标题：miR-222在膀胱癌组织中的表达及其对膀胱癌细胞增殖与迁移的影响

      作者：孙双权，陈立新，唐春华，李慧，杨谊    上海市松江区中心医院泌尿外科，上海 201600

      卷次： 2021年32卷21期

      【摘要】 目的 探究miR-222在膀胱癌与癌旁组织中的表达差异，并且初步探讨其对膀胱癌细胞增殖、迁移能力及凋亡的影响。方法 选取2018年1月至2019年6月在上海市松江区中心医院确诊并手术的45例膀胱尿路上皮癌患者，留取癌组织标本及配对癌旁组织，通过实时荧光定量PCR (RT-PCR)检测两者中miR-222的表达情况；脂质体瞬时转染miR-222 mimics、miR-222 inhibitor及miR-222 inhibitor NC至膀胱癌T24细胞，以空白T24细胞为对照组，通过MTT法、Transwell迁移实验、流式细胞术检测miR-222对T24细胞增殖、迁移能力及凋亡情况的影响；采用双荧光素酶报告基因检测验证miR-222的靶基因，Western blot分析miR-222假定靶基因CDKN1B的表达水平，并通过免疫组化检测膀胱癌及癌旁组织中的p27蛋白表达的差异。结果 miR-222在膀胱癌组织中的表达明显高于癌旁组织，差异有统计学意义(P<0.05)；体外细胞实验结果显示，与对照组相比，上调miR-222能提高膀胱癌细胞增殖能力和加快细胞迁移速率，并抑制癌细胞凋亡，差异均有统计学意义(P<0.05)；下调miR-222能抑制膀胱癌T24细胞的增殖、迁移能力并诱导细胞凋亡，差异均有统计学意义(P<0.05)；荧光素酶报告基因检测结果表明miR-222可以靶向调控CDKN1B基因的表达，Western bolt结果证实上调miR-222后膀胱癌T24细胞中CDKN1B(p27)表达水平明显下降，免疫组化染色结果证实膀胱癌组织中CDKN1B低表达，进一步印证了miR-222对CDKN1B的负调控作用。结论 miR-222在膀胱癌组织中表达明显上调，其在体外可促进膀胱癌细胞的增殖及迁移，抑制癌细胞凋亡；其机制可能与靶向调控CDKN1B的表达有关，有望成为膀胱癌检测的分子标记物和治疗靶点。
      【关键词】 膀胱癌；miR-222；细胞增殖；细胞迁移；细胞周期蛋白依赖性激酶抑制剂1B
      【中图分类号】 R737.14 【文献标识码】 A 【文章编号】 1003—6350（2021）21—2730—06

Expression of miR-222 in bladder cancer and its effect on proliferation and migration of bladder cancer cells.SUN Shuang-quan, CHEN Li-xin, TANG Chun-hua, LI Hui, YANG Yi.
Department of Urology, Songjiang District CentralHospital, Shanghai 201600, CHINA
【Abstract】 Objective To investigate the expression difference of miR-222 in bladder cancer and adjacent tis-sues, and to explore its effect on proliferation, migration and apoptosis of bladder cancer cells. Methods A total of 45patients with bladder urothelial carcinoma who diagnosed and operated in Songjiang District Central Hospital of Shanghaifrom January 2018 to June 2019 were selected. Cancer tissue specimens and paired adjacent tissues were taken, and the ex-pression of miR-222 was detected by real-time fluorescent quantitative PCR (RT-PCR). MiR-222 mimics, miR-222 inhib-itor and miR-222 inhibitor NC were transiently transfected into bladder cancer T24 cells by liposome, with blank T24cells as control group. The effects of miR-222 on proliferation, migration and apoptosis of T24 cells were detected byMTT, transwell migration test and flow cytometry. Dual luciferase reporter assay was used to verify the target gene ofmiR-222. The expression of miR-222 putative target gene CDKN1B was measured by Western blot, and the expression ofp27 protein in bladder cancer and adjacent tissues was detected by immunohistochemistry. Results The expression ofmiR-222 in bladder cancer was significantly higher than that in adjacent tissues, and the difference was statistically sig-nificant (P<0.05). The results of cell experiments in vitro showed that the upregulation of miR-222 could enhance prolif-eration of T24 cells, speed up cell migration, and inhibit the apoptosis of cancer cells compared with the control group(P<0.05). The down-regulation of miR-222 inhibited the proliferation, migration and induced apoptosis of T24 cells, andthe difference was statistically significant (P<0.05). The results of dual luciferase reporter assay showed that miR-222could regulate the expression of CDKN1B. Western blot results confirmed that the expressionof CDKN1B (p27) in blad-der cancer T24 cells was significantly decreased after up-regulation of miR-222. The results of immunohistochemicalstaining confirmed that the expression of CDKN1B was low in bladder cancer tissues, further confirming the negativeregulation effect of miR-222 on CDKN1B. Conclusion The expression of miR-222 was significantly up-regulated inbladder cancer tissues, which could promote the proliferation and migration of bladder cancer cells and inhibit the apop-tosis of cancer cells in vitro. The mechanism may be related to the targeted regulation of CDKN1B expression, which isexpected to become a molecular marker and therapeutic target for the detection of bladder cancer.
      【Key words】 Bladder cancer; miR-222; Cell proliferation; Cell migration; Cyclin-Dependent Kinase Inhibitor 1B