LncNONO-AS In Goats Can Regulates The Expression Of AR By Mediated NONO

Goats are important economic animals for human beings,mainly providing meat,milk,cashmere,and leather.Compared with other goats,Dazu black goats have a higher reproductive rate.Dazu black goat ram has the characteristics of early sexual maturity,rapid growth,strong disease resistance and genetic stability.In goat production,the development of goat testicles and the normal occurrence of sperm are key factors affecting male reproductive performance.Previous studies have confirmed that lncRNAs play an important regulatory role in testicular development and spermatogenesis.Although lncRNAs are involved in almost every step of gene regulation,the regulatory mechanisms are complex and diverse,but few of them are sequenced in goat testes.In order to investigate the role of lncRNAs in the testis of Dazu black goats,high-throughput sequencing was used to screen out lncRNA LncNONO-AS,which may be related to spermatogenesis and testicular development.The target gene is NONO.Previous studies have found that the androgen receptor?AR?may be regulated by NONO in the testis,conjecture that LncNONO-AS may regulate AR by mediating the expression of NONO.In order to verify this conjecture,based on the in vitro culture of testicular support cells,overexpression,interference,fluorescence quantification,western blot and other techniques to verify the function of lncRNA LncNONO-AS and possible regulatory relationships,the following results were obtained:1.By sequencing,a new lncRNA LTCONS₃2868 with different gene expression was found.The target gene was NONO.Bioinformatics analysis showed that LTCONS₃2868 had binding energy with NONO.BLAST alignment showed that the sequence of lncRNA and its target gene NONO 99% was complementary.Therefore,it is preliminarily speculated that this newly discovered lncRNA is an antisense lncRNA of NONO and is named LncNONO-AS.2.After overexpression of LncNONO-AS,the target gene NONO has undergone significant changes.The gene expression level of NONO in the overexpression group was 1.74 times that of the untreated control group,and the difference was extremely significant?P<0.01?.In addition,the protein expression level of the NONO overexpression group was 1.53 times that of the untreated control group,and the difference was significant?P<0.05?.It indicates that lncRNA LncNONO-AS has a regulatory effect on NONO and may be a positive regulatory effect.3.RNA pull down experiments on LncNONO-AS and NONO showed that there was indeed a combination between them.It is suggested that LncNONO-AS may regulate NONO in a direct way.4.After direct overexpression of LncNONO-AS,the gene expression of AR also changed significantly.The gene expression level of the overexpression group AR was 3.48 times that of the untreated control group,and the difference was extremely significant?P<0.01?.It indicates that lncRNA LncNONO-AS also has a regulatory effect on AR.5.Direct interference with NONO found that the expression level of AR has changed significantly.The NONO lentiviral interference vector was constructed,and three interference groups HBLV-NONO shRNA1-GFP-PURO,HBLV-NONO shRNA2-GFP-PURO,HBLV-NONO shRNA3-GFP-PURO were set.The HBLV-NONO shRNA1-GFP-PURO interference group was identified as the most efficient and could be used as a target for the NONO gene.Compared with HBV-NONO shRNA1-GFP-PURO interference group,the gene expression of AR decreased from 1.01 to 0.34,the difference was extremely significant?P<0.01?,and the protein expression decreased from 0.12 to 0.08,which decreased by 33.33.%,the difference was significant?P<0.05?.These results indicate that NONO can mediate AR expression and may affect the expression of AR by positive regulation.Based on the above results,it was concluded that lncRNA LncNONO-AS may regulate the expression of AR by mediating the expression of NONO.These data provide insight into testicular development and reproductive processes,providing valuable information for further understanding of the function of lncRNAs.