Expression Of MiR-29b In Prostate Cancer And Preliminary Study Of The Effects And Mechanism

Part I Expression of miR-29b in prostate cancer tissues and its clinical significanceObjective:To investigate the expression of miR-29b in prostate cancer (PCa) tissues as well as its clinical significance.Methods:Expression of miR-29b was determined by real time quantitative PCR in10cases of Pca and matched adjacent non-tumor tissues. The correlations between the expression level of miR-29b and the pathological staging and grading were also analyzed.Results:Compared with matched adjacent non-tumor tissues, the expression of miR-29b was lower in Pca tissues with statistical difference (P<0.05). According to the TNM staging criteria, there were7cases of T1-T2and3cases of T3b. No significant difference was identified between these two subsets(P>0.05). According to the Gleason grading criteria, there were4cases of G1-G2and6cases of G3-G4. No significant difference was neither identified between these two subsets(P>0.05).Conclusions:1. Compared with matched adjacent non-tumor tissues, the expression of miR-29b was down-regulated in Pca tissues with significant difference.2. No correlation was revealed between miR-29b level and TNM pathological staging as well as Gleason grading. Part Ⅱ Expression of miR-29b in different prostate cancer cell linesObjective:To investigate the expression of miR-29b in different prostate cancer cell lines and to reveal the correlation between the level of miR-29b and level of malignancy and androgen dependence of prostate cancer cell.Methods:Four cancer cell lines were selected, including DU-145, PC-3, LNCap-AI and LNCap, among which DU-145, PC-3and LNCap were derived from different origin while LNCap-AI and LNCap shared same origin. DU-145and PC-3have the characterastics of high malignancy and androgen independence, while LNCap has the characterastics of low malignancy and androgen dependence. LNCap-AI is a subgroup of LNCap cultured in medium without androgen. Expression of miR-29b was determined by real time quantitative PCR in these4prostate cancer cell lines.Results:The expression level of miR-29b from highest to lowest were expression in DU-145, PC-3, LNCap-AI and LNCap with significant difference between each groups (P<0.05). Expression of miR-29b in DU-145and PC-3were much higher than LNCap while miR-29b level of LNCap was higer than LNCap-AI(P<0.05).Conclusions:1. Expression level of miR-29b was different in4prostate cancer cell lines. It showed correlation between miR-29b and level of androgen dependence.2. Expression of miR-29b was low in LNCap. LNCap was selected for next research parts. Part III Effects of miR-29b on the biological behavior of prostate cancer cellObjective:To investigate the influence of miR-29b on biological behaviors, including cell proliferation, apoptosis, invasive ability and chemosensitivity of prostate cancer cell.Methods:First of all, miR-375mimics and inhibitors were constructed and transfected into LNCap cell. The expression of miR-29b in transfected LNCap was then determined by real-time PCR and the transfection efficiency was caculated under fluorescence microscope. Secondly, MTT was used to detect the effects of miR-29b on cisplatin toxicity and cell viability. FCM was used to determine the cell cycle changes and AV/PI double labeling method was utilized to detect the apoptosis of LNCap. Finally, influence of miR-29b on the invasive ability of LNCap cell was determined through Transwell invasion assay.Results:MiR-29b mimics and inhibitors were successfully transfected into LNCap with transfection effeciency over80%. Compared with negative control group, group of transfected miR-29b showed lower tumor cell survival rate. Moreover, miR-29b could increase the chemosensitity of cisplatin on LNCap with much lower cell survival rate. LNCap with transfected miR-29b revealed inceased percentage of cells at sub-GO/Gl phase and decreased percentage of cells at G0/G1phase, S phase as well as G2/M phase. Additionally, LNCap with transfected miR-29b showed increased apoptosis and decreased invasive ability.Conclusions:Increased expression of miR-29b can inhibit cell proliferation, invasion, induce apoptosis and increase chemosensitivity of PCa cells. Part IV Preliminary study of potential targeting genes of miR-29b in prostate cancerObjective:To explore potential target genes of miR-29b in prostate cancer.Methods:Bioinformatics was used to predict possible target genes of miR-29b. Western-blot was then used to detect protein level of predicted target genes.Results:Eight genes were selected as target genes of miR-29b through bioinformatics, including SOCS-1, Mcl-1, DNMT3b, MMP2, IFN-γ, SMAD3, SOCS5, AKT3. Protein level of three genes including AKT3, Mcl-1and DNMT3b decreased in group with transfected miR-29b mimics and increased in group with transfected miR-29b inhibitors.Conclusions:AKT3, Mcl-1and DNMT3b are potential target genes of miR-29b in prostate cancer. MiR-29b probably exerts its anti-tumor effects via targeting these three genes.