Study On The Mechanism Of YBX1 Mediated CDK8 Gene Affecting The Proliferation Of Sow Ovarian Granulosa Cells

As one of the most important economic traits of pigs,sow litter size has always been a research hotspot in the field of pig breeding.Mummy is an important factor leading to sow litter loss,and the reason for sow mummy production has long been attributed to disease and nutritional factors.In the actual production management,mainly to reduce the stress of pregnant sows,and do environmental control and disease prevention and control to reduce the production of mummies.Modern pig farms are relatively mature in production management,vaccination,and drug delivery procedures,and the nutritional supply of pregnant sows is also reasonable,but the phenomenon of sows producing mummies is still very common,indicating that the genetic differences of sows themselves have caused mummies.The impact cannot be ignored.Studying the genetic mechanism of sow mummy production can provide a theoretical basis for genetic improvement of pig litter size.In this study,the candidate gene CDK8 was selected based on the GWAS of the premature white sow mummy.And we used Yorkshire sow ovary tissue to explore the transcription factors that regulate the expression of CDK8 gene in ovarian tissue;using pig ovarian granulosa cells to explore the effect of transcription factor-mediated CDK8 gene on the proliferation and apoptosis of granulosa cells,and then to study the CDK8 gene on the The effect of pigs producing mummies.The results are as follows:(1)Through differential expression profiling of CDK8 gene,it was found that CDK8 gene had the highest expression abundance in ovary of Yorkshire sow,among nine tissues of heart,liver,spleen,lung,kidney,lumbar muscle,abdominal subcutaneous fat,suet,and ovary.FISH and quantitative results of CDK8 gene in ovarian tissues showed that this gene was also expressed in ovarian functional cells-granulosa cells.(2)Through the DNA pull down experiment,16 transcription factors that regulate the expression of CDK8 gene in ovarian tissue were identified.According to the comprehensive scores of the transcription factors’ i BAQ value,Score value,and P-value value,and the previous correlations of these 16 transcription factors,the transcription factor with the highest correlation coefficient with mummies produced by sows was identified—YBX1.(3)Based on STRING,the related gene network of YBX1 was drawn,and a total of 50 genes interacting with YBX1 were obtained.GO analysis and Pathway analysis of 51 interacting genes revealed that the interacting genes were mainly involved in m RNA processing,metabolism and splicing,and gene expression.And most of the 51 genes are involved in RNA-related biological processes.(4)By overexpressing the transcription factor YBX1 in granulosa cells,it was found that the expression level of CDK8 gene was significantly increased,the level of p-SMAD3 was significantly increased,the expression level of BCL-2(anti-apoptotic factor)was significantly increased,and the expression level of BAX(pro-apoptotic factor)was significantly increased.Significantly downregulated,and BCL-2 / BAX was significantly upregulated.Flow cytometry analysis results showed that this process promoted the proliferation of granular cells.(5)By interfering with the expression of the transcription factor YBX1 in granulosa cells,it was found that the expression of CDK8 gene was significantly down-regulated,the level of p-SMAD3 was significantly reduced,the expression of BCL-2(anti-apoptotic factor)was significantly down-regulated,and the expression of BAX(pro-apoptotic factor)was significantly reduced.The amount was significantly increased,and BCL-2 / BAX was significantly decreased.Flow cytometry analysis results showed that this process inhibited the proliferation of granular cells.The above results indicate that under the control of the transcription factor YBX1,the CDK8 gene interacts with the TGF-β / SMAD signaling pathway to affect the proliferation of ovarian granulosa cells.Based on the influence of ovarian granulosa cells on follicle development and embryo development,combined with previous reports about YBX1 knockdown resulting in the late embryonic death of mice and the high conservation of YBX1 between species,we speculate that due to the genetic differences of in sows with low YBX1 gene expression,the CDK8 gene expression is down-regulated,which inhibits cell proliferation and causes granulosa cell apoptosis,which in turn affects the quality of the embryo and is a genetic pathway leading to the occurrence of sow mummy.