环状RNA_0003028靶向微小RNA-498调控生殖器形成抑制基因-1/p53 信号通路对人肝癌细胞系Huh7的影响

doi:10.16098/j.issn.0529-1356.2022.05.009

摘要/Abstract

摘要：

目的探讨环状RNA（circ）_0003028对人肝癌细胞增殖、迁移和侵袭的影响及分子机制。方法人肝癌细胞系Huh7分为小干扰RNA（si）-NC组、si-circ_0003028组、微小RNA（miR）-NC组、miR-498模似物（mimics）组、si-circ_0003028+anti-miR-NC组、si-circ_0003028+anti-miR-498组；Real\|time PCR检测肝癌组织及各组细胞中circ_0003028和miR-498表达水平；MTT检测细胞增殖；Transwell检测细胞迁移和侵袭数；Western blotting检测蛋白表达；双荧光素酶报告实验检测circ_0003028和miR-498的靶向调控关系。结果肝癌组织中circ_0003028表达水平升高（0.98±0.02 vs 1.36±0.01），miR-498表达水平降低（0.98±0.02 vs 0.63±0.02）（P<0.05）。抑制circ_0003028表达或miR-498过表达后，Huh7细胞中Ki-67（0.85±0.02 vs 0.41±0.02或0.95±0.11 vs 0.37±0.02）、基质金属蛋白酶（MMP）-2（0.71±0.02 vs 0.43±0.03或0.83±0.02 vs 0.41±0.03）、MMP-9（0.74±0.02 vs 0.37±0.02或0.78±0.02 vs 0.39±0.02）蛋白表达水平降低，细胞活性（1.53±0.03 vs 1.05±0.02或1.68±0.02 vs 1.11±0.02）降低；迁移（111.40±2.12 vs 77.22±2.38或108.90±2.30 vs 78.44±1.46）和侵袭（87.89±2.18 vs 49.78±1.98或80.22±1.79 vs 38.22±1.52）细胞数减少，生殖器形成抑制基因-1(SMG-1)（0.76±0.02 vs 1.39±0.02或0.79±0.02 vs 1.39±0.02）、p53（0.77±0.02 vs 1.24±0.03或0.82±0.03 vs 1.45±0.03）、p53-ser15（0.78±0.03 vs 1.50±0.02或0.82±0.02 vs 1.49±0.04）蛋白表达水平升高（P<0.05）。Circ_0003028靶向调控miR-498，沉默miR-498逆转了抑制circ_0003028表达对Huh7细胞增殖、迁移和侵袭的影响。结论抑制circ_0003028表达通过靶向miR-498影响SMG-1/p53 信号通路而抑制人肝癌细胞增殖、迁移和侵袭。

关键词: 环状RNA_0003028, 微小RNA-498, 生殖器形成抑制基因-1/p53信号通路, 肝癌细胞系Huh7, 免疫印迹法, 人

Abstract:

Objective To investigate the effects and molecular mechanism of circular RNA(circ)_0003028 on proliferation, migration and invasion of human liver cancer cells. Methods Liver cancer cell line Huh7 were divided into small interfering RNA(si)-NC group, si-circ_0003028 group, microRNA(miR)-NC group, miR-498 mimics group, si-circ_0003028+anti-miR-NC group and si-circ_0003028+anti-miR-498 group. Real-time PCR was used to detect the expression levels of circ_0003028 and miR-498 in liver cancer tissues and cells of each groups. MTT was used to detect cell proliferation. Transwell was used to detect cell migration and invasion. Western blotting was used to detect protein expression. Dual luciferase reporter experiment was used to detect the target regulation relationship between circ_0003028 and miR-498. Results The expression level of circ_0003028(0.98±0.02 vs 1.36±0.01) increased and the expression level of miR-498(0.98±0.02 vs 0.63±0.02) decreased in liver cancer tissues (P<0.05). After inhibiting the expression of circ_0003028 or overexpression of miR-498, the expression levels of Ki-67(0.85±0.02 vs 0.41±0.02 or 0.95±0.11 vs 0.37±0.02), matrix metalloprotein(MMP)-2(0.71±0.02 vs 0.43±0.03 or 0.83±0.02 vs 0.41±0.03), and MMP-9(0.74±0.02 vs 0.37±0.02 or 0.78±0.02 vs 0.39±0.02) proteins in Huh7 cells decreased, and cell viability(1.53±0.03 vs 1.05±0.02 or 1.68±0.02 vs 1.11±0.02) decreased; the number of migration(111.40±2.12 vs 77.22±2.38 or 108.90±2.30 vs 78.44±1.46) and invasion(87.89±2.18 vs 49.78±1.98 or 80.22±1.79 vs 38.22±1.52) cells decreased, and the protein expression levels of suppressor with morphogenesis effect on genitalia-1(SMG-1) (0.76±0.02 vs 1.39±0.02 or 0.79±0.02 vs 1.39±0.02), p53(0.77±0.02 vs 1.24±0.03 or 0.82±0.03 vs 1.45±0.03), and p53-ser15(0.78±0.03 vs 1.50±0.02 or 0.82±0.02 vs1.4.04) increased (P<0.05). circ_0003028 targeted regulation of miR-498, and silencing miR-498 reversed the effects of inhibiting the expression of circ_0003028 on the proliferation, migration and invasion of Huh7 cells. Conclusion Inhibiting the expression of circ_0003028 can inhibit the proliferation, migration and invasion of liver cancer cells by targeting miR-498 to affect the SMG-1/p53 signaling pathway.

Key words: Circular RNA_0003028, MicroRAN-498, Suppressor with morphogenetic effect on genitalia-1-p53 signaling pathway, Liver cancer cell line Huh7, , Western blotting, , Human

中图分类号:

R735.7

谢宇端董国伟韩艳何生奇 . 环状RNA_0003028靶向微小RNA-498调控生殖器形成抑制基因-1/p53 信号通路对人肝癌细胞系Huh7的影响[J]. 解剖学报, 2022, 53(5): 600-606.

XIE Yu-duan DONG Guo-wei HAN Yan HE Sheng-qi . Circular RNA_0003028 targeting microRNA-498 regulating the effects of suppressor with morphogenesis effect on genitalia-1/p53 signaling pathway on liver cancer cell line Huh7[J]. Acta Anatomica Sinica, 2022, 53(5): 600-606.

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