INFLAMMATORY MACROPHAGES DERIVED EXOSOMAL MIR-222 PROMOTED THECA CELL STEROIDOGENESIS BY MODULATING MYLIP EXPRESSION

Abstract

To explore the role of M1 macrophages (M1M) in the linkage between low-grade inflammation and ovarian dysfunction in obesity. In vitro experimental laboratory study. Raw264.7 macrophage was treated with lipopolysaccharide (LPS) to establish a model of M1M. Exosome (Exo) secreted by macrophage was isolated by ultracentrifugation and identified by transmission electron microscopy (TEM), western blot, and flow nanoanalyzer. The primary murine ovary theca cell (TC) was co-incubated with PKH67 fluorescence-labeled Exo to detect whether Exo secreted by macrophage could be uptaken by TC. The expression of CYP11A1 and CYP17A1 were analyzed by qPCR and western blot. Medium testosterone and progesterone level were analyzed. High-throughput sequencing was performed to analyze the expression of exosomal miRNA (miRNA-seq). In order to select key differential exosomal miRNA, the GSE97652 (species: mouse) was downloaded from the GEO DataBases. The data set contains 7 replications in both experimental group and control group. The experimental group is obese mouse adipose tissue macrophage (ATM)-derived Exo, and the control group is control mouse ATM -derived Exo. The overlap between miRNA-seq and GSE97652 differentially expressed miRNA was analyzed according to the Fold change > 2, p < 0.01 standard, which determined miR-222 and MYLIP for subsequent experiments. Next, gain-of-function experiments were performed to examine their role in TC steroidogenesis function with the involvement of the cholesterol metabolism pathway. TEM, Flow NanoAnalyzer and western blot analysis confirmed the successful extraction of Exo. After co-cultured TC with PKH67-labeled Exo, green fluorescence could be detected in TC which demonstrated that TC was able to ingest Exo secreted by the macrophage. M1M increased the expression of CYP11A1 and the production of progesterone in TC which is dependent upon M1-Exo. The result of miRNA-seq showed that there were 30 upregulated miRNA and 112 down-regulated miRNA in M1M derived Exo compared with control Exo according to the standard of Fold change > 2(p < 0.05). A total of two differentially expressed miRNA, namely miR-222 and miR-155, were selected from the venn diagram of miRNA-seq and GSE97652. QPCR verified that miR-222 in both M1M derived Exo and M1M derived Exo treated TC were significantly upregulated. Subsequently, we predicted the target gene of miR-222 through five different databases. MYLIP, with the highest target score was selected, which has been identified as a key factor promoting LDLR degradation. M1M derived Exo was verified to display a high expression level of miR-222, and M1M derived Exo mediated TC steroidogenesis depended miR-222. The overexpression of miR-222 resulted in the down-regulation of MYLIP expression and up-regulation of LDLR expression, followed by increased cholesterol influx, CYP11A1 overexpression and progesterone overproduction. M1M derived Exo can be ingested by TC. M1M promote TC CYP11A1 overexpression and progesterone overproduction through Exo by delivering miR-222 into TC.