Abstract
MicroRNAs are a novel class of powerful endogenous regulators of gene expression. MiR-378 and miR-378* are localized in the first intron of the Ppargc1b gene that codes the transcriptional co-activator PGC-1β. The latter regulates energy expenditure as well as mitochondrial biogenesis. The miR-378:miR-378* hairpin is highly expressed in cardiac cells. To better assess their role in cardiomyocytes, we identified miR-378 and miR-378* targets via a proteomic screen. We established H9c2 cellular models of overexpression of miR-378 and miR-378* and identified a total of 86 down-regulated proteins in the presence of either one of these miRs. Functional annotation clustering showed that miR-378 and miR-378* regulate related pathways in cardiomyocytes, including energy metabolism, notably glycolysis, cytoskeleton, notably actin filaments and muscle contraction. Using bioinformatics algorithms we found that 20 proteins were predicted as direct targets of the miRs. We validated eight of these targets by quantitative RT-PCR and luciferase reporter assay. We found that miR-378 targets lactate dehydrogenase A and impacts on cell proliferation and survival whereas miR-378* targets cytoskeleton proteins actin and vimentin. Proteins involved in endoplasmic reticulum stress response such as chaperone and/or calcium buffering proteins GRP78, PPIA (cyclophilin A), calumenin, and GMMPA involved in glycosylation are repressed by these miRs. Our results show that the miR-378/378* hairpin establishes a connection among energy metabolism, cytoskeleton remodeling, and endoplasmic reticulum function through post-transcriptional regulation of key proteins involved in theses pathways.
Highlights
MicroRNAs1 are 18 –25-nucleotide noncoding RNAs that are known to regulate gene expression in a sequencespecific manner
In breast cancer cells infected with an adenovirus overexpressing the miR-378: 378* hairpin, miR-378* was found to be more abundant than miR-378 and responsible for a shift from oxidative phosphorylation to a high-rate glycolytic metabolism associated with increased lactate production, a phenomenon known as the Molecular & Cellular Proteomics 13.1
We identified 116 protein spots that were significantly altered by either miR-378 or miR-378*
Summary
Cell Culture—We used the H9c2 cardiac cell line derive from rat fetal atria (ATCC® Number: CRL-1446 TM). To screen for protein amount differences and obtain quantitative protein ratios, Two-dimensional differential in gel electrophoresis (DIGE) was performed on four samples for each group, that is, four nontransfected extracts (RNAiMAX only), four transfected with miR-378, and four with miR378* (12 nM each). Control and transfected protein extracts (50 g) were labeled with the CyDye DIGE Fluor minimal labeling kit (GE Healthcare) following the manufacturer’s recommendations. The fold change is the ratio of the mean spot value from a transfected condition (n ϭ 4) on the control nontransfected cells (n ϭ 4). Database searching parameters were the same as MALDI except the precursor mass tolerance that was set to 5 ppm and the fragment mass tolerance to 0.45 Da. RNA Extraction and Quantification—Cells were scraped in RNA reagent (Biogentex, League City, TX) according to the manufacturer’s instructions. An unpaired Student’s t test was used to determine the probability value (p value). p values of p Ͻ 0.001(***), p Ͻ 0.01 (**), and p Ͻ 0.05 (*) were considered statistically significant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
