LncRNA WNT3-IT In Goat Testis Affect The Proliferation Of Sertoli Cells By Regulating The Expression Of WNT3

The ability of proliferation and differentiation of testicular sertoli cells directly affects spermatogenesis,male reproductive development,and ram production.More and more studies on WNT family members such as WNT3 a,WNT3,WNT7,and WNT10 indicate that WNTs are involved in the regulation of various stages of cell proliferation,differentiation,and spermatogenesis.With the development of bioinformatics,the regulation of target gene expression by lncRNA in cells has become a hot research topic.However,there are few studies on goat testes,and there are only a few studies on lncRNA involved in the regulation of WNT signaling pathway.There are no studies on the presence of lncRNAs that regulate the expression of WNTs during cell proliferation and differentiation.In this study,Dazu black goats were used as experimental subjects to collect high-throughput sequencing of the testis tissues of the three stages of the newborn,prepuberty and adulthood of Dazu black goats to predict the lncRNA associated with WNTs during testicular growth and development.The final screening of lncRNA WNT3-IT may target the regulation of WNT3 expression.In order to verify the regulation of WNT3 expression,on the basis of in vitro culture of testicular sertoli cells,paraffin sections,fluorescence in situ hybridization,interference,overexpression,cytotoxicity experiments,Western blot,qPCR and other techniques,lncRNA WNT3-IT and WNT3 The expression relationship was analyzed by localization analysis,interference and overexpression analysis,and the lncRNA WNT3-IT was analyzed to sertoli cell proliferation activity and proliferation cycle,and the following results were obtained: 1.Testicular tissue paraffin section results show that the testicles of the large-footed black goats in the newborn stage contain only sertoli cells and spermatogonial stem cells;there are no sperm cells in the testis of 2 months old;sperm cells are already produced in the 9-month-old testes,and the testicular structure Completely developed.2.High-throughput sequencing revealed a lncRNA that could target the regulation of WNT3 expression,numbered LTSONS-00052549,whose sequence is in the intron of the WNT3 gene sequence.According to its position in the WNT3 gene,we named it lncRNA WNT3-IT.3.The results of fluorescence quantitative PCR analysis of WNT3-IT and WNT3 expression profiles in three age groups showed that they had a positive correlation with the expression profile of WNT3.Furthermore,the results of fluorescence in situ hybridization localization analysis indicated that lncRNA WNT3-IT is expressed by testis-sertoli cells and plays a role in sertoli cell cytoplasm.4.The expression of WNT3 mRNA and protein expression were significantly decreased after interference with WNT3-IT expression(P<0.01).However,in the transfection overexpression vector group,WNT3 mRNA expression and protein expression were both.Significantly increased(P <0.01).This indicates that WNT3-IT does positively regulate the expression of WNT3.5.Through cell viability and cell proliferation experiments,it was found that the proliferation ability and activity of sertoli cells decreased significantly after interference with WNT3-IT(P<0.01),while the sertoli cells of the overexpressing group showed completely opposite results.It indicates that the expression of WNT3-IT helps to sertoli cell proliferation.Based on the above results,it was concluded that lncRNA WNT3-IT can affect the proliferation of sertoli cells by positively regulating the expression of WNT3.This study provides basic data for exploring lncRNAs that affect cell proliferation,and lays a foundation for studying the molecular mechanisms underlying WNT3-IT regulation of WNT3.