miR-205靶向调控PLCε抑制膀胱癌T24细胞EMT和迁移的作用机制研究

Study on the action mechanism of miR-205 targeted regulation of PLCεinhibiting EMT and migration of bladder cancer T24 cells in vitro

目的探讨miR-205调控磷脂酰肌醇特异性磷脂酶Gepsilon(PLCε)对膀胱癌T24细胞上皮间质转化(EMT)和迁移的影响。方法qRT-PCR检测miR-205在膀胱癌患者癌组织及癌旁组织的表达,将miR-205 inhibitors、miR-205 mimics和阴性对照转染至人膀胱癌细胞系T24,qRT-PCR检测miR-205的表达量,划线法检测细胞的迁移能力,Transwell法检验细胞的侵袭能力,Western blot检测E-Cadhein、Vimentin、Slug的表达量,荧光素酶检测miR-205与PLCε的关系。结果qRT-PCR结果显示细胞癌组织中miR-205的表达量显著低于癌旁组织,差异有统计学意义(P<0.05);miR-205 inhibitors组中穿透细胞数明显高于对照组,差异有统计学意义(P<0.05),miR-205 mimics组中穿透细胞数明显低于对照组,差异有统计学意义(P<0.05);miR-205 inhibitors组T24迁移率明显高于对照组,差异有统计学意义(P<0.05),miR-205 mimics组T24迁移率明显低于对照组,差异有统计学意义(P<0.05);Western blot检测结果显示miR-205 inhibitors组中上皮标志物E-Cadhein的表达明显低于对照组(P<0.05),间质标志物Vimentin、Slug的表达明显高于对照组(P<0.05),miR-205 mimics组上皮标志物E-Cadhein的表达明显高于对照组(P<0.05),间质标志物Vimentin、Slug的表达明显低于对照组,差异有统计学意义(P<0.05);双荧光素酶报告基因结果显示,转染miR-205后,野生型PLCε的荧光素酶活性被抑制(P<0.05),突变型PLCε的荧光素酶活性无明显变化(P>0.05),说明PLCε和miR-205具有靶向调控关系。Western blot检测上调miR-205细胞系和空白细胞对照中PLCε结果表明,上调miR-205后,PLCε表达水平显著下降(P<0.05),证实miR-205可以特异性结合PLCε并发挥负向调控作用。结论miR-205在膀胱癌中作为抑癌基因,能够靶向调控PLCε抑制膀胱癌T24细胞EMT和迁移的作用。

Objective To investigate the effects of miR-205 in regulating PLCεon EMT and migration of bladder cancer T24 cells in vitro.Methods The qRT-PCR was used to detect the expression levels of miR-205 in cancer tissues and adjacent tissues of patients with bladder cancer.The miR-205 inhibitors,miR-205 mimics and negative controls were transfected into human bladder cancer cell line T24,then qRT-PCR was used to detect the expression levels of miR-205,and streaking method was used to detect the migration ability of cells.Moreover the Transwell method was used to detect the invasion ability of cells,and Western Blot was used to detect the expression levels of E-Cadhein,Vimentin and Slug,and luciferase was used to analyze the correlation between miR-205 and PLCε.Results The qRT-PCR results showed that the expression levels of miR-205 in cell carcinoma tissues were significantly lower than those in cancer adjacent tissues(P<0.05).The number of penetrating cells in miR-205 inhibitors group was significantly higher than that in control group(P<0.05).The T24 mobility in miR-205 inhibitor group was significantly higher than that in control group(P<0.05),and the T24 mobility in miR-205 mimics group was significantly lower than that in control group(P<0.05).Western blot results showed that the expression levels of epithelial marker E-Cadhein in miR-205 inhibitor group were significantly lower than those in control group(P<0.05),however,the expression levels of interstitial markers-Vimentin and Slug were significantly higher than those in control group(P<0.05).The expression levels of epithelial marker E-Cadhein in miR-205 mimics group were significantly higher than those in control group(P<0.05),however,the expression levels of interstitial markers-Vimentin and Slug were significantly lower than those in control group(P<0.05).Moreover the results of double luciferase reporter gene detection showed that after transfection of miR-205,the luciferase activity of wild-type PLCεwas obviously inhibited(P<0.05),but,the luciferase activity of mutant PLCεhad no significant changes(P>0.05),which suggested that PLCεand miR-205 had targeted regulation correlation.Western blot results showed that PLCεexpression levels were decreased significantly(P<0.05)after up-regulating miR-205,which confirmed that miR-205 could specifically bind PLCε,which played a negative regulatory role.Conclusion The miR-205 as umor suppressor gene can inhibit EMT and migration of bladder cancer T24 cellsin vitro through the target regulating of PLCε.