) Cell lysates were disrupted by sonication and pre-cleared with agarose beads at 4 ℃.
(4) The probes targeting circRBM33 and the negative control probes were hybridized with the cell lysate and incubated with streptavidin magnetic beads.
(5) The pulled down RNA and protein samples were subjected to qRT-PCR and WB assays, respectively.
The detection results are shown in FIG. 21. The result shows that RN A separated from the Churp sample is circRM33, and protein separated from the Churp sample is found to be one of RBP of circRBM33 by WB detection. The presence of molecular binding between FMR1 and circRBM33 was subsequently confirmed by RIP-able detection of anti-FMR1 (see FIG. 22).
Since METTL3 is one of the most important regulatory factors in the m6A modification, it was investigated whether the circRBM33 interacts with FMR1 in an m 6A-dependent manner by silencing expression of METTL3 using sh-METTL3 (sequence ccggGCTGCACTTCAGACGAATTATctcgagATAATT CGTCTGAAGTGCAGCtttttt). The results showed that when METTL3 expression levels were down-regulated, neither M1 nor M2 containing the potential binding site sequence of circRBM33 could be detected in the MeRIP samples, indicating that METTL3 was involved in the M6A modification of circRBM33 (see FIG. 23). Subsequent FMR1-RIP experiments showed that inhibiting METTL3 expression disrupts the binding between FMR1 and circRBM33 (see fig. 24), from which it can be concluded that MET TL3 is involved in the interaction of FMR1 and circRBM33, demonstrating that circRBM33 interacts with FMR1 in an m6A dependent manner.
Example 5
In the previous examples, it was verified that the interaction of circRBM33 with FMR1, in order to determine whether FMR1 would affect prostate cancer progression, FMR1 expression silenced cells were constructed by siRNA (si-1, si-2, where si-1 is CCAGAAGACUUACGGCAAAT T, si-2 is GCAUCAAAUGCUUCUGAAATT, and si-NC is a blank) transfection in 22Rv1 and DU145 cells (see FIG. 25). The effect of FMR 1-targeted siRNA on prostate cancer cell proliferation, colony formation and migration was then investigated, see example 3 for specific procedures.
The results are shown in FIGS. 26-28. The cell proliferation experimental result shows that after the SiR NA is utilized to silence FMR1, the proliferation capacity of the prostate cancer cells is obviously weakened, the growth of the prostate cancer cells can be obviously inhibited, the proliferation activity of the prostate cancer cells is obviously reduced, and the difference has statistical significance. The results of the clonogenic experiments showed that, compared to the blank vector si-NC group, the clonogenic capacity of prostate cancer cells was significantly reduced after silencing FMR1 with siRNA, which significantly inhibited the clonogenic capacity of prostate cancer cells, and the differences were statistically significant (p < 0.001, p < 0.0001). Cell migration experiments showed that, for both 22Rv1 cells and DU145 cells, the ability of the cells to migrate was significantly inhibited after inhibition of intracellular FMR1 expression levels using siRNA compared to the blank vector si-NC group, the differences were statistically significant (< 0.01, 0.001).
Through analysis of clinical samples, patients with low in vivo FMR1 expression levels were found to possess better DFS than patients with high FM R1 expression levels, suggesting that FMR1 is associated with a poor prognosis for prostate cancer (see fig. 29). Further analysis found that the expression level of circRBM33 was significantly higher in the tumor tissue of the prostate cancer patient than in the normal tissue, and that the higher the Gleason score, the higher the expression level of circRBM33 (see fig. 30). The immunohistochemical results showed that FMR1 levels were significantly higher in tumor tissues than in normal tissues, and that the more severe the malignancy, the higher the FMR1 expression level (see fig. 31). In addition, there was a significant difference in FMR1 scores between the circRBM33 low-expression group and the circRBM33 high-expression group (see fig. 32). Tumor tissue of the patients in the circRBM33 high expression group had a higher FMR1 score than tumor tissue of the patients in the circRBM33 low expression group. In this regard, the clear positive correlation of circRBM33 with FMR1 in prostate cancer was confirmed using the chi-square test (see fig. 33). It is assumed that the circR BM33 may co-exert its tumorigenic effect in PCa by interacting with FMR1 to form a complex, and that inhibition of circRBM33 and/or FMR1 would be effective in inhibiting prostate cancer progression.
To verify the above hypothesis, FMR1 was knocked out by using siRNA in the circumrbm 33 overexpressed prostate cancer cells to investigate the role of FMR1 in the circumrbm 33 mediated malignant phenotype, as shown in fig. 34, the results showed that PDHA1 expression in circumrbm 33 overexpressed cells could be significantly inhibited after FMR1 knockdown. A series of cell experiments prove that the reduction of the FMR1 expression can effectively inhibit the proliferation, colony formation and migration capacity of the prostate cancer cells, and has obvious reversal effect on the functional change of the prostate cancer cells caused by the overexpression of the circRBM33 (see figures 35-37).
In view of the above results, the invention fully reveals the role of the circRBM33 in cancers, especially prostate cancer cells and the target genes regulated and controlled by the circRBM33, clarifies the new mechanism of regulating and controlling the prostate cancer by the circRBM33, and finds out the key targets for inhibiting the prostate cancer. The invention carries out intensive research on the function of the circR BM33 in the prostate cancer progression process, and defines the influence of the circR BM33 on the proliferation and migration capacity of prostate cancer cells and the influence of the circR BM33 on ARSIs treatment. The invention makes a brand-new elucidation on the progress mechanism of the prostate cancer, provides a brand-new thought for later-stage related drug development and clinical diagnosis and treatment, and has great social significance and market prospect.
The above detailed description describes the analysis method according to the present invention. It should be noted that the above description is only intended to help those skilled in the art to better understand the method and idea of the present invention, and is not intended to limit the related content. Those skilled in the art may make appropriate adjustments or modifications to the present invention without departing from the principle of the present invention, and such adjustments and modifications should also fall within the scope of the present invention.
Claims (10)
- Use of a circrbm33 inhibitor for the preparation of a product for the prevention and/or treatment of prostate cancer.
- 2. The use according to claim 1, wherein the circRBM33 inhibitor is selected from shRNA based on the circRBM33 gene design.
- 3. Use of an agent for detecting the expression level of circRBM33 in the preparation of a product for use in the assisted diagnosis and/or prognosis evaluation of prostate cancer.
- 4. The use according to claim 3, wherein the reagent for detecting the expression level of circRB M33 comprises a primer pair for detecting the expression level of the circRBM33 gene.
- Use of a circrbm33 inhibitor for the manufacture of a medicament for promoting sensitivity of an androgen receptor signaling pathway inhibitor to cancer treatment.
- 6. The use according to claim 5, wherein the cancer is selected from prostate cancer.
- 7. The use according to claim 5, wherein the androgen receptor signaling pathway inhibitor is selected from one or more of enzal Lu An, darunamine, apatamide, abiraterone.
- 8. The use according to claim 5, wherein the circRBM33 inhibitor is selected from shRNA based on the circRBM33 gene design.
- 9. A pharmaceutical composition for preventing and/or treating prostate cancer, comprising a circRBM33 inhibitor and an FMR1 inhibitor.
- 10. The pharmaceutical composition of claim 9, wherein the cir cRBM33 inhibitor is selected from shRNA designed based on the circRBM33 gene; the FMR1 inhibitor is selected from siRNA designed based on FMR1 gene.
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