Background: Aberrant expression of microRNAs (miRNAs) has been implicated in human carcinogenesis where they function as oncogenes or tumor suppressor genes. Overexpression of HB-EGF, member of the epidermal growth factor (EGF) family, plays pivotal role in ovarian carcinogenesis. However the molecular mechanisms underlying miRNA(s) mediated aberrant function of HB-EGF in ovarian carcinoma remain unresolved. Here, we investigated the miRNA(s) mediated deregulation of HB-EGF in human ovarian carcinogenesis.
Methods: Web-based bioinformatics algorithms were used to identify candidate miRNAs with putative binding sites on target hHB-EGF mRNA. Expression of miRNAs and mRNA were checked by real-time PCR whereas protein expression was investigated by western blotting assay. Luciferase reporter assay was carried out to examine miR-27a target on 3´UTR of hHB-EGF mRNA. Changes in the oncogenic effects of HB-EGF were studied after gain/loss -of-function of the miR-27a using soft agar colony formation assay and 3D collagen culture. In vivo tumor growth potential of miR-27a was measured in BALB/c nude mice.
Results: By using web-based bioinformatics algorithms 11 miRNAs ((miR-27a/b, -29a/b/c, -96, -128, -132, -182,-194, -212) having putative binding sites on the 3′UTR of hHB-EGF mRNA have been selected for further analysis. Among the putative candidates miR-27a showed maximum reciprocal expression with hHB-EGF and thus we selected miR-27a as promising candidate for hHB-EGF regulation. Overexpression of miR-27a significantly inhibited the hHB-EGF expression in SKOV3 cells by almost 50% and miR-27a inhibitors (anti-miR-27a) upregulated the expression of endogenous hHB-EGF mRNA whereas miR-27a overexpression decreased hHB-EGF protein level in MCAS cells suggesting that miR-27a modulate endogenous hHB-EGF expression both at mRNA and protein level. Luciferase reporter assay revealed that miR-27a directly targets 3´UTR of hHB-EGF mRNA at the predicted site. Ectopic expression of miR-27a suppresses in-vitro growth of SKOV3 ovarian cancer cells. In addition, in-vivo tumorigenecity of SKOV3 cells was also inhibited by miR-27a treatment.
Conclusion: The current study has identified novel miR-27a that negatively regulates hHB-EGF expression by directly targeting 3`UTR of its mRNA. This miR-27a might act as oncosupressor in human epithelial ovarian cancer.
Citation Format: Md. Mustafizur Rahman, Razia Sultana, Ryo Iwamoto, Eisuke Mekada. Micro-RNA mediated deregulation of hHB-EGF in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4424.