The microRNA212 regulated PEA15 promotes ovarian cancer progression by inhibiting of apoptosis

Abstract

PEA15 (Proliferation And Apoptosis Adaptor) is a 15kDa multifunctional phosphoprotein involved in various essential biological processes such as proliferation and apoptosis of cancer cells. Previous studies have demonstrated that PEA15 can promote the progression of many malignancies. In the present study, the expression of PEA15 in ovarian cancer and normal tissues analyzed in several databases and PEA15 was found to be significantly up-regulated in OC tissues compared to normal tissues. Immunochemical assays performed using 171 OC tissue specimens proved that the expression of PEA15 was remarkably positively correlated with the FIGO stage and associated with histologic subgroups of ovarian cancer. IHC assay for the two phosphorylation sites of PEA15 S116 and S104 was also performed. PEA15 high expression predicted a poor prognosis in OC patients analysed from K-M plot dataset. In addition, we proved knockdown of PEA15 inhibits OC cell proliferation and induces cell apoptosis by Bcl2 downregulation and Bax and cleaved Caspase-3 upregulation. Overexpression of PEA15 promotes the proliferative capacity of OC cells. Moreover, this study first discovered PEA15 expression in OC can be negatively regulated by microRNA212. Overexpression of miR-212 in ovarian cancer cells could cause downregulated the expression of PEA15 expression. Overexpression of miR-212 was found to exerted similar effects on the proliferation, and apoptosis of the ovarian cancer cells as that of PEA15 suppression. Additionally, overexpression of PEA15could at least partially abolished the effects of miR-212 on the proliferation, and apoptosis of ovarian cancer cells. In conclusion, our findings revealed PEA15 appears as a novel predictive biomarker, thus providing a valuable therapeutic target in OC treatment strategy.

Keywords: Apoptosis; OC; PEA15; Proliferation; miR212.

Figure 1

The expression of PEA15 is significantly upregulated in ovarian cancer tissue. A: The mRNA expression of PEA15 is upregulated in different tumor types compared with the normal tissues based on the Oncomine dataset. B-C: The mRNA expression of PEA15 is upregulated in tumor compared with non-tumor tissue as revealed by the TCGA and GEO datasets. D: PEA15 mRNA expression is elevated in 20 paired OC tissues and normal tissues obtained from the first affiliated hospital of Wannan Medical College.

Figure 2

PEA15 expression in OC tissue samples and immunohistochemical staining of two phosphorylation sites S116 and S104 of PEA15in normal and tumor tissues. A-D: Representative images of PEA15 expression in OC are shown at 200x magnification. A: OC, scored as (-); B: OC, scored as (+); C: OC, scored as (++); D: OC, scored as (+++). E: Normal ovarian tissue with negative PEA15-Ser104 staining and tumor tissue with positive PEA15-Ser104 staining. F: Normal ovarian tissue with negative PEA15-Ser116 staining and tumor tissue with positive PEA15-Ser116 staining.

Figure 3

Kaplan-Meier analysis of overall survival in OC patients based on K-M plotter dataset. A: PEA15 expression is negatively correlated with overall survival (OS) and progression free survival (PFS) in OC patients; B-D: Correlation between PEA15 expression and overall survival is independent of clinical stage (B), type (C) and Grade (D). P-values were calculated by log-rank test.

Figure 4

Silencing of PEA15 suppresses ovarian cancer cell proliferation in vitro and tumor growth in vivo. A: RT-PCR and western blotting experiments showing the PEA15 expression at mRNA and protein level in 5 OC cell lines. B: PEA15 knockdown efficiency was confirmed by RT-PCR in OVCAR8 and 3AO cells. C: The cell proliferation of Nc and sh-groups in OVCAR8 and 3AO cells were evaluated by CCK8 assay at 0, 24, 48, 72, 96h. The results showed that knockdown of PEA15 significantly inhibited the proliferation of OVCAR8 and 3AO cells in vitro (P<0.01). D: Plate colony formation assay of OVCAR8/PEA15-sh and 3AO/PEA15-sh and Nc cells on regular culture plates after 14 days of culture. Relative colony numbers of PEA15-sh cells were significantly lower than that of Nc cells. E: Photographs of tumors from mice inoculated with OVCAR8/Nc and OVCAR8/sh3 cells. F: Tumor weights of Nc and sh3 groups shown in figure 4F, n = 5

Figure 5

Effect of PEA15 on cell apoptosis in OC cells. A-B: Downregulation of PEA15 significantly increased apoptosis in OVCAR8 and 3AO cells, the statistical results are shown on the left (*P < 0.05, **P < 0.01). C-D: The expression of Bcl2, Bax and cleaved caspase3 were determined by RT-PCR and western blotting analysis in OVCAR8 and 3AO cells.

Figure 6

PEA15 is negatively regulated by miR212 and silencing of PEA15 abolishes the effects of miR212 in OC cell lines. A: Analysis of TargetScan, miRwalk, picTar and miRBse datasets identified 3 miRNAs as potential target of PEA15. B-D: Effect of 3 miRNAs on the luciferase activity of WT PEA-15 3′UTR in OVCAR8 cells by luciferase reporter assay. Blank PEA-15 negative vector, Mock 3 miRNAs plus PEA-15 negative vector. Error bars mean ± SEM. E: The expression of miR212 is downregulated in ovarian tumor tissues compared with normal tissues. F: The expression of miR212 in 5 ovarian cancer cells. G: The protein expression of PEA-15 is down-regulated when miR-212 is overexpressed. H: Effect of reintroduction of PEA-15 on miR-212-induced cell proliferation by CCK-8 assay (P < 0.01 with two-way ANOVA). I: Effect of reintroduction of PEA-15 on miR-212-induced cell apoptosis by Flow analysis (P < 0.01 for both groups).