miR-204-5p通过靶向负性调控RAB22A促进膀胱癌细胞的恶性生物学行为

doi:10.12122/j.issn.1673-4254.2024.11.21

摘要/Abstract

摘要：

目的探讨MiR-204-5p靶向RAB22A对膀胱癌细胞生物学行为的影响及其分子机制。方法 TCGA数据库进行了miR-204-5p的生存分析和临床病理性状的相关性分析；检测膀胱癌组织和癌旁组织以及正常尿路上皮细胞和膀胱癌细胞中miR-204-5p的表达水平；过表达/敲低miR-204-5p后检测细胞增殖、迁移和侵袭、和凋亡等变化；转录组测序，数据库分析和多种实验证实miR-204-5p靶向抑制RAB22A基因调控膀胱癌细胞的生物学行为。结果 TCGA数据库中MiR-204-5p高表达患者的中位生存期较低，表现出较差的预后（P<0.05）；miR-204-5p在膀胱癌组织和细胞表达明显上调（P<0.05）；过表达miR-204-5p可促进细胞增殖（P<0.05）、迁移和侵袭（P<0.05），减少细胞凋亡（P<0.05），敲低后则相反；转录组测序，数据库分析和双荧光素酶实验提示RAB22A是miR-204-5p下游关键因子，qRT-PCR，Western blotting证实过表达miR-204-5p可抑制RAB22A的表达（P<0.05）；过表达RAB22A可部分逆转miR-204-5p对膀胱癌细胞生长的促进作用（P<0.05）。结论 miR-204-5p通过靶向负性调控RAB22A促进膀胱癌细胞的恶性生物学行为。

关键词: miR-204-5p, RAB22A, 膀胱癌

Abstract:

Objective To explore the regulatory effect of miR-204-5p on biological behaviors of bladder cancer cells and its molecular mechanism. Methods Survival analysis and correlation analysis were performed using TCGA database to explore the association of miR-204-5p expression with survival outcomes and clinicopathological parameters of bladder cancer patients. The expression level of miR-204-5p was detected in bladder cancer and adjacent tissues and in normal uroepithelial cells and bladder cancer cells. In cultured bladder cancer cells, the effects of miR-204-5p overexpression and knockdown on cell proliferation, migration, invasion, and apoptosis were analyzed. Transcriptome sequencing, bioinformatics analysis and dual-luciferase assay were carried out to confirm targeted inhibition of RAB22A by miR-204-5p to promote malignant biological behaviors of bladder cancer cells. Results Patients with high miR-204-5p expressions had lowered median survival time and poor prognosis (P<0.05). The expression of miR-204-5p was significantly up-regulated in bladder cancer tissues and cells (P<0.05). In bladder cancer cells, miR-204-5p overexpression significantly promoted cell proliferation, migration and invasion and reduced cell apoptosis. Transcriptome sequencing, bioinformatics analysis and dual-luciferase assay all suggested that RAB22A was a key downstream factor of miR-204-5p. Overexpression of miR-204-5p significantly inhibited RAB22A expression in bladder cancer cells, and overexpression of RAB22A partially reversed miR-204-5p overexpression-induced enhancement of bladder cancer cell proliferation. Conclusion High expression of miR-204-5p promotes proliferation, migration and invasion and reduces apoptosis of bladder cancer cells by negatively regulating RAB22A expression.

Key words: miR-204-5p, RAB22A, bladder cancer

李立强, 郭园园, 王成勇, 常睿, 孙巍, 高五岳, 王超, 刘贝贝. miR-204-5p通过靶向负性调控RAB22A促进膀胱癌细胞的恶性生物学行为[J]. 南方医科大学学报, 2024, 44(11): 2235-2242.

Liqiang LI, Yuanyuan GUO, Chengyong WANG, Rui CHANG, Wei SUN, Wuyue GAO, Chao WANG, Beibei LIU. High expression of miR-204-5p promotes malignant behaviors of bladder cancer cells by negatively regulating RAB22A[J]. Journal of Southern Medical University, 2024, 44(11): 2235-2242.

图/表 7

表1 引物序列

Tab.1 Primer sequences for RT-qPCR

Gene	Primer sequence (5'-3')
miR-204-5p-F	GCTACAGTCTTTCTTCATGTG
U6-F	CGCTTCGGCAGCACATATACTAA
Micro-R	AGTGCAGGGTCCGAGGTATT
RAB22A-F	GTCCCTTAGCACCAATGTACTATC
RAB22A-R	AATGGCAACTACAATATTAGGTGG
GAPDH-F	GGAGCGAGATCCCTCCAAAAT
GAPDH-R	GGCTGTTGTCATACTTCTCATGG

图1 miR-204-5p的高表达与膀胱癌患者预后不良有关

Fig.1 High expression of miR-204-5p is associated with poor prognosis of patients with bladder cancer. A: Kaplan-Meier plotter curves of patients with high and low expression of miR-204-5p. B-F: Correlation of miR-204-5p expression with clinical grade, stage, T-stage, N-stage and M-stage of the patients. G: Forest plot showing the results of the univariate Cox regression analysis. H: Forest plot showing the results of the multivariate Cox regression analysis.

图2 miR-204-5p在膀胱癌中的表达水平

Fig.2 Verification of miR-204-5p expression level in bladder cancer tissue and cells. A: miR-204-5p expressions in normal tissues and bladder cancer tissues. B: miR-204-5p expression in bladder cancer cell lines T24, J82, 5637 and EJ and normal SV-HUC-1 cells (n=3, **P<0.01).

图3 miR-204-5p对膀胱癌细胞增殖、迁移、侵袭及凋亡的影响

Fig.3 Effects of miR-204-5p knockdown and over-expression on proliferation, migration, invasion and apoptosis of bladder cancer cells. A: CCK8 assay for assessing proliferation ability of T24 cells with miR-204-5p knockdown or overexpression. B: Flow cytometry for analyzing apoptosis of T24 cells with miR-204-5p knockdown or overexpression. C: Transwell assay for assessing migration and invasion abilities of T24 cells with miR-204-5p knockdown or overexpression (Original magnification: ×100). *P<0.05, **P<0.01 (n=3).

图4 miR-204-5p与RAB22A的靶向关系

Fig.4 Targeting relationship between miR-204-5p and RAB22A. A: Result of transcriptome sequencing. B: Wayne diagram of the 3 major databases. C: Binding site between miR-204-5p and RAB22A. D: Overexpression of miR-204-5p reduces mRNA expression level of RAB22A as detected by qRT-PCR. E, F: Overexpression of miR-204-5p reduces protein expression level of RAB22A as detected by Western blotting (n=3, **P<0.01).

图5 双荧光素酶报告实验的结果

Fig.5 Result of the dual-luciferase reporter assay (n=3, **P<0.01).

图6 miR-204-5p靶向RAB22A调控膀胱癌细胞增殖、迁移、侵袭及凋亡

Fig.6 Rescue experiment confirms that miR-204-5p targets RAB22A to regulate proliferation, migration, invasion and apoptosis of bladder cancer cells. A: Overexpression of RAB22A partially reverses the effect of miR-204-5p overexpression on migration and invasion of T24 cells. B: Overexpression of RAB22A partially reverses the effect of miR-204-5p overexpression on apoptosis of T24 cells. C: Overexpression of RAB22A partially reverses the effect of miR-204-5p overexpression on the proliferation of T24 cells (×100). *P<0.05, **P<0.01 (n=3).

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