Transcriptomic studies provide insights into the tumor suppressive role of miR-146a-5p in non-small cell lung cancer (NSCLC) cells.

Non-small cell lung cancer (NSCLC) is a complex disease in need of new methods of therapeutic intervention. Recent interest has focused on using microRNAs (miRNAs) as a novel treatment method for various cancers. miRNAs negatively regulate gene expression post-transcriptionally, and have become attractive candidates for cancer treatment because they often simultaneously target multiple genes of similar biological function. One such miRNA is miR-146a-5p, which has been described as a tumor suppressive miRNA in NSCLC cell lines and tissues. In this study, we performed RNA-Sequencing (RNA-Seq) analysis following transfection of synthetic miR-146a-5p in an NSCLC cell line, A549, and validated our data with Gene Ontology and qRT-PCR analysis of known miR-146a-5p target genes. Our transcriptomic data revealed that miR-146a-5p exerts its tumor suppressive function beyond previously reported targeting of EGFR and NF-κB signaling. miR-146a-5p mimic transfection downregulated arachidonic acid metabolism genes, the RNA-binding protein HuR, and many HuR-stabilized pro-cancer mRNAs, including TGF-ß, HIF-1α, and various cyclins. miR-146a-5p transfection also reduced expression and cellular release of the chemokine CCL2, and this effect was mediated through the 3' untranslated region of its mRNA. Taken together, our work reveals that miR-146a-5p functions as a tumor suppressor in NSCLC by controlling various metabolic and signaling pathways through direct and indirect mechanisms.

Regulation of COX-2 expression by miR-146a in lung cancer cells.

Prostaglandins are a class of molecules that mediate cellular inflammatory responses and control cell growth. The oxidative conversion of arachidonic acid to prostaglandin H2 is carried out by two isozymes of cyclooxygenase, COX-1 and COX-2. COX-1 is constitutively expressed, while COX-2 can be transiently induced by external stimuli, such as pro-inflammatory cytokines. Interestingly, COX-2 is overexpressed in numerous cancers, including lung cancer. MicroRNAs (miRNAs) are small RNA molecules that function to regulate gene expression. Previous studies have implicated an important role for miRNAs in human cancer. We demonstrate here that miR-146a expression levels are significantly lower in lung cancer cells as compared with normal lung cells. Conversely, lung cancer cells have higher levels of COX-2 protein and mRNA expression. Introduction of miR-146a can specifically ablate COX-2 protein and the biological activity of COX-2 as measured by prostaglandin production. The regulation of COX-2 by miR-146a is mediated through a single miRNA-binding site present in the 3' UTR. Therefore, we propose that decreased miR-146a expression contributes to the up-regulation and overexpression of COX-2 in lung cancer cells. Since potential miRNA-mediated regulation is a functional consequence of alternative polyadenylation site choice, understanding the molecular mechanisms that regulate COX-2 mRNA alternative polyadenylation and miRNA targeting will give us key insights into how COX-2 expression is involved in the development of a metastatic condition.

RHAPA: a new method to quantify alternative polyadenylation.

3' end formation of eukaryotic messenger RNAs (mRNAs) is an essential process that influences mRNA stability, turnover, and translation. Polyadenylation is the process by which mRNAs are cleaved at specific sites in response to specific RNA sequence elements and binding of trans-acting protein factors; these cleaved mRNAs subsequently acquire non-templated poly(A) tails at their 3' ends. Alternative polyadenylation occurs when multiple poly(A) signals are present in the primary mRNA transcript, in either the 3' untranslated region (3'UTR) or other sites within the mRNA, resulting in multiple transcript variants of different lengths. We demonstrate here a new method, termed RHAPA (RNase H alternative polyadenylation assay), that employs conventional RT-PCR with gene-specific oligonucleotide hybridization and RNase H cleavage to directly measure and quantify alternatively polyadenylated transcripts. This method gives an absolute quantified expression level of each transcript variant and provides a way to examine poly(A) signal selection in different cell types and under different conditions. Ultimately, it can be used to further examine posttranscriptional regulation of gene expression.

Regulation of genes in the arachidonic acid metabolic pathway by RNA processing and RNA-mediated mechanisms.

Arachidonic acid (AA) is converted by enzymes in an important metabolic pathway to produce molecules known collectively as eicosanoids, 20 carbon molecules with significant physiological and pathological functions in the human body. Cyclooxygenase (COX) enzymes work in one arm of the pathway to produce prostaglandins (PGs) and thromboxanes (TXs), while the actions of 5-lipoxygenase (ALOX5 or 5LO) and its associated protein (ALOX5AP or FLAP) work in the other arm of the metabolic pathway to produce leukotrienes (LTs). The expression of the COX and ALOX5 enzymes that convert AA to eicosanoids is highly regulated at the post- or co-transcriptional level by alternative mRNA splicing, alternative mRNA polyadenylation, mRNA stability, and microRNA (miRNA) regulation. This review article will highlight these mechanisms of mRNA modulation.