miR-216a-5p和WASL在子宫内膜癌组织中的表达及其调控子宫内膜癌细胞增殖、迁移和侵袭的分子机制

doi:10.13481/j.1671-587x.20200429

摘要/Abstract

摘要： 目的：探讨微小RNA-216a-5p（miR-216a-5p）靶向湿疹血小板减少伴免疫缺陷综合征样蛋白（WASL）对子宫内膜癌细胞增殖、迁移和侵袭的影响，阐明miR-216a-5p与WASL基因的靶向关系及其作用机制。方法：收集行手术切除的47例子宫内膜癌患者癌组织和癌旁组织；将miR-216a-5p模拟物（miR-216a-5p）、模拟物阴性对照（miR-con）、沉默对照（si-con）、沉默WASL的小干扰RNA（si-WASL）、抑制物阴性对照（anti-miR-con）、miR-216a-5p抑制物（anti-miR-216a-5p）、miR-216a-5p及空载质粒（pcDNA）和miR-216a-5p及WASL过表达质粒（pcDNA-WASL）分别转染子宫内膜癌HEC-1-B细胞作为miR-216a-5p组、miR-con组、si-con组、si-WASL组、anti-miR-con组、anti-miR-216a-5p组、miR-216a-5p+pcDNA组和miR-216a-5p+pcDNA-WASL组。采用逆转录实时荧光定量PCR（Real-time RT-qPCR）和Western blotting法分别检测子宫内膜癌组织、癌旁组织和各组HEC-1-B细胞中miR-216a-5p和WASL mRNA及蛋白表达水平。采用MTT法检测各组细胞增殖活性，Transwell法检测各组迁移和侵袭细胞数。采用双荧光素酶报告基因法检测各组细胞双荧光素酶活性，以此确定WASL是否为miR-216a-5p的靶基因。结果：与癌旁组织比较，子宫内膜癌组织中miR-216a-5p表达水平明显降低（P<0.05），WASL mRNA和蛋白表达水平明显升高（P<0.01），两者呈负相关关系（r=-0.317，P<0.01）。与miR-con组和si-con组比较，miR-216a-5p组和si-WASL组细胞中WASL蛋白表达水平（P<0.01）和细胞增殖活性均明显降低（P<0.05），迁移和侵袭细胞数明显降低（P<0.05）。双荧光素酶报告基因和Western blotting检测，WASL为miR-216a-5p的靶基因。与miR-216a-5p+pcDNA组比较，miR-216a-5p+pcDNA-WASL组细胞增殖活性、迁移细胞和侵袭细胞数均明显升高（P<0.05或P<0.01）。结论：miR-216a-5p在子宫内膜癌组织中呈低表达，其可能通过靶基因WASL抑制子宫内膜癌细胞的增殖、迁移和侵袭能力，可能是子宫内膜癌治疗的潜在靶点。

关键词: 微小RNA-216a-5p, 湿疹血小板减少伴免疫缺陷综合征样蛋白, 子宫内膜癌, 细胞增殖, 细胞迁移, 细胞侵袭

Abstract: Objective: To investigate the effects of miR-216a-5p targeting Wiskott-Aldrich syndrome like (WASL) protein on the proliferation, migration and invasion of endometrial carcinoma cells, and to clarify the targeting relationship between miR-216a-5p and WASL gene and its mechanism. Methods: A total of 47 cases of endometrial cancer tissue and normal tissue adjacent to the cancer surgically removed were collected. The miR-216a-5p group,minics negative control(miR-con group) si-con (si-con group), si-WASL (si-WASL group), anti-miR-con (anti-miR-con group), anti-miR-216a-5p (anti-miR-216a-5p group), miR-216a-5p+pcDNA (miR-216a-5p+pcDNA group) and miR-216a-5p+pcDNA-WASL (miR-216a-5p+pcDNA-WASL group) were transfected into the endometrial carcinoma HEC-1-B cells. The expression levels of miR-216a-5p and WASL mRNA and protein in the endometrial carcinoma tissue the adjacent tissue and the HEC-1-B cells in various groups were detected by RT-qPCR and Western blotting methods. MTT assay was used to detect the proliferation activities of the cells in various groups. Transwell assay was used to detect the number of migrated and invasive cells in various groups. The dual luciferase reporter gene assay was used to detect the double luciferase activities in various groups and to determine whether WASL was the target gene of miR-216a-5p. Results: Compared with the adjacent tissue, the expression level of miR-216a-5p in the endometrial carcinoma tissue was significantly decreased (P<0.05), the expression level of WASL mRNA and protein were significantly increased (P<0.05),and they had negative relationship (r=-0.317, P<0.01). Compared with miR-con group and si-con group, the expression level of WASL protein (P<0.01),the proliferation activity of cells (P<0.05) and the number of migrated and invasive cells(P<0.05) in miR-216a-5p group and si-WASL group were significantly decreased. The results of double luciferase reporter gene assay and Western blotting method showed that WASL was the target gene of miR-216a-5p. Compared with miR-216a-5p + pcDNA group, the proliferation activity of cells and the number of migrated and invasive cells in miR-216a-5p + pcDNA-WASL group were significantly increased (P<0.05). Conclusion: The expression level of miR-216a-5p in endometrial carcinoma tissue is decreased. MiR-216a-5p may inhibit the proliferation, migration and invasion of endometrial carcinoma cells through WASL gene, which may be a potential target for the treatment of endometrial carcinoma.

Key words: miR-216a-5p, Wiskott-Aldrich syndrome like protein, endometrial carcinoma, cell proliferation, cell migration, cell invasion

中图分类号:

R737.33

秦巧红, 张楠, 赵书君, 李红雨. miR-216a-5p和WASL在子宫内膜癌组织中的表达及其调控子宫内膜癌细胞增殖、迁移和侵袭的分子机制[J]. 吉林大学学报(医学版), 2020, 46(04): 844-850.

QIN Qiaohong, ZHANG Nan, ZHAO Shujun, LI Hongyu. Expressions of miR-216a-5p and WASL in endometrial carcinoma tissue and their molecular mechanisms of regulating proliferation, migration and invasion of endometrial carcinoma cells[J]. Journal of Jilin University(Medicine Edition), 2020, 46(04): 844-850.

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