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.