Upregulation of miR-22-3p contributes to plumbagin-mediated inhibition of Wnt signaling in human colorectal cancer cells

Abstract

The emergence of resistance and side effects for currently available drugs warrant the need for an alternative treatment regime for colorectal cancer (CRC). Several natural compounds, including plumbagin, have exhibited promising anti-cancer effects in different cancer models, although the molecular mechanisms underlying their effects remain at large. We previously reported plumbagin-mediated inhibition of Wnt signaling in CRC cells and to unravel the actual mechanism, we hypothesized the involvement of miRNAs as they have emerged as critical regulators of gene expression. We performed miRNA-microarray to check if plumbagin-mediated effects are through alteration of miRNA expression and found 32 DE-miRNAs (11 upregulated and 21 downregulated), and KEGG enrichment analysis indicated that their target mRNAs are associated with several pathways relevant to CRC and Wnt signaling. miRNA-mRNA network analysis by miRNET revealed a highly upregulated miRNA upon plumbagin treatment, miR-22-3p, having multiple Wnt-associated mRNAs interacting partners. 3′UTR luciferase assays revealed that miR-22-3p directly targeted the 3′UTR of BCL9L (a coactivator of CTNNB1). miR-22-3p inhibited Wnt signaling by downregulating the levels of BCL9L and that of CTNNB1 and several Wnt-associated proteins (TCF4, c-Myc, CCND1, Snail, Slug, and vimentin) in CRC cells. We also demonstrated that both miR-22-3p and plumbagin reduced colony formation and caused apoptotic cell death (detected by Annexin V/PI dual staining), possibly through increased ROS (detection by Dihydroethidium staining) and decreased MMP (detection by MitoTracker™ Orange staining) in CRC cells. These effects of plumbagin were partially rescued by antimiR-22-3p, suggesting the involvement of miR-22-3p in plumbagin-mediated effects. The present study revealed that alteration in miR-22-3p levels by plumbagin contributes to the induction of apoptosis and its inhibitory effects on Wnt signaling and colony formation, thus providing a mechanistic basis behind its anti-cancer potential.