miR-152-3p靶向DDX6/CBLB在肥胖相关子宫内膜癌发生过程中的作用及机制研究

Role and mechanism of miR-152-3p targeting DDX6/CBLB in the developmentof obesity-related endometrial cancer

摘要

目的 明确miR-152-3p是否通过靶向DDX6和CBLB在肥胖相关子宫内膜癌发生发展过程中发挥作用,并探究其可能的分子机制。方法 体外培养子宫内膜癌细胞系Ishikawa;qRT-PCR、ELISA和Western Blot检测患者血浆、子宫内膜组织和ECa细胞中miR-152-3p及其下游靶基因的表达水平;生物信息学方法及双荧光素酶报告基因实验,筛选和验证miR-152-3p下游与肿瘤发生密切相关的靶基因;CCK-8、EdU增殖检测、细胞划痕和Transwell等实验,明确miR-152-3p及其下游靶基因对ECa细胞生物学行为的影响。稳定过表达miR-152-3p的ECa细胞进行裸鼠成瘤实验,明确miR-152-3p及其下游靶基因对ECa细胞体内成瘤能力的影响;SPSS 20.0软件进行统计学分析,P＜0.05定义为差异具有统计学意义。结果 与正常体重ECa患者相比,miR-152-3p在肥胖伴ECa患者血浆和肿瘤组织中表达水平显著降低;双荧光素报告基因实验证实DDX6和CBLB是miR-152-3p的下游靶基因,并且在肥胖伴ECa患者血浆和肿瘤组织中表达水平显著增加;过表达miR-152-3p后,DDX6和CBLB的表达水平显著降低,并且显著抑制了ECa细胞的增殖和迁移能力;过表达miR-152-3p的ECa细胞在裸鼠体内的成瘤能力显著降低,并且肿瘤组织中DDX6和CBLB表达水平显著降低。结论 miR-152-3p可通过靶向下调DDX6/CBLB的表达,抑制子宫内膜癌细胞的生物学行为,并在肥胖相关肿瘤微环境中降低其成瘤能力。

Abstract

Objective To determine whether miR-152-3p plays a role via in the development of obesity-related endometrial cancer targeting DDX6 and CBLB, and to investigate its potential molecular mechanisms. Methods The endometrial cancer cell line Ishikawa was cultured in vitro. qRT-PCR, ELISA, and Western Blot were used to detect the expression levels of miR-152-3p and its downstream target genes in patient plasma, endometrial tissues, and ECa cells. Bioinformatics approaches and dual-luciferase reporter gene assays were employed to screen and validate the tumor-related downstream target genes of miR-152-3p. CCK-8, EdU proliferation detection, cell scratch, and Transwell experiments were conducted to determine the effects of miR-152-3p and its downstream target genes on the biological behaviors of ECa cells. Tumorigenesis experiments in nude mice were performed using ECa cells with stable overexpression of miR-152-3p to clarify the effects of miR-152-3p and its downstream target genes on the tumorigenic capacity of ECa cells in vivo. Statistical analysis was performed using SPSS 20.0 software, and P<0.05 indicates statistically significant differente. Results Compared with Eca patients with normal weight, miR-152-3p expression levels in plasma and tumor tissues of obese ECa patients were signification decreased. Dual-luciferase reporter gene assays validated that DDX6 and CBLB were downstream target genes of miR-152-3p, both of which were significantly upregulated in plasma and tumor tissues of obese ECa patients. Overexpression of miR-152-3p significantly suppressed DDX6 and CBLB expression, and concomitantly inhibited ECa cell proliferation and migration. In vivo tumorigenesis assays demonstrated that stable overexpression of miR-152-3p had significantly decreased tunorigenicity in nude mice, with corresponding downregulation of DDX6 and CBLB expression in tumor tissues. Conclusions These findings demonstrate that miR-152-3p suppresses malignant behaviors of endometrial cancer cells via targeted downregulation of DDX6 and CBLB, and reduces tumorigenicity in obesity-associated tumor microenvironment.

关键词

子宫内膜癌 / miR-152-3p / DDX6 / CBLB

Key words

endometrial cancer / miR-152-3p / DDX6 / CBLB

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基金资助

国家自然科学基金项目(82160496,82160156),新疆生产建设兵团重点领域科技项目(2022ZD001,2022AB022)