Estradiol deficiency and skeletal muscle apoptosis : Possible contribution of microRNAs

Background Menopause leads to estradiol (E2) deficiency that is associated with decreases in muscle mass and strength. Here we studied the effect of E2 deficiency on miR-signaling that targets apoptotic pathways. Methods C57BL6 mice were divided into control (normal estrous cycle, n = 8), OVX (E2 deficiency, n = 7) and OVX + E2 groups (E2-pellet, n = 4). Six weeks following the OVX surgery, mice were sacrificed and RNA isolated from gastrocnemius muscles. miR-profiles were studied with Next-generation sequencing (NGS) and candidate miRs verified using qPCR. The target proteins of the miRs were found using in silico analysis and measured at mRNA (qPCR) and protein levels (Western blot). Results Of the apoptosis-linked miRs present, eleven (miRs-92a-3p, 122-5p, 133a-3p, 214-3p, 337-3p, 381-3p, 483-3p, 483-5p, 491-5p, 501-5p and 652-3p) indicated differential expression between OVX and OVX + E2 mice in NGS analysis. In qPCR verification, muscle from OVX mice had lower expression of all eleven miRs compared with OVX + E2 (p < 0.050). Accordingly, OVX had higher expression of cytochrome C and caspases 6 and 9 compared with OVX + E2 at the mRNA level (p < 0.050). At the protein level, OVX also had lower anti-apoptotic BCL-W and greater pro-apoptotic cytochrome C and active caspase 9 compared with OVX + E2 (p < 0.050). Conclusion E2 deficiency down regulated several miRs related to apoptotic pathways thus releasing their targets from miR-mediated suppression, which may lead to increased apoptosis and contribute to reduced skeletal muscle mass. Abbreviations AIFApoptosis inducing factorBCL2B-cell lymphoma-2 regulator proteinBCL-XLB-cell lymphoma-extra-large regulator proteinBCL-WB-cell lymphoma-like protein 2CASPCaspasecytCCytochrome CE2EstradiolFasLFas ligandGAPDHGlyceraldehyde 3-phosphate dehydrogenaseHSPHeat shock proteinmiRmicroRNANGSNext-generation sequencingOVXOvariectomy