MiR-145 supports cancer cell survival and shows association with DDR genes, methylation pattern, and epithelial to mesenchymal transition

Siddharth Manvati,Pawan Kumar Dhar,Rupali Chopra,Sunil Kumar Saini,Kailash Chandra Mangalhara,Monika Kaushik,Rakesh Kumar,Rameshwar Nath Koul Bamezai,Ankita Arora,Ponnuswamy Kalaiarasan,Usha Kumari,Gaurav Agarwal

doi:10.1186/s12935-019-0933-8

Abstract

BackgroundDespite several reports describing the dual role of miR-145 as an oncogene and a tumor suppressor in cancer, not much has been resolved and understood.MethodIn this study, the potential targets of miR-145 were identified bio-informatically using different target prediction tools. The identified target genes were validated in vitro by dual luciferase assay. Wound healing and soft agar colony assay assessed cell proliferation and migration. miR-145 expression level was measured quantitatively by RT-PCR at different stages of breast tumor. Western blot was used to verify the role of miR-145 in EMT transition using key marker proteins.ResultWound healing and soft agar colony assays, using miR-145 over-expressing stably transfected MCF7 cells, unraveled its role as a pro-proliferation candidate in cancerous cells. The association between miR-145 over-expression and differential methylation patterns in representative target genes (DR5, BCL2, TP53, RNF8, TIP60, CHK2, and DCR2) supported the inference drawn. These in vitro observations were validated in a representative set of nodal positive tumors of stage 3 and 4 depicting higher miR-145 expression as compared to early stages. Further, the role of miR-145 in epithelial–mesenchymal (EMT) transition found support through the observation of two key markers, Vimentin and ALDL, where a positive correlation with Vimentin protein and a negative correlation with ALDL mRNA expression were observed.ConclusionOur results demonstrate miR-145 as a pro-cancerous candidate, evident from the phenotypes of aggressive cellular proliferation, epithelial to mesenchymal transition, hypermethylation of CpG sites in DDR and apoptotic genes and upregulation of miR-145 in later stages of tumor tissues.

Highlights

Despite several reports describing the dual role of miR-145 as an oncogene and a tumor suppressor in cancer, not much has been resolved and understood
This role of miR-145 in facilitating cell proliferation and survival, as observed in wound healing assay and the features associated with epithelial–mesenchymal transition (EMT) (Fig. 5), found support in the epigenetic study of a select set of apoptotic and DNA damage response (DDR) genes in a representative set of sporadic breast tumours (Figs. 3, 4)
In stage 3 and 4 tumours, was the expression of miR-145 high (Fig. 2) but it showed similar features of induction of epigenetic imprint in the studied genes, as was observed in miR-145 over-expressing cells in vitro (Figs. 3, 4). These findings along with the bio-informatics pathway analysis, identifying cross talk with molecules involved in growth and EMT (Additional file 2: Figure S1), supported that regulation of SMAD3, DR5 and BRCA2 by miR-145 plays a significant role in uncontrolled cellular proliferation with relevance in cancer

Summary

Introduction

Despite several reports describing the dual role of miR-145 as an oncogene and a tumor suppressor in cancer, not much has been resolved and understood. Suggestions of miR-145 playing a role in the development of colon and rectal cancers but not in its progression [39] have been made, raising a question about its exact role in tumour initiation and development. It was, pertinent to understand the dual role of miR-145 in cancer, using both in vitro and cellular assays

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Results

Discussion

Conclusion