Departmemt of Kidney Transplantation, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2022 Apr 28;47(4):407-415. doi: 10.11817/j.issn.1672-7347.2022.210702.
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. Sex-determining 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 SOX4 and 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.
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.
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 mRNA and SOX4 protein levels in bladder cancer tissues was also found (=-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.
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.
膀胱癌是最常见的尿路上皮肿瘤之一，具有较高的发病率和死亡率。尽管已有研究表明 microRNA（miR）-133b 可调节膀胱癌的发生，但具体机制尚不清楚。性决定区 Y 框转录因子 4（SOX4）在肿瘤发生中具有重要作用，但尚不清楚 SOX4 和 miR-133b 是否与膀胱癌发病机制的调节有关。本研究旨在检测膀胱癌组织和细胞中 SOX4 和 miR-133b 的表达情况，探讨其对膀胱癌细胞增殖、集落形成和侵袭能力的影响，并研究 miR-133b 与 SOX4 调节膀胱癌细胞生物学特性的关系。
收集 2015 年 1 月至 6 月中南大学湘雅二医院 10 例膀胱癌手术患者的膀胱癌及癌旁组织标本。采用实时 PCR 检测 miR-133b 的水平，Western blot 检测 SOX4 蛋白在膀胱癌组织、配对癌旁组织和细胞系中的表达。分析膀胱癌组织中 miR-133b 表达与 SOX4 表达的相关性。利用在线数据库 TargetScan 预测 SOX4 与 miR-133b 的关系。通过 Lipofectamine 2000 将 miR-133b 模拟物、miR-133b 抑制剂和短发夹 RNA（shRNA）-SOX4 转染至 T24 细胞。通过双荧光素酶报告基因实验进一步验证 miR-133b 与 SOX4 的关系。采用细胞计数试剂盒（CCK-8）检测培养 0、12、48、72 和 96 h 的 T24 细胞的增殖情况。14 天后检测膀胱癌细胞的集落形成能力，Transwell 侵袭实验检测细胞侵袭能力。
与配对癌旁组织和正常膀胱上皮细胞相比，膀胱癌组织和膀胱癌细胞中 miR-133b 水平较低，SOX4 水平较高，且膀胱癌组织中 miR-133b 与 SOX4 蛋白水平呈负相关（=-0.84）。在线数据库 TargetScan 的结果显示，miR-133b 靶向 SOX4，过表达 miR-133b 可显著抑制 T24 细胞中 SOX4 的表达。过表达 miR-133b 和敲低 SOX4 均可显著抑制膀胱癌细胞的体外增殖、集落形成和侵袭能力。SOX4 下调可恢复 miR-133b 抑制剂对 T24 细胞增殖、集落形成和侵袭能力的影响。
SOX4 的上调促进了膀胱癌的进展，miR-133b 可通过抑制 SOX4 来调节膀胱癌细胞的增殖、集落形成和侵袭能力。
