论文部分内容阅读
目的:探讨miR-145调控PLCε对膀胱癌细胞T24上皮间质转化(EMT)和迁移的影响及可能的分子机制。方法:(1)腺病毒感染T24细胞,划痕实验和Transwell检测细胞的迁移能力;RTPCR、Western blot分别检测PLCε及EMT相关分子的表达;为探究其分子机制,Western blot检测GSK-3β磷酸化(Ser9位点)和Snail的表达情况。(2)利用生物信息学技术预测可能调控PLCε的miRNA,结合文献报道的膀胱癌microRNA表达谱结果筛选出miR-145;转染miR-145 mimics至T24,q PCR检测miR-145、PLCε的表达,Western blot检测PLCε的表达。(3)转染miR-145 mimics,Western blot检测EMT相关分子及p-GSK-3β、Snail;划痕实验、Transwell检测过表达miR-145后细胞的迁移能力。结果:(1)干扰PLCε表达能显著抑制细胞的迁移,同时,使T24细胞中E-cadherin表达上调,N-cadherin和Vimentin表达下调;干扰PLCε后,GSK-3β磷酸化(Ser9位点)水平下降,Snail表达降低。(2)转染miR-145 mimics可使T24细胞中miR-145表达增高,且明显抑制T24细胞中PLCε的表达。(3)在T24中过表达miR-145,细胞迁移能力显著下降,EMT标志分子的表达情况与沉默PLCε结果一致。同时,与阴性对照组相比,转染miR-145 mimics组p-GSK-3β和Snail表达显著减少。结论:PLCε通过GSK-3β/Snail信号通路促进膀胱癌细胞T24发生EMT及迁移,miR-145可以逆转PLCε诱导膀胱癌EMT的发生,从而阻止膀胱癌细胞的迁移。
Objective: To investigate the effects of miR-145 on the regulation of epithelial-to-mesenchymal transition (EMT) and the migration of bladder cancer cell line T24 and its possible molecular mechanisms. Methods: (1) Adenovirus-infected T24 cells, scratch assay and Transwell assay of cell migration; RTPCR and Western blot were used to detect the expression of PLCε and EMT related molecules; To investigate the molecular mechanism, GSK-3βphosphorylation (Ser9 site) and Snail expression. (2) Bioinformatics techniques were used to predict the miRNAs that may regulate PLCε, and the miR-145 was screened based on the microRNA expression profiles of bladder cancer reported in the literature. The miR-145mimics were transfected into T24 and the expression of miR- Western blot was used to detect the expression of PLCε. (3) Mimics were transfected with miR-145mimics, and EMT related molecules and p-GSK-3β, Snail were detected by Western blot. Scratch assay and Transwell assay were used to detect the migration ability of miR-145. Results: (1) Interference of PLCε expression significantly inhibited cell migration, and at the same time, up-regulated E-cadherin expression and down-regulated expression of N-cadherin and Vimentin in T24 cells. After interference with PLCε, phosphorylation of GSK-3β Decline, Snail expression decreased. (2) Transfection of miR-145 mimics could increase the expression of miR-145 in T24 cells and significantly inhibit the expression of PLCε in T24 cells. (3) Overexpression of miR-145 in T24 significantly decreased cell migration, and the expression of EMT marker was consistent with that of silencing PLCε. Meanwhile, compared with the negative control group, the expression of p-GSK-3β and Snail in miR-145 mimics group was significantly decreased. CONCLUSION: PLCε promotes EMT and migration of bladder cancer cell line T24 through GSK-3β / Snail signaling pathway. MiR-145 can reverse the PLCε-induced EMT of bladder transitional cell carcinoma and prevent the migration of bladder cancer cells.