| Follicle-stimulating hormone(FSH)is key gonadotropin synthesized and secreted by the pituitary gland and plays a very important role in animal reproduction.Therefore,it is very necessary to explore the potential molecular mechanisms that regulate the synthesis and secretion of FSH in the pituitary gland.Long non-coding RNA(lncRNA),micro RNA(miRNA)are endogenous non-coding RNA,which play an important role in regulating metabolism,disease occurrence,and reproduction and development.Studies have shown that lncRNA and miRNA can compete with each other and regulate the expression of downstream target genes through the ce RNA mechanism.However,there are still few studies on the ce RNA mechanism involved in regulating animal reproductive development,especially the synthesis and secretion of FSH.In this study,a new lncRNA was screened and named lncRNA-m18as1 based on the results of whole-transcriptome sequencing of the pituitary gland before and after sexual maturation in rats.The analysis of related websites showed that this lncRNA has no coding ability.Rat primary pituitary gland cells were cultured according to the method described in the group's previously published article,and all experiments were performed in this cell.The results of lncRNA-m18as1 overexpression plasmid and si RNA transfection experiments showed that lncRNA-m18as1 has a positive regulatory effect on FSH synthesis and secretion.Fluorescence in situ hybridization(FISH)results showed that lncRNA-m18as1 was distributed in both the cytoplasm and the nucleus.In order to further explore the molecular mechanism by which lncRNA-m18as1 regulates the synthesis and secretion of FSH in the adenohypophysis,we first predicted the miRNAs that could target lncRNA-m18as1.It was found that miR-18a-5p among these miRNAs can also target Smad2,which has been reported to play an important role(p<0.05)in the regulation of FSH formation and secretion.The results of FISH experiments showed that lncRNA-m18as1 and miR-18a-5p could co-localize.The dual-luciferase reporter system and AGO RIP-q PCR experiments showed that lncRNAm18as1 could interact with miR-18a-5p,and the dual-luciferase reporter system also showed that miR-18a-5p could also interact with Smad2,so lncRNA-m18as1 may be able to regulate the expression of FSH through the Smad2/3 pathway by competing with miR-18a-5p.Furthermore,in this experiment,the lncRNA-m18as1 overexpression plasmid was transfected,and the expression changes of miR-18a-5p and Smad2 were detected.The results showed that the overexpression of lncRNA-m18as1 inhibited the expression of miR-18a-5p,and Smad2 was detected at the same time.protein levels were significantly up-regulated.In addition,the transfection results of miR-18a-5p mimic and inhibitor showed that miR-18a-5p could inhibit the gene expression of lncRNA-m18as1 and the protein expression of Smad2.Simultaneous overexpression of lncRNA-m18as1 and miR-18a-5p as a rescue experiment,WB detected that overexpression of miR-18a-5p attenuated the up-regulation of Smad2 protein level caused by overexpression of lncRNA-m18as1,and also reduced the expression of lncRNA-m18as1 Promotion of FSH secretion.After the Smad2/3 pathway was inhibited with a Smad2/3 pathway inhibitor,the secretion of FSH induced by lncRNA-m18as1 was also inhibited.In summary,this study analyzed that lncRNA-m18as1,as a molecular sponge of miR-18a-5p,regulates the expression of Smad2 protein by competitively binding with miR-18a-5p,thereby regulating the expression of FSH in the ratpituitary gland,and exploring the potential molecular mechanism of lncRNA involved in regulating the synthesis and secretion of FSH in the rat gland pituitary gland will provide a certain theoretical basis for the study of regulating pituitary hormone secretion in rats. |
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