miR-424 suppresses proliferation and promotes apoptosis of human ovarian granulosa cells by targeting Apelin and APJ expression

Abstract

Objective: Polycystic ovary syndrome (PCOS) is associated with alteration of Apelin signaling in ovarian granulosa cells (GCs). However, the molecular mechanisms regulating Apelin expression remain poorly understood. This study aims to investigate the role of miR-424 in modulating Apelin expression and GC functions.

Methods: miRNA expression in GCs was altered by transfection with specific miR-424 mimics and inhibitors. Apelin level was determined by ELISA. miR-424 and mRNA expression were analyzed by quantitative RT-PCR. Protein abundance was measured by western blotting. Genomic sequence targeted by miR-424 was validated by dual-luciferase reporter assay. Apelin gene was overexpressed by transfection of LV-003 vector carrying its cDNA. GC proliferation was analyzed by MTS method, and its cell cycle progression and apoptosis were measured by flow cytometry.

Results: Apelin concentration was increased in serum and follicular fluid from PCOS patients, accompanied by upregulated APJ (Apelin receptor) expression and suppressed miR-424 expression in GCs. miR-424 mimics suppressed Apelin and APJ expression in KGN cells by targeting 3' UTR of Apelin and APJ, whereas miR-424 inhibitors had the opposite effects. miR-424 inhibited KGN cell proliferation and cell cycle progression by down-regulating Cyclin-D/E expression. Moreover, miR-424 promoted KGN cell apoptosis by increasing truncated Caspase-3 level. The regulation of KGN cell proliferation and apoptosis by miR-424 was mediated by directly suppressing Apelin gene expression, instead of inhibiting Apelin peptide activity.

Conclusion: miR-424 suppresses proliferation and promotes apoptosis of human ovarian granulosa cells by directly targeting and inhibiting Apelin and APJ expression.

Keywords: APJ; Apelin; apoptosis; granulosa cell; miR-424; proliferation.

Figure 1

Differential expression of Apelin, APJ, and miR-424 in PCOS patients. A. Elevated expression of Apelin in serum from PCOS patients. Apelin concentration in human serum was determined by ELISA. B. Elevated expression of Apelin in follicular fluid collected from patients with PCOS. Apelin concentration in follicular fluids was measured by ELISA. C. Decreased miR-424 levels in granulosa cells (GCs) from follicular fluid of PCOS patients. D. Increased expression of APJ in GCs purified from follicular fluid in PCOS patients. PCOS: polycystic ovary syndrome; APJ: Apelin receptor; NOR: normal control; * indicates P < 0.05.

Figure 2

Suppression of Apelin and APJ expression in KGN cells by miR-424. (A) qRT-PCR was used to measure the expression of miR-424. (B) Decreased expression of Apelin in KGN cells transfected with miR-424 mimics. Apelin concentration in KGN cells was determined by ELISA. (C) Elevated expression of Apelin in KGN cells transfected with miR-424 inhibitors. Apelin concentration in KGN cells was measured by ELISA. (D) Alterations of APJ expression in KGN cells transfected with miR-424 mimics and inhibitors. qRT-PCR was used to analyze APJ gene expression. (E, F) APJ protein abundance in KGN cells transfected with miR-424 mimics (E) and miR-424 inhibitors (F). APJ: Apelin receptor; NC: negative control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; * and ** indicates P < 0.05 and < 0.01, respectively.

Figure 3

miR-424 directly targets 3’-UTR of Apelin and APJ genes. A. The 3’-UTR of the Apelin gene used for construction of recombinant plasmids for dual-luciferase reporter assays. B. Direct targeting of the 3’ UTR of Apelin gene by miR-424 in human KGN cells. C. The binding sites of miR-424 on the Apelin gene was predicted by TargetScanHuman 7.1. D. The 3’-UTR of the APJ gene used for construction of recombinant plasmids for dual-luciferase reporter assays. E. Direct targeting of 3’-UTR of APJ by miR-424 in human KGN cells, shown by dual-luciferase reporter assays. F. Binding of miR-424 to 3’ UTR of APJ gene predicted by TargetScanHuman 7.1 software. APJ: Apelin receptor; NC: negative control; WT: wild type; MUT: mutant; UTR: untranslated region; * and ** indicates P < 0.05 and < 0.01, respectively.

Figure 4

Suppression of KGN cell cycle progression and proliferation by miR-424. A. KGN cell cycle progression after transfection with miR-424 mimics and inhibitors. More KGN cells at S stage were observed using flow cytometry after miR-424 mimics transfection. B. Quantitation of KGN cells at different stages of cell cycle following transfection of miR-424 mimics and inhibitors. C. Relative mRNA levels of ERK1/2, CCND1, CCNE, and PCNA genes in KGN cells transfected with miR-424 mimics and inhibitors. Gene expression was analyzed by quantitative RT-PCR. D. Protein abundances of p-AMPK, p-ERK1/2, CCND1, and CCNE in KGN cells transfected with miR-424 mimics and inhibitors. E. Proliferation of KGN cells transfected with miR-424 mimics and inhibitors by MTS. F. PCNA protein abundance in KGN cells transfected with miR-424 mimics and inhibitors, determined by Western blotting. NC: negative control; ERKs: extracellular signal-regulated kinases; CCND1: cyclin D1; CCNE: cyclin E; PCNA: Proliferating cell nuclear antigen; AMPK: AMP-activated protein kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *P < 0.05.

Figure 5

miR-424 promotes KGN cell apoptosis by inducing Caspase-3 truncation. A. Alteration of KGN cell apoptosis induced by transfection with miR-424 mimics and inhibitors. Cell apoptosis was analyzed by flow cytometry. B. Quantitation of apoptotic KGN cells transfected with miR-424 mimics and inhibitors. C. The abundance of Caspase-3 protein and truncated Caspase-3 protein in KGN cells transfected with miR-424 mimics and inhibitors. Western blotting was performed to analyze protein abundance. NC: negative control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *P < 0.05.

Figure 6

miR-424 modulates KGN cell proliferation and apoptosis by suppressing Apelin expression. A. Changes in KGN cell proliferation rate after being treated with multiple combinations of miR-424 inhibitors, LV003-Apelin overexpression construct, and Apelin 13 peptides. Cell proliferation was analyzed by MTS. B, C. Alterations in KGN cell cycle progression after being treated with multiple combinations of miR-424 inhibitors, LV003-Apelin construct, and Apelin 13 peptides. Cell cycle progression was assessed by flow cytometry. D, E. Regulation of KGN cell apoptosis by multiple combinations of miR-424 inhibitors, LV003-Apelin construct, and Apelin 13 peptides. Cell apoptosis was evaluated by flow cytometry. NC: negative control; ns: non-significant difference; * and ^# indicate P < 0.05.