232 Examination of Mirna Mimics and Inhibitors on Satellite Cell Proliferation in Vitro

Abstract

Abstract The objective of this study was to examine how the use of specific miRNA mimics or inhibitors alters ovine satellite cell proliferation in vitro. Satellite cells were isolated from a Texel x Suffolk cross lamb at d 2 of age and cell purity was assessed with PAX7 protein staining and the population was 95% pure. miRNA candidates were selected from miRNA sequencing of the longissimus muscle at several time points from gestational d85 to market weight. Satellite cells were plated and allowed to reach 60% confluence, then treated with a miRNA mimic (miR-127 or 299a) at 50 or 100nM or a miRNA AntagomiR (miR-22-3p, 29a, 133, or 27) at 500nM using lipofectamine transfection reagent. Cells were allowed to proliferate for 4d following transfection and samples were collected daily for proliferation analysis using DNA quantification. Experiments were replicated and data analyzed using a priori contrasts with miRNA treatment in the model. DNA quantification showed that miR-127 mimic-100nM and miR-22-3p AntagomiR increased (P< 0.05) DNA content after 4d of proliferation following transfection. miR-127 mimic-100nM treatment increased (P< 0.05) miR-127 expression relative to controls at d1 and d4 post transfection. miR-22-3p AntagomiR transfection tended (P< 0.10) to reduce miR-22-3p expression on d4 post-transfection. miR-22-3p AntagomiR did not alter (P>0.05) PAX7, MYOD, or MYOG expression compared with the control. miR-127 mimic-100nM treatment did not alter (P>0.05) PAX7 expression, while it did increase (P< 0.05) MYOD expression on d4 and reduced (P< 0.05) MYOG expression compared to the control. mRNA sequencing was performed on d4 samples to determine possible miR-127 and 22-3p targets. IGF1, fibroin and other extra cellular matrix proteins were identified from miR-127 treated samples and CNDP2, involved in carnosine hydrolysis, was identified from miR-22-3p treated samples. miR-127 and 22-3p enhanced satellite cell proliferation and therefore could be used to promote muscle fiber hypertrophy.