miR-143 promotes cell proliferation, invasion and migration via directly binding to BRD2 in lens epithelial cells

Abstract

Objective: Cataract causes the greatest number of blindnesses worldwide. This study aims to investigate the role of miR-143 in lens epithelial cells.

Methods: Clustering analysis was conducted to systematically compare miRNA expression levels across cataract and myopia. The levels of miR-143 and Bromodomain containing 2 (BRD2) were determined using real-time quantitative PCR (RT-qPCR) assay in lens epithelial cells. Transwell and wound healing assays were conducted to detect cell invasive and migratory abilities. The regulation relationship between MiR-143 and BRD2 was assessed using dual-luciferase reporter gene assays. BRD2 was knocked down using siRNA-BRD2, and siRNA-BRD2, and miR-143 inhibitors were transfected into cells with lipofectamine 2000.

Results: Through retrieving five databases, 2690 miRNAs were selected. Volcano plot results demonstrated that 200 miRNAs were differentially expressed between cataract and myopia, in which 152 miRNAs were upregulated and 48 miRNAs downregulated in myopia compared with cataract. MiR-143 was upregulated in cataract compared with myopia (P<0.05). MiR-143 inhibitor suppressed the proliferation, invasion and migration of lens epithelial cells (all P<0.05). Luciferase reporter assays confirmed that BRD2 was a miR-143 target gene in SRA01/04 cells. Knockdown of BRD2 promoted SRA01/04 cell proliferation, invasion and migration (all P<0.05). In addition, silencing of BRD2 partially reversed the functions of miR-143 inhibitor on proliferation, invasion and migration (all P<0.05).

Conclusion: MiR-143 suppresses lens epithelial cell proliferation, invasion and migration by regulating BRD2, which may support a novel therapeutic strategy for cataract patients.

Keywords: BRD2; Cataract; clustering analysis; invasion; miR-143.

Figure 1

Identification of dysregulated miRNAs in cataract. A: Veen results showed that 2690 miRNAs were filtered out from five databases; B: Volcano results revealed that 200 miRNAs were differentially expressed between cataract and myopia, in which 152 miRNAs were upregulated and 48 miRNAs were downregulated in myopia compared with cataract.

Figure 2

Bioinformatics analysis of cataract and myopia. A: The differentially expressed miRNAs were analzyed in the clusters of cataract patients and the myopia patients; B: Heatmap was conducted to select the top 40 differentially expressed genes (DEGs) between cataract and myopia.

Figure 3

The top 5 upregulated miRNAs in cataract compared to myopia. A-E: miR-1, miR-10b, miR-23b, miR-143 and miR-145.

Figure 4

The top 5 downregulated miRNAs in cataract compared to myopia. A-E: miR-9, miR-124, miR-129, miR-219a and miR-654.

Figure 5

Knockdown of miR-143 inhibited cell proliferation and metastasis. A: MiR-143 inhibitor was employed to silence miR-143 in human lens epithelial SRA01/04 cells; B: Cell proliferation was found to be suppressed after transfecting the miR-143 inhibitor; C and D: Cell invasive and migratory activities were calculated using transwell (200×) and wound healing (100×) assays. NC: normal control. *P<0.05, **P<0.01, ***P<0.001.

Figure 6

Network graphs indicated the target genes of miRNAs. The target genes of miRNAs were predicted. A: The target genes of some upregulated miRNAs in cataract compared to myopia; B: The target genes of some downregulated miRNAs in cataract compared to myopia.

Figure 7

BRD2 was proved to be a target of miR-143. A: Tagetscan predicted the binding sequences between mIR-143 and BRD2, and 3’-UTR of BRD2 mRNA was mutated from UGAUGUCA to AGUAGAG in SRA01/04 cells; B: Luciferase reporter gene assay was performed to verify miR-143 direct binding to BRD2; C: The mRNA level of BRD2 after transfecting miR-143 inhibitor; D: The protein level of BRD2 after transfecting miR-143 inhibitor. NC: normal control; WT: wild type. *P<0.05, **P<0.01.

Figure 8

Silencing of BRD2 partially reversed the functions of miR-143 inhibitor on proliferation and metastasis. A: The mRNA and protein levels of BRD2 were detected after co-transfection of miR-143 inhibitor and si-BRD2; B: Co-transfection of miR-143 inhibitor and si-BRD2 reversed the effect of miR-143 inhibitor on cell proliferation; C and D: Knockdown of BRD2 reversed the effects of miR-143 inhibitor on cell invasion (200×) and migration (100×). NC: normal control. *P<0.05, **P<0.01, ***P<0.001.