Effect Of Progesterone On MicroRNA Expression Of Dorsal Root Ganglia In Female SD Rats

progesterone(PROG)is an important steroid hormone needed to maintain pregnancy.It is secreted mainly by luteal cells of ovary,testis and adrenal cortex.The target cells of progesterone are mainly found in uterus.There are a large number of progesterone target cells in the brain,pituitary gland,mammary gland,ovary and peripheral nervous system,which can induce biological effects mediated by specific binding of progesterone receptors(PRs).Previous work at the dorsal root ganglion(DRG)has confirmed the distribution of progesterone receptors,suggesting that progesterone may play a neuroregulatory role by binding to receptors on the DRG,but there is little evidence from molecular biology.Micrornas(mirnas)are a class of non-coding single-stranded RNA molecules encoded by endogenous genes with a length of about 20-24 nucleotides.They can regulate protein expression through RNA-induced silencing complex to inhibit the translation of target genes or degrade m RNA.miRNA high-throughput sequencing technology has been increasingly applied in the study of transcriptional mechanisms of various biological phenomena,and has achieved important results.In order to explore the effect of progesterone on the regulation of miRNA in DRG tissues,this study studied the effect of ovary removal and different doses of progesterone(0,8,16,32 mg/kg)on miRNA expression in DRGS of ovariectomized rats by establishing the model of ovary removal rats and the sham operation model.High-throughput sequencing technology was used to establish the differential expression profile.The differentially expressed genes were screened,and the sequencing results were verified by real-time quantitative PCR.Bioinformatics analysis techniques were used to analyze GO and KEGG signaling pathways of predicted target genes,and luciferase assay was used to verify miRNA and target gene loci,aiming to provide evidence from the molecular level that progesterone may exert neuroendocrine feedback regulation mechanism through binding to receptors on DRG.It provides new ideas for further study of neuroendocrine network.The research contents and main results are as follows:1.The effect of ovarian extraction on miRNA expression in DRG neurons:(1)The models of rats in the ovarian extraction group and the sham operation group were constructed,with 6 rats in each group.Two weeks after recovery,the DRGS of the rats were extracted,RNA was extracted,cDNA libraries of the two groups were established,and 939 miRNAs were identified by high-throughput sequencing.Of these,807 were known miRNAs and 132 were newly predicted miRNAs.The expression profile of differentially expressed miRNAs was established.Compared with the sham operation group,77 mirnas were differentially expressed in DRGS of ovariectomy SD rats(P < 0.05),42 were up-regulated(UP)and 35 were down-regulated(Down),and the expression of rno-miR-146a-5p was significantly different after ovariectomy.(2)In order to verify the accuracy of the sequencing results,real-time fluorescence quantitative detection was performed to detect the expression level of DRG tissues of rats in the ovarian extraction group and sham operation rats in the control group.The results were consistent with the high-throughput sequencing results,and the differential expression results were credible.(3)Target genes were predicted and analyzed.A total of 16,378 GO terms were obtained by GO analysis through GO and KEGG signaling pathway analysis,among which 2,134 GO terms were significantly expressed and were mainly involved in biological processes such as phosphorylation and positive regulation of gene expression.KEGG signaling pathway is mainly involved in the regulation of MAPK signaling pathway and axon-directed signaling pathway.(4)Results of luciferase reporter gene report showed that rno-miR-146a-5p targeted Pak1 gene.2.Effects of progesterone on miRNA differential expression in DRG neurons after ovarian removal:(1)A total of 24 rat models of ovarian extraction were constructed and divided into 4 groups with 6 rats in each group,including the ovarian extraction group,low-dose progesterone group,medium-dose progesterone group and high-dose progesterone group,respectively.After two weeks of postoperative recovery and continuous administration for 9 days,DRGS were extracted and RNA was extracted on the 10 th day to construct 4 groups of cDNA libraries for high-throughput sequencing.A total of 985 miRNAs were identified in the low-dose progesterone group,and 233 miRNAs were differentially expressed(P < 0.05).Among the 233 differentially expressed miRNAs,136 were up-regulated and 97 were down-regulated.A total of 934 miRNAs were identified in the medium-dose progesterone group.221 miRNAs were differentially expressed(P < 0.05).Among the 221 differentially expressed miRNAs,119 were up-regulated and 102 were down-regulated.A total of 922 miRNAs were identified in the high-dose progesterone group.184 miRNAs were differentially expressed(P < 0.05).Among the 184 differentially expressed miRNAs,94 were up-regulated and 90 down-regulated.rno-let-7a-5p,rno-let-7c-5p,rno-miR-9a-5p,rno-miR-434-5p,suggesting that these five mirnas play an important role in the neuronal reactivity of progesterone,and there is no dose relationship between progesterone effect.Among them,only the miRNA differentially expressed in the low-dose progesterone group was rno-miR-409a-5p;miRNA differentially expressed only in the medium-dose progesterone group :rno-miR-26b-3p;miRNA differentially expressed only in the high-dose progesterone group :rno-miR-148b-3p.It is suggested that rno-miR-409a-5p,rno-miR-26b-3p and rno-miR-148b-3p are affected by dose in the reactivity of progesterone on neurons.(2)Real-time fluorescence quantitative detection of expression levels was consistent with high-throughput sequencing results,and the differential expression results were credible.(3)Prediction and analysis of target genes.Through GO and KEGG signaling pathway analysis,GO analysis related target genes were mainly involved in biological processes such as phosphorylation and positive regulation of DNA template transcription.KEGG signaling pathway is mainly regulated by MAPK signaling pathway,axon-directed signaling pathway,and synaptic vesicle cycle.(4)Luciferase gene reporting confirmed the relationship between rno-let-7a-5p and Sema4 f.The above experimental results provide evidence that progesterone may exert neuroendocrine feedback regulation mechanism through binding to receptors on DRG at the molecular level,and provide new ideas for further study of new ways of neuroendocrine network.