Sex dimorphic response to osteocyte miR21 deletion in murine calvaria bone as determined by RNAseq analysis

Abstract

Low levels of microRNA (miR) 21 may explain the higher osteocyte apoptosis with Cx43-deficient and aged female mice. However, miR21 exerts a sex-divergent role in osteocytes, regulating bone mass and architecture through non-cell autonomous effects on osteoblasts and osteoclasts, via sex-specific regulation of osteocyte cytokine production. miR21 deficiency improves bone strength in females, and, to a higher extent, in male miR21-deficient mice. To understand the molecular basis for the effects of miR21 deletion, mRNA was isolated from miR21fl/fl (controls) or miR21-deficient (by deletion in cells expressing Cre recombinase under the control of the 8 kb fragment of the DMP1 promoter: miR21ΔOt mice). miR21 was 50% lower in miR21ΔOt whole calvaria bone compared to control mice of the corresponding sex. RNAseq was performed in 4 samples/sex and genotype. There were 152 genes with <.05 P-value and >1 absolute log2 fold change in the male data analysis, and expression of most genes was higher in the miR21fl/fl group. Two of the genes, Actn3 and Myh4, had a false discovery rate < 0.1. Gene enrichment analysis of significant genes on both KEGG pathways and gene ontology (GO) gene sets shows that the significant genes were enriched in muscle contraction. Some muscle-related genes like Actn3 were included in multiple significant pathways. For females, only 65 genes had P-value <.05 and >1 absolute log2 fold change. Yet, no significant KEGG or GO pathways, including ≥5 significant genes, were seen, and no overlap of significant genes was found between male and female samples. Therefore, deletion of miR21 has a stronger effect on male transcriptome in calvaria, compared to females. Further, no enrichment of any pathway was detected in female samples. Thus, either there are no differences between 2 groups in female or the effect size is small, and a larger sample size is needed to uncover miR21-dependent differences.