Researches On The Function Of MiR-375and MTDH In Prostate Cancer Cell

Part I Expression of miR-375and MTDH in prostate cancer and clinical significanceObjective:To study the expression of miR-375and MTDH in Prostate cancer (PCa) and clinical significance.Methods:The Expressions of miR-375and MTDH were detected by real-time PCR in10cases of PCa and matched adjacent non-tumor tissue.The relevance between the expressions of miR-375and MTDH and the pathologic staging and grading were also studied.Results:The relative expression of miR-375in PCa is significantly down-regulated than that in the matched adjacent non-tumor tissue (p<0.01),The relative expression of MTDH in PCa was higher than that in the matched adjacent non-tumor tissue with significant difference (p<0.01).The relative expression of miR-375and MTDH was significantly different in tumors according to T1-T2and T3a pathologic staging (P<0.05);Grouping G1-G2and G3-G4according to pathologic grading,the expression of miR-375had no significant difference (p>0.05),the expression of MTDH had significant difference (p<0.05). There is a negative correlation between the expression of miR-375and MTDH in prostate cancer tissue, the correlation coefficient r=-0.79(p <0.01).Conclusions:1. Compared with matched adjacent non-tumor tissue,the expression of miR-375was down-regulated and MTDH was up-regulated in PCa, suggesting that miR-375and MTDH may participate in the pathogenesis of PCa.2. The expression of miR-375and MTDH had significant correlation with pathologic staging,and the expression of MTDH had significant correlation with grading in PCa.miR-375and MTDH expression may be associated with PCa prognosis.3. miR-375and MTDH expression in prostate cancer tissues had negative correlation,suggesting that the relationship of regulation may exist between the miR-375and MTDH. Part II miR-375, MTDH expression in different prostate cancer cell linesObjective:To study the expression of miR-375and MTDH in PC3,DU145,LNCap Prostate cancer(PCa)cell lines.Cell line with relatively low miR-375expression and high MTDH expression was selected for further experiments.Methods:Expressions of miR-375and MTDH was detected by real-time PCR in these PCa cell lines. Protein expressions of MTDH were detected by Western Bolt.Results:The relative expression of miR-375in DU145and LNCap were lower than in PC3with significant differences (p<0.01);The relative expression of MTDH in DU145and LNCap were higher than in PC3with significant differences(p<0.01);The relative expression of MTDH protein in DU145and LNCap were higher than in PC3with significant differences(p<0.01).Expression of miR-375and MTDH in DU145and LNCap had no significant differences(p>0.05).In LNCap cell lines, miR-375expression was relatively low, MTDH expression was relatively high.Conclusions:1. miR-375and MTDH expression in prostate cancer cell lines have no evident correlation with the malignant degree PCa cells. 2. LNCap cell lines had relatively low miR-375expression and high MTDH expression, thus were chosen for further experiments. Part Ⅲ The relations of regulation between miR-375and MTDH in prostate cancerObjective:To explore the relations of regulation between miR-375and MTDH in prostate cancer.Methods:Using LipofectamineTM2000transfect miR-375mimics fragment and Empty vector in LNCap cells.The expression of miR-375and MTDH mRNA in LNCap cells was detected by real-time PCR. Expressions of MTDH protein was detected by Western Bolt.Results:In LNCap cells, the relative expression of miR-375in NC group was lower than the effective transfection group with significant differences(p<0.01);MTDH in NC group was higher than the effective transfection group with significant differences (p<0.01);MTDH protein in NC group was higher than the effective transfection group with significant differences (p<0.01).Conclusions:1. miR-375mimics fragment could be effectively transfected to PCa cells, resluting in upregulating the expression of miR-375.2. Upregulation expression of miR-375induce downregulation expression of MTDH in PCa cells. partIV Effects of miR-375and MTDH on the biological behavior of LNCap cellObjective:To explore the influence of miR-375and MTDH on biological behaviors of the cell proliferation, apoptosis and invasive ability in LNCap cell.Methods:1. Construct miR-375mimics fragment and inhibitors of MTDH,transfect them into LNCap cells.2. The expression of miR-375and MTDH in LNCap cells was detected by real-time PCR, and expressions of MTDH protein and PI3K/Akt pathway proteins was detected by Western Bolt.3. Cell viability was determinded by means of MTT. Apoptosis of LNCap cells was detected by AV/PI Double Labeling Method FCM. The influence of miR-375and MTDH on the invasive ability of LNCap cells was determined through cell invasion assay.4. The MTT assay cisplatin toxicity experiments were applied to select the optimal drug concentration.5. Cells were devived into6groups:Group A:Normally developed LNCap cells;Group B:LNCap cells+miR-375mimics fragment;Group C:LNCap cells+miR-375mimics fragment+cisplatin;Group D:LNCap cells+cisplatin;Group E:LNCap cells+inhibitors of MTDH;Group F: LNCap cells+inhibitors of MTDH+cisplatin Results:1. miR-375mimics fragment and inhibitors of MTDH was constructed and successfully transfected into LNCap cells,the transfection rate was over80%.2. Tumor cell survival rate was83%after treated with1μg/ml cisplatin for24h, this drug concentration was used in following experiments.3. The cell viability of cells in B, C, D, E, F group was obviously lower than that of A group, and the difference has statistical significance(P <0.05). The cell viability was markedly lower in group C and F than that of group A, and the difference had statistical significance(P<0.01); The cell viability of group C and F was obviously lower than that of group D, and the difference had statistical significance(P<0.01).Group B and C had obviously lower viability than that of Group E and F respectively, and the difference had statistical significance(P<0.01).4. The apoptosis rate of Cell in B, C, D, E, F group was obviously higher than that of A group, and the difference had statistical significance(P <0.01).C, F group was obviously higher than that of A group, and the difference had statistical significance(P<0.01);C, F group were obviously higher than that of D group,and the difference had statistical significance(P<0.01).B, C group were obviously higher than that of E、F group respectively, and the difference had statistical significance(P <0.01). 5. The number of cell invasion in B, C, D, E, F group was obviously less than that of A group, and the difference had statistical significance(P <0.01).C, F group were obviously less than that of A group, and the difference had statistical significance(P<0.01);C, F group were obviously less than that of D group, and the difference had statistical significance(P <0.01);B,C group were obviously less than that of E,F group respectively, and the difference had statistical significance(P<0.01).6. Expression of MTDH,p-PI3K-p85,Akt and p-Akt in A group were higher than that of E group,and the difference had statistical significance(P<0.01).Conclusions:1. The up-regulated expression of miR-375and down-regulated expression of MTDH can inhibit the proliferation, invasion and induce apoptosis in PCa cells.2. The up-regulated expression of miR-375and down-regulated expression of MTDH can increase the sensitivity of PCa cells to cisplatin chemotherapy.3. MTDH promote the development of prostate cancer by P13K/AKT pathway.4. miR-375may down-regulate MTDH to inhibit the development of prostate cancer...