High expression of miR-204-5p promotes malignant behaviors of bladder cancer cells by negatively regulating RAB22A

doi:10.12122/j.issn.1673-4254.2024.11.21

Abstract

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

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.

Figures/Tables 7

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

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.

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).

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).

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).

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

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).

References 25

1	Dyrskjøt L, Hansel DE, Efstathiou JA, et al. Bladder cancer[J]. Nat Rev Dis Primers, 2023, 9(1): 58.
2	Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6): 394-424.
3	Kamat AM, Hahn NM, Efstathiou JA, et al. Bladder cancer[J]. Lancet, 2016, 388(10061): 2796-810.
4	Andreassen BK, Grimsrud TK, Haug ES. Bladder cancer survival: women better off in the long Run[J]. Eur J Cancer, 2018, 95: 52-8.
5	Cumberbatch MGK, Jubber I, Black PC, et al. Epidemiology of bladder cancer: a systematic review and contemporary update of risk factors in 2018[J]. Eur Urol, 2018, 74(6): 784-95.
6	Choi W, Ochoa A, McConkey DJ, et al. Genetic alterations in the molecular subtypes of bladder cancer: illustration in the cancer genome atlas dataset[J]. Eur Urol, 2017, 72(3): 354-65.
7	Li HT, Duymich CE, Weisenberger DJ, et al. Genetic and epigenetic alterations in bladder cancer[J]. Int Neurourol J, 2016, 20(): S84-S94.
8	Fabian MR, Sonenberg N. The mechanics of miRNA-mediated gene silencing: a look under the hood of miRISC[J]. Nat Struct Mol Biol, 2012, 19(6): 586-93.
9	Bartel DP. MicroRNAs: target recognition and regulatory functions[J]. Cell, 2009, 136(2): 215-33.
10	邹震海, 程琪, 李中, 等. MiR-let-7g-3p靶向HMGB2调控膀胱癌细胞的增殖、迁移、侵袭和和凋亡[J]. 南方医科大学学报, 2022, 42(9): 1335-43.
11	郭晓波, 赵宇峰, 赵波, 等. BRD4抑制剂JQ1及miR-141对膀胱癌细胞增殖和凋亡的影响及其机制[J]. 中国老年学杂志, 2022, 42(22): 5621-5.
12	McLoughlin LC, O'Halloran S, Tjong M, et al. The prognostic value of urinary cytology after trimodal therapy (TMT) for muscle-invasive bladder cancer[J]. Urol Oncol, 2022, 40(7): 346. e9-16.
13	Hong BS, Ryu HS, Kim N, et al. Tumor suppressor miRNA-204-5p regulates growth, metastasis, and immune microenvironment remodeling in breast cancer[J]. Cancer Res, 2019, 79(7): 1520-34.
14	薛无涯. MiR-204-5p对结直肠癌细胞增殖、迁移及侵袭的作用及机制的初步探讨[D]. 泸州: 西南医科大学, 2018.
15	Yin Y, Zhang BB, Wang WL, et al. MiR-204-5p inhibits proliferation and invasion and enhances chemotherapeutic sensitivity of colorectal cancer cells by downregulating RAB22A[J]. Clin Cancer Res, 2014, 20(23): 6187-99.
16	He HH, Chen K, Wang F, et al. MiR-204-5p promotes the adipogenic differentiation of human adipose-derived mesenchymal stem cells by modulating DVL3 expression and suppressing Wnt/β-catenin signaling[J]. Int J Mol Med, 2015, 35(6): 1587-95.
17	Antoni S, Ferlay J, Soerjomataram I, et al. Bladder cancer incidence and mortality: a global overview and recent trends[J]. Eur Urol, 2017, 71(1): 96-108.
18	刘晨溪, 赵红娟, 田伟, 等. MiR-199b-3p对肾癌细胞增殖、凋亡、侵袭的影响及其机制探讨[J]. 山东医药, 2022, 62(4): 26-30, 35.
19	Yang S, Chen BN, Zhang BG, et al. MiR-204-5p promotes apoptosis and inhibits migration of gastric cancer cells by targeting HER-2[J]. Mol Med Rep, 2020, 22(4): 2645-54.
20	Cai KT, Liu AG, Wang ZF, et al. Expression and potential molecular mechanisms of miR-204-5p in breast cancer, based on bioinformatics and a meta-analysis of 2, 306cases[J]. Mol Med Rep, 2019, 19(2): 1168-84.
21	He M, Shen LH, Jiang CW, et al. Rab22a is a novel prognostic marker for cell progression in breast cancer[J]. Int J Mol Med, 2020, 45(4): 1037-46.
22	Zhou Y, Wu B, Li JH, et al. Rab22a enhances CD147 recycling and is required for lung cancer cell migration and invasion[J]. Exp Cell Res, 2017, 357(1): 9-16.
23	Magadán JG, Barbieri MA, Mesa R, et al. Rab22a regulates the sorting of transferrin to recycling endosomes[J]. Mol Cell Biol, 2006, 26(7): 2595-614.
24	Mosesson Y, Mills GB, Yarden Y. Derailed endocytosis: an emerging feature of cancer[J]. Nat Rev Cancer, 2008, 8(11): 835-50.
25	Abella JV, Park M. Breakdown of endocytosis in the oncogenic activation of receptor tyrosine kinases[J]. Am J Physiol Endocrinol Metab, 2009, 296(5): E973-84.

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