miR-301a靶向调控Smad4介导高糖促进前列腺癌细胞增殖的作用及其机制

目的观察miR-301a靶向调控Smad4介导高糖促进前列腺癌细胞增殖的作用及其机制。方法预测miR-30la的靶基因并转染miR-301a模拟物,分别采用实时定量聚合酶链反应、蛋白质印迹法对miR-30la.Smad4的表达进行检测,并采用流式细胞术对细胞增殖及周期进行分析。结果通过Pic Tar数据库及TargetScan数据库对miR-30la的靶基因进行寻找,结果显示,Smad4是miR-301a的靶基因。双荧光素酶实验也提示.miR-301a的结合位点是Smad4。miR-30la可与Smad4的野生型3:UTR结合(但未与Smad4中发生突变的3:UTR结合),对荧光素酶活性造成抑制。miR-30la过表达后,可抑制Smad4蛋白,但不影响Smad4的mRNA。加入高糖后会导致Smad4蛋白表达水平明显降低,与miR-30la表达相似,miR-301a抑制物注入到DU145和PC3细胞可改变其抑制作用。转染Smad4 siRNA后DU145和PC3细胞中的Smad4 mRNA均呈低水平表达,致使Cyclin E和Cyclin DI表达呈上升趋势,可增加磷酸化Rb蛋白(ppRb)。上调miR-30la的表达同时转染过表达Smad4的质粒。采用CCK-8实验检测,结果显示,转染96 h后,DU145和PC3细胞的增殖能力明显上升(P<0.05)。在PC3细胞中,转染miR-30la前的细胞周期比例为G0/GI期63.05%,S期27.93%,02/M期9.37%;转染后的G0/CI期49.96%,s期40.97%,G2/M期9.04%;转染miR-30la前、后的细胞周期中G0和S期的比例比较,差异有统计学意义(P<0.05);在DU145细胞中,转染miR-301a前的G0/GI期为65.22%,SD期为24.62%,G2/M期为10.35%;转染后为G0/GI期为53.45%,S期为37.65%,G2/M期为10.03%;转染miR-30la前、后的C0和S期比例比较,差异有统计学意义(P<0.05)。沉默DU145.PC3细胞中的Smad4与过表达miR-301a,均可导致细胞周期CI期缩短,S期延长。另外,miR-301a诱导的G1/S期转化可被Smad4过表达阻断。结论Smad4是milR-301a的靶基因,miR-30la靶向调控Smad4介导高糖促进前列腺癌细胞增殖的作用机制主要是通过对Smad4和miR-301a通路进行抑制.对高i血u糖诱导的前列腺癌细胞生长起到阻断作用。

miR-301a regulates targeting Smad4-mediated high glucose promotes proliferation of prostate cancer cells and its mechanism

Objective To observe the effect and mechanism of miK-30la in regulating and targeting Smad4 to promote the proliferation of prostate cancer cells by high glucose.Methods The target genes of miR-30la were predicted,miR-301a mimics were transfected,and the expressions of miR-30la and Smad4 were detected by real-time quantitative polymerase-linked reaction and W estern boting.Flow cytomelry was used to detect the expression of miR 301a and Smad4.Cycle analysis.Results The target gene of miR-301a was searched through Pic Tar database and TargetScan data-base.The results showed that Smad4 is the target gene of miR 301a.The dual lueiferase experiment also suggests that the binding site of miR-301a is Smad4.miR-301a can bind to the wild-type 3-UTR of Smad4,but it does not bind to the 3-UTR that is mutated in Smad4,which can inhibit luciferase ac-tivity.After miR-301a is overexpressed,Smad4 protein can be suppressed,but it does not afect Smad4 mRNA.The addition of high sugar will result in a signifcant decrease in the expression level of Smad4 protein,similar to the expression of miR-301a.Adding DU145 and PC3 cells to miR-30la can change is production.After transfection of Smad4 siRNA,the expression of Smad4 mRNA in DU145 and PC3 cells was low,causing the expression of Cyclin E and Cyelin DI to inerease,which can in-crease phosphorylated Rb protein(ppRb).The expression of miR-301a was up-regulated and trans-fected with a plasmid overexpressing Smad4.The CCK-8 experiment showed that the proliferation abili-ty of DU145 and PC3 cells increased significantly by 162%at 96 hours after transfection,a significant difference(P<0.05).In PC3 cells,the cell cycle ratio before miR-301a transfection was G0/G 163.05%,S 27.93%,G2/M 9.37%;after transfection G0/G149.96%,S 40.97%,G2/M 9.04%.The ratio of G0 and S phases in the cell cycle before and after miR-301a transfection was sig-nificantly different(P<0.05).In DU-145 cells,before miR-301a transfection G0/GI 65.22%,S 24.62%,G2/M 10.35%,G0/GI 53.45%,S 37.65%,G2/M 10.03%after transfection,G0 and S phases proportionsbefore and after miR-301a transfeetion were compared,the difference was signifi-cant(P<0.05).Sileneing DU-145 cells and PC3 cells with Smad4 and overexpression of miR-301a both shortened the Gl phase of the cell cycle and prolonged the s phase.In addition,miR-301a-in-duced GI/S transition could be blocked by Smad4 overexpression.Conclusions Smad4 is the larget gene of miR-301a.The mechanism of miR 301a regulating and targeting Smad4 to mediate high glucose and promote the proliferation of prostate cancer cells is to suppress the Smad4 and miR-301a pathways and inhibit the growth of prostate cancer cells induced by hyperglycemia play a blocking role.