| As the core subunit of the SWI/SNF chromatin remodeling complex,SMARCA2（SWI/SNF related,matrix associated,actin dependent regulator of chromatin,subfamily a,member 2）plays a key role in regulating gene expression,cell function,and disease occurrence.Our previous simplified genome sequencing results showed that SM4RCA2 gene was located in differentially selected regions（DSRs）between Landrace,a famous western pig breed,and Erhualian and Meishan,two Chinese indigenous pig breeds with high reproductive performance.It was revealed that SMARCA2 may be the major gene that affected the sow fertility.However,the role of SMARCA2 in mammalian ovarian function and female fertility is unknown.In order to explore the relationship between SMARCA2 and sow reproductive traits,we investigated the polymorphism in the 3’-UTR of the SMARCA2 gene.The tissue expression patterns and ovarian cell expression characteristics of porcine SMARCA2 gene were also analyzed.In addition,we investigated the effect of SMARCA2 on porcine granulosa cells（pGCs）apoptosis and explored the transcriptional regulation mechanism of SMARCA2 gene in pGCs.The main research results of the full text are as followed:（1）Identification and analysis of the 3’-UTR of the porcine SMARCA2 geneThe full sequence of porcine SMARCA2 3’-UTR,a total length of 916 bp,was obtained by RACE experiment for the first time.Homology analysis found that the 3’-UTR nucleotide sequence of porcine SMARCA2 gene was highly in accordance with that in mammals,such as human（91.88%,GenBank ID:NM001289396.1）and sheep（89.89%,GenBank ID:XM027964526.1）.Like most encoding genes,the 3’-UTR of the porcine SMARCA2 gene also contained typical polyA signals（PAS）（AAUAAAA）,GU-rich elements（GRE）（UUGUU）,AU-rich elements（ARE）（AUUUA）and polyA tail（PAT）.In addition,the 3’-UTR of porcine SMARCA2 gene contained a large number of miRNA response elements（MRE）,such as miR-29c,miR-135a-5p,miR-128-3p,miR-19a-3p and miR-216a-3p.（2）SMARCA2 was a candidate gene for sow litter size traitsA novel 1-bp indel mutation was found in a ployA region from nt*640*650 of the 3’-UTR of SMARCA2 gene by DNA pooled sequencing and named*640ANV.Three genotypes were detected in the Large White pig population.The genotype frequence were 0.686（11A/11A）,0.202（11A/12A）and 0.112（12A/12A）.To determine the effect of mutation*640ANV in SMARCA2 3’-UTR on sow litter size traits in the Large White pig population,association analysis was carried out by using a mixed model.The results showed that the total number of piglets born（TNB）and the total number of piglets born alive（NBA）of sows carried with genotype 11A/12A was significantly higher than that of sows carried with other genotypes in multiparities.At the same time,the TNB and NBA of sows carried with genotype 11A/12A in primiparity were 0.69 and 0.52 more per litter than 12A/12A individuals.Athough the diffence did not reach the significant level,it still had certain significance in the actual production process of pig breeding.（3）SMARCA2 inhibited apoptosis of pGCsBy using RT-PCR and immunohistochemical techniques to explore the expression characteristics of pig SMARCA2 gene,we found that SMARCA2 gene was highly expressed in pig ovarian tissue and was closely related to follicular atresia.Subsequently,specific small interfering RNA（siRNA）was used to knock down the expression of SMARCA2 in pGCs cultured in vitro to analyze its effect on pGCs apoptosis.Results showed that SMARCA2-siRNA can effectively knock down the mRNA and protein levels of SMARCA2 in pGCs.Flow cytometry revealed that the apoptosis rate of pGCs was significantly increased.In addition,anti-apoptotic Bcl-2 mRNA levels were significantly decreased,and Bcl-2/Bax ratio was significantly decreased.The above results indicate that SMARCA2 participated in the regulation of follicular atresia by inhibiting pGCs apoptosis.（4）SMARCA2 was a functional target gene of miR-29c in pGCsCombined with bioinformatics website predictions and our previous lab miRNA chip results,we found that miR-29c is not only a candidate miRNA targeting the SMARCA2 gene but also a proapoptosis factor.Luciferase assay confirmed that miR-29c could directly target the 3’-UTR of porcine SMARCA2 gene.And over-expression of miR-29c significantly decreased the mRNA and protein levels of SMARCA2 in pGCs,while inhibition of miR-29c increased the mRNA and protein levels of SMARCA2.Flow cytometry revealed that inhibition of miR-29c significantly decreased apoptosis rate of pGCs,while the inhibitory effect of miR-29c inhibitor on pGCs apoptosis was reversed by SMARCA2-siRNA.Results above indicate that miR-29c regulates cell apoptosis via inhibiting SMARCA2 expression in pGCs.（5）SMARCA2 gene transcription is regulated by linc-NORFA/SMAD4/miR-29c axis in pGCsLuciferase activity analysis and ChIP assay confirmed that SMAD4 could be as a transcription factor to directly target the miR-29c promoter to inhibit its transcription in porcine GCs,and its epigenetic regulatory factor linc-NORFA contained the MRE element of miR-29c.In porcine GCs cultured in vitro,overexpression of SMAD4 significantly increased the mRNA and protein levels of SMARCA2,while upregulation of miR-29c reversed SMAD4 induces up-regulation of SMARCA2 expression.After knocking down NORFA in porcine GCs,miR-29c levels were significantly up-regulated,SMARCA2 expression levels were significantly down-regulated,and miR-29c interference could significantly rescue the inhibitory effect of SMARCA2 after NORFA knockdown.The above results indicate that porcine SMARCA2 gene is regulated by linc-NORFA/SMAD4/miR-29c axis in pGCs.In addition,linc-NORFA can not regulate the expression of SMARCA2,a miR-29c target gene,in porcine ovarian GCs by binding to miR-29c.In conclusion,our findings demonstrate that SMARCA2 is a candidate gene for sow litter size traits through regulation of follicular atresia and GC apoptosis,and regulated by linc-NORFA/SMAD4/miR-29c axis. |
