Functional analysis of microRNA-181a: identification of target proteins and application in HCC therapy

Abstract

Hepatocellular carcinoma (HCC), or the cancer of the liver, is of great concern due to its poor patient outcome despite the various treatments available. It is imperative, therefore, that a novel, viable treatment method is developed such that patient survival rates may be improved from current statistics of less than 50%. The role of miRNAs in the regulation of gene expression and cellular development makes it an important player in cancer development process, as it is found that the aberrant expression of miRNAs is a typical feature of cancer cells or even pre-disposed cancer cells. MiR-181a has been shown to be an important miRNA involved in HCC. In this study, we investigated the potential effects of miR-181a in HepG2 cells and the mechanisms in which it works in controlling cell fate. As chemotherapy is widely used in liver cancer treatment, we also study the use of miR-181a along with chemotherapy (i.e. Cisplatin). Using iTRAQ-coupled 2D LC-MS/MS analysis, we report here the study of protein profile of HepG2 cells transfected with miR-181a and its inhibitor respectively. Three main types of cellular proteins including metabolic enzymes, protein binding and stress proteins displayed changes. The changes in the level of proteins (14-3-3σ, Hsp-90β and NPM1) involved in important cancer processes like cell growth were further supported by a Western blot analysis. MiR-181a was subsequently found to significantly increase HepG2 cell viability while inhibiting it displayed the opposite effect. Inhibiting miR-181a also sensitized HepG2 cells to cisplatin treatment and retards cell cycle progression by decreasing the proportion of cells in S and G2/M phases. We next investigated the reasons behind these observations at a molecular level. As miRNAs are known to regulate genes by binding to and targeting mRNAs, we first used bioinformatics to screen out potential cellular targets. Two important genes identified, cyclin-dependent kinase inhibitor 1B (CDKN1β) and transcriptional factor E2F7 (E2F7), which are involved in cell cycle and cell proliferation, were chosen to be further experimentally studied. In vitro validation via surface plasmon resonance (SPR) technique showed a positive binding between miR-181a and the seed regions of the 3’UTRs of the two putative mRNA targets, with dissociation constants being 272.5 ± 0.008 nM and 1.186 ± 0.009 uM for CDKN1β and E2F7 respectively. In vivo luciferase assay studies further validated the miR-181a:mRNA bindings, in both cases displaying significant decrease in luciferase activity when HepG2 cells were co-transfected with the 3’UTR-containing reporter plasmids and miR-181a. A positive binding, however, may not necessarily lead to a lowered expression of protein levels. A Western blot study on the expression levels of the two proteins, however, showed a decrease in the levels of CDKN1β and E2F7. Lastly, to gain an insight into the overall effects miR-181a has in HepG2 cells, a microarray analysis was performed. Cellular pathways important in cancer were studied and results show that miR-181a significantly activated the MAPK/JNK pathway by increasing the expression levels or activity of transcription factor activator protein 1 (AP-1). Inhibiting miR-181a, on the other hand, abolished this observation and significantly decreased expression levels or activity of hypoxia-inducible factors (HIF) and also significantly upregulated the expression levels or activity of SMAD2/3/4 proteins, possibly inducing a cancer-suppressing effect. Overall, miR-181a appears to activate mainly cancer-promoting pathways, and may act as an oncogene in HepG2 cells. Inhibiting it, on the other hand, activates mainly the tumour-suppressing pathways, making it a possible option for therapy. A separate microarray analysis on gene expression showed that one way in which miR-181a could have activated the SMAD, NFκB and MAPK pathways is via the significant increase in gene expression of bone morphogenetic protein receptor type II (BMPR2), a cellular receptor that mediates the signal transduction of these pathways. Our findings provide a new platform of identifying miRNA targets, in the process offering molecular evidence on the mechanism of action of miR-181a, including the beneficial effects of inhibiting miR-181a in HCC therapy.