| Background and purpose:Mutiple myeloma(MM)is a worldwide common malignant hematological malignancy.Abnormal proliferation of monoclonal plasma cells in the bone marrow is the main pathological change of MM.Monoclonal plasma cells secrete large amount of monoclonal immunoglobulin,which damages important organs and results in poor prognosis.MM is the second largest tumor in hematopoietic system,and the incidence rate is about 10% of the malignant tumors of hematopoietic system.The incidence rate is increasing in recent years,and the age of onset is becoming younger.The pathogenesis of MM is complicated.Although the clinical application of new research and development drugs has improved the prognosis of MM patients in recent years,drug resistance and disease recurrence still challenge the clinical treatment of MM patients.Numerous studies found that epigenetics was involved in the progression of MM.Epigenetics presented to be a new treatment strategy for MM patients.Epigenetics may provide a new approach for individualized gene therapy in patients with multiple myeloma.MicroRNAs(miRNAs)are the major form of epigenetics.MiRNAS is a class of highly conserved non coding small molecule RNAs with a length of 22 nucleotides.MiRNAs specifically binds to the 3’UTR region of the target gene,regulating approximately 60% of human genes at post transcriptional levels.MiRNAs play a role in cell proliferation,cell cycle,embryonic development and immunity.Recent studies have shown that miRNAs are specifically expressed in a variety of hematopoietic malignancies.MiRNAs play an important role in the progression of hematopoietic malignancies by targeting the expression of tumor suppressor genes or the expression of oncogenes.Recent studies indicate that circulating miRNAs may present as a new molecular biomarker for the early diagnosis,efficacy monitoring and targeted gene therapy of multiple myeloma.MicroRNA-195(miR-195),is located on the human chromosome 17p13.1,belongs to the miR-15 family.It is specifically expressed in many malignant tumors such as prostate cancer,hepatocellular cancer,gastric cancer,ovarian cancer,oral verrucous carcinoma and so on.MiR-195 plays an important role in tumor cell proliferation,invasion,angiogenesis,and apoptosis.However,its role in multiple myeloma has not been established.Currently,there are no research reports about the role of miR-195 in multiple myeloma.The aim of this study is to investigate the serum expression of miR-195 in multiple myeloma patients and analyze the correlation between the expression and clinicopathological parameters of MM patients.As well as the molecular mechanism of miR-195 inhibits multiple myeloma cell proliferation and angiogenesis.Furthermore,we also investigate the molecular mechanism of miR-195 regulates target gene and downstream signaling pathway.This study is divided into three parts: Part one: The expression of circulating miR-195 in multiple myeloma and the relation between miR-195 and the patients clinicopathological features;Part two: The potential role of miR-195 expression on proliferation and apoptosis of multiple myeloma in vitro,in vivo;Part three: The miR-195 targeted gene and downstream signaling pathways.Part one: The expression of circulating miR-195 in multiple myeloma and the relation between miR-195 and the clinicopathological features Purpose:By detecting the expression serum level of miR-195 in multiple myeloma,and combining patient’s clinical data,we try to identify the relation between circulating miR-195 and patients’ clinicopathological features.Methods:1.We detect the circulating serum expression of miR-195 in 30 healthy population and 96 multiple myelomapatients by qRT-PCR assay,compare circulating serum expression levels of miR-195 mRNA in multiple myeloma with healthy population expression levels.The level of miR-195 is divided into two groups: the ratio of expression in healthy population and multiple myeloma is ≤ 2 for high expression,ratio> 2 for the low expression group.2.Correlation analysis for the relationship between miR-195 expression and clinicopathological features.Results:1.qRT-PCR results showed that the expression of miR-195 in multiple myeloma were 2.634±0.231、1.748±0.135.miR-195 was significantly up-regulated in healthy population compared with multiple myeloma(P = 0.0015).2.The expression of miR-195 mRNA in multiple myeloma and clinical ISS stage is correlated(P<0.05),but are not significant correlation with age,monoclonal immunoglublin types(P>0.05).miR-195 expressing in ISS I multiple myeloma was higher than ISS Ⅱ and Ⅲ(P=0.032).Summary:1.Circulating miR-195 is downregulated in multiple myeloma may be involved in myeloma initiation and development.2.Expression of miR-195 in late clinical stage patients with multiple myeloma was significantly lower,indicating that decreased miR-195 have some relation with myeloma developing.Part two: The potential role and mechanism of miR-195 on proliferation and apoptosis in multiple myeloma in vitro and in vivo Purpose:In vitro and in vivo study detected the potential mechanisms of miR-195 and exploring its role in inhibiting the proliferation and inducing the apoptosis of myeloma cells.Methods:1.RT-PCR was used to detect the expression of miR-195 mRNA in different multiple myeloma cell lines KMS-11,RPMI8226 and H929.2.pGCMV/EGFP/miR/Blasticidin lentiviral vector to construct the H929 cell line with miR-195 over-expression and RPMI8226 cell line with miR-195 low-expression.Then we identified the successfully constructed by RT-PCR.3.CCK-8 assay was applied to identify the role of miR-195 myeloma cell proliferation.4.AnnexinV/PI assay was applied to identify the role of miR-195 over-expression and low-expression on the proliferation and apoptosis of multiple myeloma cells.5.We established xenografts mice model and detected the role of miR-195 over-expression and low-expression on xenografts proliferation in mice model.Results:1.RT-PCR indicated that compared with normal plasma cells from bone marrow,the miR-195 mRNA expressed in KMS-11,RPMI8226 and H929 were 0.190±0.028,0.613±0.018 and 0.410±0.029,respectively.Therelative miR-195 mRNA in multiple myeloma cells was significantly decreased compared with normal bone marrow plasma cells(P=0.0002,P=0.012 and P=0.0022,respectively).The miR-195 was the lowest in H929 cells and was the highest inRPMI8226 cells.Since H929 and RPMI8226 cell lines can establish solid myeloma in nude mice model,we select them to construct mice model to identify the role of miR-195 on multiple myeloma cells in vivo.2.qRT-PCR demonstrated that compared with the H929 cells with empty vector transfecting,the miR-195 was significantly upregulated in H929 cells infected with miR-195 lentivirus(0.173±0.015 vs 1.858±0.118,P=0.0003).Compared with RPMI8226-NC cells,the miR-195 mRNA was significantly downregulated in RPMI8226-anti-miR195 cells(0.531±0.039 vs 0.179±0.029,P=0.0019).3.CCK-8 results indicated thatcompared with H929-NC cells,the growth of H929-miR195 cells appeared weakened(P=0.034).Compared with RPMI8226-NC cells,the number of RPMI8226-anti-miR195 cells significantly increased(P=0.044).4.AnnexinV FITC/PI showed that compared with H929-NC cells the apoptosis in H929-miR195 cells apoptosis rate significantly increased(16.24±0.054% vs 3.32±0.136%,P=0.00013).Compared withRPMI8226-NC cells,the apoptosis rate inRPMI8226-miR195 cells significantly decreased(2.08±0.048%vs 6.860±0.049%,P=0.00016).5.The size of subcutaneous tumor in xenograft mice models showed that the tumor size in miR-195 overexpressed H929-miR195 nude mice significantly smaller than H929-NC group(P=0.00013).The miR-195 was significantly higher in RPMI8226-NC group than in RPMI8226-anti-miR195 group(P=0.024)and increase with tumor volume increasing.Summary:1.In vitro and in vivo examinations demonstrated that miR-195 significantly inhibited proliferation and induced cell apoptosis of myeloma cells.Part three: MiR-195 targeted gene and related signaling pathway onproliferation and apoptosis of multiple myeloma Purpose:To study the mechanism of miR-195 and its potential targeted genes and related downstream signaling pathways in inhibiting the proliferation of multiple myeloma cells.Methord:1.Using bioinformatics data analysis software to predict the target genes of miR-195.2.luciferase reporter gene assay system,Western Blot and qRT-PCR SYBR green were applied to identify that VEGF was the target gene of miR-195.qRT-PCR SYBR green was applied to detect the circulating VEGF in miR-195 low expressed and high expressed multiple myeloma patients and analysis the relation between VEGF and miR-195 mRNA expression.3.Lentiviral vectors were used to construct VEGF overexpressed lentiviral(pVEGF)plasmids and VEGFinterferedlentiviral(shVEGF)plasmids respectively.Western Blot was used to verify whether VEGF overexpressed and VEGF interfered lentiviral plasmid were successfully constructed.4.CCK-8 was applied to detect the role of miR-195 in negatively regulating the VEGF gene expression on the proliferation of myeloma cells.5.Annexin V FITC/PI was applied to detect the effect of MiR-195 regulated VEGF expression on myeloma cells apoptosis.6.Western Blot,qRT-PCR and immunohistochemistry were used to detect the effect of miR-195 in regulating VEGF and its related VEGFR2 signaling pathway.Results:1.Bioinformatics analysis software was used to analyze and predict the genes that miR-195 genes might regulate.The VEGF gene,which is related to myeloma cell proliferation and angiogenesis,was selected as a possible target gene for miR-195.MiR-195 is involved in the proliferation and apoptosis of multiple myeloma cells.2.The dual luciferase reporter assay results showed that the luciferase activity of the cells after co-transfection of miR-195 expression plasmid and VEGFAwt plasmid was significantly reduced(P=0.034);the luciferase activity was significantly enhanced(P=0.026)in the cells co-transfected with interference miR-195 plasmid and VEGFAwt plasmid.MiR-195 expression plasmid and VEGFAmut plasmid co-transfection,and miR-195-negative plasmid,VEGFwt,VEGFmut plasmid co-transfection showed no significant difference in luciferase activity(P>0.05).This suggested miR-195 might bind to VEGF 3’UTR region and dowen-regulate the expression of VEGF gene.3.The results of Western blotting and Real-time quantitative PCR showed that,compared with H929-NC and RPMI8226 control group,the expressions of VEGF mRNA and protein were significantly decreased(P=0.00018,P=0.026)in H929-miR-195 cells;and compared with RPMI8226-anti-NC control group,he expressions of VEGF mRNA and protein were significantly increased(P=0.0061,P=0.043),suggested that,with connected with VEGF’s 3’UTR,after transcription to suppress VEGF protein translation,miR-195 can cause VEGFmRNA degradation.4.With LV5 lentiviral vector to construct VEGF overexpressed vector,With pGLVH6/luci05/Puro lentiviral vector to construct VEGF interfered vector,The protein expression levels afterRPMI8226 cells overexpressing VEGF significantly increased(P<0.001),while H929 cells interfere with protein expression levels after VEGF was significantly lower(P<0.001),all these results suggested that interference and overexpression VEGFvector was successfully constructed.5.The results of CCK-8 showed that,compared with H929-miR-195 group,growth capacities ofH929-miR195-pVEGF group were significantly increased(P=0.043);compared with H929-miR-195 cells,proliferation of H929-shVEGF group were no significant difference(P>0.05).Compared with RPMI8226-anti-miR195 cells RPMI8226-anti-miR195-shVEGF cells was significantly weakened(P=0.036).Compared with RPMI8226-anti-miR195 cells,proliferation of RPMI8226-shVEGF cells was significantly enhanced(P=0.0003).6.The results of Apoptosis assay showed that,compared with H929-miR-195 cells,H929-miR-195-pVEGF cells apoptosis rate was significantly lower(P=0.00016);and H929-miR-195 cells compared apoptosis rates H929-shVEGF cells was no significant difference(P>0.05).Compared with RPMI8226-anti-miR-497 cells,RPMI8226-anti-miR-195-shVEGF group apoptosis rate was significantly increased(P=0.043).Compared with RPMI8226-shVEGF cells,RPMI8226-anti-miR195 apoptotic cells was significantly lower,the difference was statistically significant(P=0.0047).7.RT-PCR showed that the VEGFR2,RAF1,ERK and PI3 K mRNA levels lower in H929-miR195 cells than H929-NC group(P=0.035,P=0.041,P=0.039,and P=0.0037,respectively)and caspase-9 and BAD mRNA levels higher than the H929-NC group(P=0.0043,and P=0.0041,respectively).The VEGFR2,RAF1,ERK and PI3 K mRNA levels significantly increased in H929-miR195-pVEGF than H929-miR195 cells(P=0.034,P=0.042,P=0.035,and P=0.037,respectively)and caspase-9 and BADmRNA levels lower than the H929-NC(P=0.034 and P=0.036,respectively).After interference miR-195 expression,VEGFR2,RAF1,ERK and PI3 K mRNA levels in RPMI8226-anti-miR195 cells increased compared with RPMI8226-NC cells(P=0.041,P=0.046,P=0.044 and P=0.043,respectively)and caspase-9 and BAD mRNA levels lower than RPMI8226-NC group(P =0.044,and P=0.046,respectively).While inhibiting the VEGFR2,RAF1,ERK and PI3 K mRNA levels reduced in RPMI8226-anti-miR195-shVEGF cells compared with RPMI8226-anti-miR195 cells(P=0.048,P=0.045,P=0.046 andP=0.043,respectively)and caspase-9 and BAD mRNA levels lower than the RPMI8226-anti-miR195 group(P=0.0029 and P=0.0026,respectively).Spearman correlation analysis showed that VEGFR2 multiple myelomapatients’ blood,expression level of miR-195 showed a negative correlation(r=-0.30,P=0.0018).Conclusion:1.miR-195 is downregulated in multiple myeloma and act as tumor suppressor.2.VEGF,pivotal angiogenic factors,is confirmed as the downstream target gene of miR-195.3.miR-195 negatively regulates the expression of VEGF,affects the activity of VEGFR2 signaling pathway,inhibits proliferation,and induces apoptosis of multiple myeloma cell. |
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