[microRNA let-7g-3p regulates proliferation, migration, invasion and apoptosis of bladder cancer cells by targeting HMGB2]

Abstract

Objective: To explore the molecular mechanism by which microRNA let-7g-3p regulates biological behaviors of bladder cancer cells.

Methods: The expression levels of let-7g-3p in bladder cancer and adjacent tissues, normal bladder epithelial cells (HUC cells) and bladder cancer cells (T24, 5637 and EJ cells) were detected using qRT- PCR. T24 cells were transfected with let-7g-3p mimic or inhibitor, and the changes in cell proliferation, migration, invasion, and apoptosis were examined. Transcriptome sequencing was carried out in cells overexpressing let-7g-3p, and the results of bioinformatics analysis, double luciferase reporter gene assay, qRT-PCR and Western blotting confirmed that HMGB2 gene was the target gene of let-7g-3p. The expression of HMGB2 was examined in HUC, T24, 5637 and EJ cells, and in cells with HMGB2 knockdown, the effect of let-7g-3p knockdown on the biological behaviors were observed.

Results: qRT-qPCR confirmed that let-7g-3p expression was significantly lower in bladder cancer tissues and cells (P < 0.01). Overexpression of let-7g-3p inhibited cell proliferation, migration and invasion, and promoted cell apoptosis, while let-7g-3p knock-down produced the opposite effects. Bioinformatics and transcriptome sequencing results showed that HMGB2 was the key molecule that mediate the effect of let-7g-3p on bladder cancer cells. Luciferase reporter gene assay, qRT-PCR and Western blotting all confirmed that HMGB2 was negatively regulated by let-7g-3p (P < 0.01). Knocking down HMGB2 could partially reverse the effect of let-7g-3p knockdown on the biological behaviors of the bladder cancer cells.

Conclusion: The microRNA let-7g-3p can inhibit the biological behavior of bladder cancer cells by negatively regulating HMGB2 gene.

Keywords: HMGB2; bladder cancer; let-7g-3p.

图 1

let-7g-3p在膀胱癌组织中的表达水平 Expression level of let-7g-3p in bladder cancer tissues (n=5, ^**P < 0.01).

图 2

let-7g-3p在膀胱癌细胞中的表达水平 Expression level of let-7g-3p in bladder cancer cells lines (n=3). ^**P < 0.01 vs SV-HUC-1.

图 3

let-7g-3p和HMGB2转染效率的验证 Expression of let-7g-3p and HMGB2 in bladder cancer cells. A: let-7g-3p was successfully knocked down or over-expressed in bladder cancer cells. B: HMGB2 was successfully knocked down in bladder cancer cells. ^**P < 0.01 (n=3).

图 4

let-7g-3p对膀胱癌细胞增殖、迁移、侵袭及凋亡的影响 Effects of let-7g-3p on proliferation, migration, invasion and apoptosis of bladder cancer cells. A, B: CCK8 assay for assessing the proliferation ability of T24 cells with let-7g-3p knockdown or overexpression. C, D: Transwell assay for assessing migration and invasion ability of T24 cells with let-7g-3p knockdown or overexpression (Original magnification: ×100). E: Flow cytometry for assessing apoptosis of T24 cells with let-7g-3p knockdown or overexpression. ^**P < 0.01 vs NC (n=3).

图 5

let-7g-3p与HMGB2的靶向关系 Targeting relationship between let-7g-3p and HMGB2. A: Result of transcriptome sequencing. B: Wayne diagram of the major databases. C: Expression level of HMGB2 in each group of samples in the results of transcriptome sequencing. D: Binding site between let-7g-3p and HMGB2.

图 6

双荧光素酶报告实验的结果 Results of the double luciferase reporter gene assay (n=3, ^**P < 0.01).

图 7

过表达let-7g-3p可以显著降低HMGB2的表达水平 Overexpression of let-7g-3p significantly reduces the expression level of HMGB2. A: Overexpression of let-7g-3p reduces the mRNA expression level of HMGB2 detected by qRT-PCR. B, C: Overexpression of let-7g-3p reduces protein expression level of HMGB2 detected by Western blotting. D: Overexpression of HMGB2 does not affect the expression level of let-7g-3p detected by qRT-PCR. ^**P < 0.01, ^***P < 0.001 (n=3).

图 8

HMGB2在膀胱癌细胞中的表达水平 Expression level of HMGB2 in bladder cancer cells. ^**P < 0.01 vs SV-HUC-1 (n=3).

图 9

let-7g-3p靶向HMGB2调控膀胱癌细胞增殖、迁移、侵袭及凋亡 let-7g-3p targets HMGB2 to regulate the proliferation, migration, invasion and apoptosis of bladder cancer cells. A: Knockdown of HMGB2 partially reverses the effect of let-7g-3p knockdown on migration and invasion of T24 cells. B, C: Knock-down of HMGB2 partially reverses the effect of let-7g-3p knockdown on proliferation of T24 cells (×100). D: Knock-down of HMGB2 partially reverses the effect of let-7g-3p knockdown on apoptosis of T24 cells. ^**P < 0.01 (n=3)

图 10

GO和KEGG通路富集分析的结果 Enrichment analysis of GO and KEGG pathways. A: Histogram of GO enrichment analysis. B: Bubble diagram of KEGG pathways enrichment analysis.