Study Of LncRNA-m433s1 As MiR-433 Sponge Regulating FSH Secretion In Male Rat Pituitary

The role of reproductive hormones runs through every link of mammalian reproduction.The reproductive endocrine system composed of the hypothalamicpituitary-gonadal axis finely regulates the reproduction process of animals.The secretion of follicle-stimulating hormone(FSH)in the downstream pituitary is regulated by Gonadotropin-releasing hormone(GnRH)secreted by the hypothalamus.It is essential for regulating the reproduction process of animals.FSH plays an important role in the reproduction of both female and male mammals.Therefore,clarifying the molecular mechanism that regulates the secretion of FSH from the adenohypophysis has important reference significance for enriching the basic theories of reproductive endocrinology,and then realizing the artificial regulation of animal reproduction.microRNA(miRNA)and long non-codingRNA(lncRNA)are members of the non-codingRNA family,both of which can participate in the regulation of a variety of biological processes,including epigenetics,cell differentiation,transcriptional regulation,development and diseases,etc.,have received widespread attention.Recent studies have shown that lncRNA can regulate the expression of miRNA target genes through the ceRNA mechanism by acting as a miRNA molecular sponge.However,there are few studies on the regulation of non-codingRNA in animal reproduction,especially the mechanism of its regulation of FSH secretion.In order to clarify how miRNAs are involved in regulating the secretion of FSH in the rat adenohypophysis,we used the Target Scan website to predict miRNAs that may target Fshβ.The dual luciferase reporter system was used to screen miRNAs that may act on the Fshβ gene,and 3 miRNAs(miR-21-3p,miR-433 and miR-186-5p)were randomly selected for functional verification.After overexpressing and knocking down candidate miRNAs,respectively,the changes in mRNA and protein levels of Fshβ are detected.The results indicate that miR-21-3p,miR-433 and miR-186-5p can target the3’UTR region of Fshβ to inhibit the expression of Fshβ,thereby reducing the secretion of FSH.Next,in order to fully reveal the role of lncRNA in regulating the reproductive development of mammals,we performed lncRNA sequencing on the rat pituitary gland after GnRH treatment.Finally,23742 lncRNAs were identified and their distribution on the chromosomes were clarified;the differences in the number of exons,transcript length,and expression levels of mRNA and lncRNA were compared.A total of 704 differentially expressed lncRNAs were screened,of which 338 were down-regulated and 366 were up-regulated.The KEGG pathway enrichment and GO classification analysis were performed on the target genes regulated by lncRNA cis.Fluorescence quantification verifies the accuracy of the sequencing results.Use lncRNATargets software to predict the lncRNA that can interact with Fshβ,and construct an interaction network between Fshβ and lncRNA.In order to further explore whether lncRNA can regulate the secretion of FSH through the ceRNA mechanism,we predict the lncRNA that can interact with the above three miRNAs based on the results of lncRNA sequencing.According to the length and coding ability of lncRNA,a new lncRNA(lncRNA-m433s1)was finally confirmed for further experiments.lncRNA-m433s1 is an intergenic lncRNA located in the cytoplasm.Next,the MS2-RIP experiment proved that lncRNA-m433s1 can interact with miR-433.In addition,after overexpression and knockdown of lncRNA-m433s1,the expression changes of lncRNA-m433s1,miR-433 and Fshβ,the changes of FSH concentration and the level of apoptosis were detected.The results show that lncRNA-m433s1 can upregulate the expression level of Fshβ.Rescue experiments showed that lncRNAm433s1 can reduce the inhibitory effect of miR-433 on Fshβ,and further regulate FSH secretion.In summary,this study enriched the differential expression profile of lncRNA in rat pituitary after GnRH treatment,analyzed the molecular mechanism of miRNA regulating FSH secretion by targeting Fshβ gene,and proved the molecular mechanism of lncRNA-m433s1 as the molecular sponge of miR-433 to regulate FSH secretion in rat adenohypophysis.The research results will provide a theoretical basis for revealing the molecular mechanism of FSH secretion regulation.