MiR-133b通过抑制SOX4调节膀胱癌细胞的增殖、克隆形成和侵袭能力

MiR-133b regulates the proliferation,colony formation,and invasion of bladder cancer cells via inhibiting SOX4

目的:膀胱癌是泌尿系统最常见的恶性肿瘤之一,但其发病机制仍不十分清楚。虽然研究表明miR-133b可调控膀胱癌的发生,但其机制尚未明确。性别决定基因-区域转录因子4(sex determining region Y-box transcriptim factor 4,SOX4)在肿瘤的发生中起至关重要的作用,目前尚不清楚在膀胱癌中miR-133b与SOX4是否存在相互作用。本研究旨在探究膀胱癌组织和细胞中miR-133b和SOX4的表达情况及其对膀胱癌细胞增殖、克隆形成和侵袭能力的调控作用,并探讨在调节膀胱癌细胞生物特性过程中miR-133b与SOX4的相互关系。方法:获取2015年1至6月在中南大学湘雅二医院行手术切除的10例患者的膀胱癌及邻近癌旁组织标本,分别采用real-time PCR和蛋白质印迹法检测膀胱癌组织、癌旁组织、膀胱癌细胞系及正常膀胱上皮细胞中miR-133b的mRNA表达水平和SOX4的蛋白质表达水平。利用数据库TargetScan预测SOX4与miR-133b的相关性。利用脂质体Lipofectamine 2000为载体,将miR-133b模拟物、抑制物或SOX4的短发夹RNA(short hairpin RNA,shRNA)转染至T24细胞。应用双荧光素酶报告基因检测系统检测高表达miR-133b对T24细胞表达SOX4的影响。体外培养T24细胞14 d,计数培养克隆数并计算克隆形成率。使用CCK-8试剂盒检测T24细胞在不同时间点(0、12、24、48、72、96 h)的增殖情况。应用Transwell检测T24细胞的侵袭能力。结果:与癌旁组织和正常膀胱上皮细胞比较,膀胱癌组织和膀胱癌细胞系均低表达miR-133b、高表达SOX4,并且miR-133b mRNA与SOX4蛋白质在膀胱癌组织中的表达水平呈负相关(r=-0.84)。TargetScan数据库预测SOX4存在miR-133b结合位点,miR-133b模拟物转染可抑制膀胱癌T24细胞内SOX4的表达。MiR-133b模拟物或shRNA-SOX4转染均能抑制膀胱癌T24细胞的体外增殖、克隆形成和侵袭能力。相对于转染miR-133b抑制物,miR-133b抑制物和shRNA-SOX4共转染能够显著抑制膀胱癌T24细胞的增殖、克隆形成和侵袭能力。结论:MiR-133b和SOX4均能参与调控膀胱癌细胞的增殖、克隆形成和侵袭能力,并且miR-133b可以通过抑制SOX4的表达从而抑制膀胱癌细胞的增殖、克隆形成和侵袭能力。

Objective:Bladder cancer is one of the most common urothelial tumors with high incidence and mortality rates.Although it has been reported that microRNA(miR)-133b can regulate tumorigenesis of bladder cancer,the mechanism remains unclear.Sexdetermining region Y-box transcription factor 4(SOX4)exhibits an important role in tumorigenesis,but it is unclear whether SOX4 and miR-133b are associated with regulation of pathogenesis of bladder cancer.This study aims to determine the expressions of SOX4and miR-133b in bladder cancer tissues and cells,investigate their effects on the proliferation,colony formation,and invasion of bladder cancer cells,and to explore the association between miR-133b and SOX4 in regulating biological featurss of bladder cancer cells.Methods:The bladder cancer and adjacent tissue samples of 10 patients who underwent surgical resection in the Second Xiangya Hospital of Central South Universty from Januray to June 2015 were obtained.The levels of miR-133b were tested by real-time PCR,and the protein levels of SOX4 were evaluated using Western blotting in bladder cancer tissues,matched adjacent tissues,and cell lines.The correlation between miR-133b expression and SOX4 expression in bladder cancer tissues was analyzed.Using the online database TargetScan,the relationship between SOX4 and miR-133b was predicted.MiR-133b mimics,miR-133b inhibitor,and short hairpin RNA(shRNA)-SOX4 were transfected into T24 cells by Lipofectamine 2000.The relationship between miR-133b and SOX4 was also verified by a dual-luciferase reporter assay.The proliferation of T24 cells cultured for 0,12,48,72,and 96 h was evaluated by cell counting kit-8(CCK-8)assay.The colony formation capacity of bladder cancer cells was tested after 14-day culture,and cell invasion capacity was evaluated with Transwell invasion assay.Results:Bladder cancer tissue and bladder cancer cells had low level of miR-133b but high level of SOX4,compared with matched adjacent tissues and normal bladder epithelial cells.A negative correlation between miR-133b m RNA and SOX4 protein levels in bladder cancer tissues was also found(r=-0.84).The results of online database TargetScan showed that miR-133b targets at SOX4,and overexpression of miR-133b significantly attenuated the expression of SOX4 in T24 cells.Both overexpression of miR-133b and knockdown of SOX4 significantly inhibited the proliferation,colony formation,and invasion capacity of bladder cancer cells in vitro.SOX4 down-regulation restored the effects of miR-133b inhibitor on the proliferation,colony formation,and invasion capacity of T24 cells.Conclusion:The up-regulation of SOX4 contributes to the progression of bladder cancer,and miR-133b can regulate the proliferation,colony formation,and invasion of bladder cancer cells via inhibiting SOX4.