靶向调控miR-21-5p上调TIMP3抑制膀胱癌SCSP-571细胞ECM和EMT的机制研究

目的探讨靶向调控miR-21-5p上调金属蛋白酶组织抑制因子(TIMP3)抑制膀胱癌细胞外基质降解(ECM)和上皮间质转化(EMT)的作用效果与机制。方法 96孔板培养膀胱癌SCSP-571细胞,分对照组、空载质粒组、miR-21-5p沉默组、TIMP3沉默组、miR-21-5p+TIMP3沉默组。对照组常规培养,空载质粒组加空载质粒共培养,miR-21-5p沉默组加入miR-21-5p inhibitor;TIMP3沉默组加入TIMP3-shRNA慢病毒感染质粒;miR-21-5p+TIMP3沉默组同时加入miR-21-5p inhibitor和TIMP3-shRNA慢病毒感染质粒。培养72 h,采用荧光定量PCR实验测定各组miR-21-5p和TIMP3的mRNA表达,WB实验测定各组ECM和EMT标志物的表达,Transwell小室实验测定各组细胞迁移能力和侵袭能力。结果与对照组相比,miR-21-5p沉默组E钙粘蛋白和TIMP3蛋白表达水平升高,同时N钙粘蛋白、纤维蛋白、MMP-2、MMP-7、MMP-9蛋白表达水平,细胞迁移与侵袭能力降低;TIMP3沉默组E钙粘蛋白和TIMP3蛋白表达水平降低,同时N钙粘蛋白、纤维蛋白、MMP-2、MMP-7、MMP-9蛋白表达水平,细胞迁移与侵袭能力升高,差异有统计学意义(均P<0.05)。miR-21-5p+TIMP3沉默组的各项指标介于miR-21-5p沉默组与TIMP3沉默组之间,差异有统计学意义(P<0.05)。结论靶向调控miR-21-5p能够起到抑制膀胱癌细胞侵袭与转移的效果,作用机制可能与提高了TIMP3表达,从而抑制了MMPs介导的ECM与EMT有关。

Mechanism for upregulation of TIMP3 by suppressing the overexpression of miR-21-5p to inhibit ECM and EMT in SCSP-571 cells

Objective To investigate the effect and mechanism of up-regulating metalloproteinase tissue inhibitors (TIMP3) by targeted regulation of miR-21-5p to suppress matrix degradation (ECM) and epithelial mesenchymal transformation (EMT) in bladder cancer cells. Methods Bladder cancer SCSP-571 cells were cultured on 96-well plates and divided into Control group, Plasmid group, miR-21-5p silencing group, TIMP3 silencing group and miR-21-5p+TIMP3 silencing group. The cells in Control group were cultured as normal, cells in Plasmid group were co-cultured with plasma, cells in miR-21-5p silencing group were co-cultured with miR-21-5p inhibitor, cells in TIMP3 silencing group were co-cultured with TIMP3 -shRNA plasma, and cells in miR-21-5p+TIMP3 silencing group were co-cultured with both miR-21-5p inhibitor and TIMP3 -shRNA plasma. When cells were cultured for 72 h, the mRNA expression of miR-21-5p and TIMP3 in each group was determined by fluorescence quantitative PCR and the expression of ECM and EMT markers in each group was determined by WB experiment. Cell migration and invasion were measured by Transwell assay. Results Compared with Control group and Plasmid group, the expression levels of E cadherin and TIMP3 in miR-21-5p silencing group were increased, while the expression levels of N cadherin, fibrin, MMP-2, MMP-7 and MMP-9 were decreased, and the cell migration and invasion count were decreased. The expression levels of E cadherin and TIMP3 in TIMP3 silencing group were decreased, while the expression levels of N cadherin, fibrin, MMP-2, MMP-7 and MMP-9 were decreased, and cell migration and invasion were increased. The difference had statistical significance (P＜0.05). The indexes of miR-21-5p+TIMP3 silencing group were between miR-21-5p silencing group and TIMP3 silencing group, and the differences were statistically significant (P<0.05). Conclusion The targeted regulation of miR-21-5p could inhibit the invasion and metastasis of SCSP-571 cells, and the mechanism may be related to the improvement of TIMP3 expression, thereby inhibiting the MMPs mediated ECM and EMT.