MiR-596 activated by EP300 controls the tumorigenesis in epithelial ovarian cancer by declining BRD4 and KPNA4

Abstract

Background: Epithelial ovarian cancer (EOC), a subclass of ovarian cancer (OC), is usually diagnosed at advanced stages due to the lack of effective screening means. Mounting reports have disclosed the vitally important roles of microRNAs (miRNAs) in carcinogenesis. Here, we aimed to find out possible miRNAs participating in EOC development.

Methods: qRT-PCR ad western blot respectively examined the mRNA and protein levels of studied genes. CCK-8, colony formation, flow cytometry, TUNEL and spheroid formation assays were appropriately employed for examining cell proliferation, cell cycle, apoptosis and stemness. The interaction between molecules was affirmed by luciferase reporter, RNA pull down and ChIP assays.

Results: In consistent with the observation of a past study, miR-596 expression was relatively low in EOC cells. Up-regulating miR-596 suppressed EOC cell proliferation and stemness. EP300 transcriptionally activated miR-596 to serve as a tumor-repressor in EOC. Then BRD4 and KPNA4, whose knockdown led to restraining effects on cell growth and stemness, were both revealed to be targeted by miR-596 in EOC. Lastly, rescue assays affirmed the tumor-restraining role of miR-596-BRD4/KPNA4 axis in EOC.

Conclusion: EP300-activated miR-596 hampered cell growth and stemness via targeting BRD4 and KPNA4 in EOC, proofing miR-596 as a promising therapeutic target in treating EOC patients.

Keywords: BRD4; EP300; Epithelial ovarian cancer (EOC); KPNA4; miR-596.

Fig. 1

Up-regulation of miR-596 suppressed the proliferation of EOC cells by arresting cell cycle and stimulating cell apoptosis. a The enhanced expression of miR-596 in EOC cell lines (SKOV3, OVCAR3, A2780) compared to human ovarian surface epithelial cell line (HOSEpiC) was validated by qRT-PCR. b qRT-PCR confirmed the overexpression of miR-596 in A2780 and OVCAR3 cells by transfecting with miR-596 mimics. c, d The hindered proliferation of A2780 and OVCAR3 cells under miR-596 elevation was indicated by CCK-8 and colony formation assays. e, f Flow cytometry analyses evidenced that cell cycle was arrested at G0/G1 phases and apoptosis rate was increased under miR-596 upregulation. g TUNEL staining assay detected that the percent of TUNEL-positive cells was augmented in miR-596 mimics group relative to miR-NC group. Scale bar = 50 μm. h Western blot analyzed that miR-596 enhancement fortified the level of Bax and cleaved PARP and reduced the level of Bcl2. **P < 0.01

Fig. 2

EP300 transcriptionally stimulated miR-596 to restrain the growth of EOC cells. a qRT-PCR and western blot verified that EP300 expression in A2780 and OVCAR3 cells was silenced by shEP300 but elevated by pcDNA3.1/EP300 (EP300). b Dual-luciferase reporter assay detected that the luciferase activity of miR-596 promoter was decreased or increased after EP300 was inhibited or overexpressed. c ChIP assay validated that the high H3K27ac status in miR-596 promoter in EOC cells compared with that in normal HOSEpiC cells. d EP300 expression at mRNA and protein levels was reduced in EOC cells relative to HOSEpiC cells, as assessed by qRT-PCR and western blot. e qRT-PCR confirmed that miR-596 level was inhibited or enhanced in EOC cells in response to EP300 depletion or overexpression, separately. f qRT-PCR proved the successful inhibition of miR-596 in EOC cells by miR-596 inhibitor. g Cell viability was controlled by EP300 upregulation but recovered after miR-496 suppression, as monitored by CCK-8 assay. h, i Flow cytometry analyses indicated that EP300 induced cell cycle arrest and apoptosis, while such effects were offset by inhibited miR-596. j TUNEL assay results proved that EP300 augmented the percentage of TUNEL-positive cells, while inhibiting miR-596 could counteract such influence. Scale bar = 50 μm. **P < 0.01

Fig. 3

BRD4 and KPNA4 acted as two targets of miR-596 in EOC. a As indicated in the heatmap, qRT-PCR analysis unveiled the expression pattern of possible miR-596 targets in EOC cells relative to HOSEpiC cells. b The outcomes of qRT-PCR proved that only BRD4 and KPNA4 were targeted by miR-596 in both A2780 and OVCAR3 cells. c Heightened protein levels of BRD4 in EOC cells was disclosed by western blot. d, e The results of qRT-PCR and western blot testified the enhanced or reduced mRNA and protein levels of BRD4 in A2780 and OVCAR3 cells responding to miR-596 inhibitor or miR-596 mimics. f The sequences of BRD4 3′UTR with wild-type or mutant miR-596 binding sites were designed for dual-luciferase reporter assays. g As examined by luciferase reporter assay, the luciferase activity of BRD4 (WT) was declined whereas that of BRD4 (Mut) was not affected in EOC cells with miR-596 upregulation. h The high enrichment of BRD4 in the complex pulled down by biotinylated miR-596 sense was examined by RNA pull-down assay. i Elevated protein levels of KPNA4 in EOC cells was validated by western blot. j, k The outcomes of RT-PCR and western blot uncovered that KPNA4 expression in EOC cells was enhanced by miR-596 inhibitor and decreased by miR-596 mimics. l The sequences of KPNA4 3′UTR containing wild-type or mutant miR-596 binding sites were designed for dual-luciferase reporter assays. m Luciferase reporter assay results indicated that miR-596 interacted with KPNA4 at the predicted binding sites. n The high enrichment of KPNA4 in the complex captured by biotinylated miR-596 sense was validated by RNA pull down assay plus qRT-PCR analysis. *P < 0.05 and **P < 0.01

Fig. 4

BRD4 or KPNA4 knockdown served restraining effects on the growth of EOC cells. a The transfection of shBRD4 suppressed BRD4 expression at mRNA and protein levels in A2780 and OVCAR3 cells, as detected via qRT-PCR and western blot. b–g The results of functional assays demonstrated that depleted BRD4 impaired EOC cell proliferation, blocked cell cycle progression, and facilitated cell apoptosis. h As examined by qRT-PCR and western blot, the expression of KPNA4 was weakened by shKPNA4 compared to that in the control group (shNC). i–n The suppressive impact of KPNA4 knockdown on the growth of A2780 and OVCAR3 cells was assessed by CCK-8, colony formation and TUNEL assays and flow cytometry and western blot analyses. **P < 0.01

Fig. 5

MiR-596 regulated cell proliferation, cell cycle and apoptosis in EOC via targeting BRD4 and KPNA4. a, b Elevation on the mRNA and protein levels of BRD4 or KPNA4 severally induced by transfection of pcDNA3.1/BRD4 (BRD4) or pcDNA3.1/KPNA4 (KPNA4) in A2780 cells was determined by qRT-PCR and western blot. c, d The results of CCK-8 and colony formation assays proved that miR-596 upregulation-restrained cell proliferation was reversed by overexpressing BRD4 or KPNA4. e–h The normalization of BRD4 or KPNA4 overexpression on cell cycle and apoptosis of miR-596-enhanced A2780 cells were confirmed by flow cytometry, TUNEL staining (Scale bar = 50 μm) and western blot. **P < 0.01