Abstract C22: Overexpression of miR-181b and underlying molecular mechanisms in aggressive progression of African American colorectal cancers

Background: MicroRNAs (miRs) are 19 to 24 nucleotide sequences that regulate cell proliferation, differentiation, and apoptosis. mir-181b is involved in tumor initiation and progression of several human malignancies, including colorectal cancers (CRCs). Our recent study of CRCs has demonstrated that increased expression of miR-181b is an indicator of poor patient prognosis, especially for African American patients with Stage III CRCs [ Clin Cancer Res; 19(14); 1–11. 2013 ]. In the current study, we investigated molecular mechanisms of miR-181b over-expression in aggressive progression of CRCs. Methods: The effects of miR-181b over-expression on cell migration, apoptosis, and the cell cycle were analyzed. Putative molecular targets of miR-181b were identified by in silico analysis. miR-181b mediated, post-transcriptional regulation of these targets was assessed in CRC cell lines (HCT116 wt/wt , HCT116 p53 -/- and SW480 p53 mut/mut ) subjected to transient transfection. The targets were validated by measuring their mRNA expression levels, by qRT-PCR, in Stage III CRC tissues (n=18), which exhibited elevated levels of miR-181b. Further, luciferase assays were performed to assess a direct interaction between miR-181b and the 3′UTR of its target genes. Results: Transfection studies indicated that, in CRC cells, over-expression of miR-181b enhanced the rate of migration, diminished apoptosis, and shifted CRC cells from the G1 to the G2/M phase in a p53-dependent manner. Fourteen putative molecular targets of miR-181b were identified by in silico analysis, and miR-181b mediated post-transcriptional regulation of these targets was validated in CRC cell lines by qRT-PCR. Upon over-expression of miR-181b, integrin alpha-3 (cell surface adhesion molecule that mediates cell-adhesion to extra cellular matrix) was up-regulated, and BCL2L11 (apoptosis facilitator) and TNF-alpha (cytokine that has been implicated in tumor regression and also causes apoptosis) were down-regulated. Also, these targets were validated in human CRC tissues that exhibited elevated miR-181b levels (n=8). Finally, the direct interaction of miR-181b with the target gene 3′UTRs was confirmed by luciferase assays. Conclusions: These results demonstrate that, in CRCs, miR-181b post-transcriptionally regulates expression of integrin alpha-3, BCL2L11, and TNF-alpha and also regulates the cell cycle, migration, and apoptosis. This study was funded in part by the National Cancer Institute of the National Institute of Health UAB/TU/MSM Partnership grant (U54 CA118948). Citation Format: Balananda-Dhurjati Kumar Putcha, Brittany Michelle Holt, Liselle Bovell, Trafina Jadhav, Upender Manne. Overexpression of miR-181b and underlying molecular mechanisms in aggressive progression of African American colorectal cancers. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C22. doi:10.1158/1538-7755.DISP13-C22