The Mechanism Of GSTM2-STAT1 Regulating Pre-implantation Embryo Recognition In Uterus

Glutathione-S-transferases(GSTs) which is a kind of key detoxifying enzymes in vivo are composed of two homologous dimer subunit. In this family, mu subunit of Glutathione-S-transferase(GST) has showed significant influence on reproductive performance of sow. A nonsense mutation of one base in the 5 exon of GSTM2 induces nonsense mediated m RNA decay(NMD). In addition, this result could lead to early abortion of sow as well as death of piglets possibly. In order to find out the mechanism of this phenomenon, we knockdown GSTM2 gene by RNAi in ST cells then got the data of RNA-seq in previous study. We did GO analysis based on data of RNA-seq and interestingly, we found some genes related to immune and adhesion biological process. In addition, the results of KEGG pathway and Networks analysis were corresponding to the GO analysis conclusion. As the consequence, GSTM2 may play an important role in pre-implantation. STAT1 gene which was selected form differentially expressed genes was promisingly interacting with GSTM2 to regulate the process of pre-implantation. To dig out the interaction between two genes, we overexpressed GSTM2 in ST cells and investigated the expression of genes related to embryo implantation. Then we showed the interaction between two genes by co-immunoprecipitation technology. At the meantime, we overexpressed STAT1 and knockdown STAT1 by RNAi to show the regulation and effects to downstream genes of STAT1. We first showed the mechanism of GSTM2-STAT1 regulating pre-implantation embryo recognition in uterus. The main research results are as follows:1. Investigation of differentially expressed genes by q PCR. We verified the 12 genes of DEGs by q PCR and the results were corresponding to the RNA-seq.2. We extracted RNA from endometrium samples which are from different pregnancy stage(12d, 15 d, 18 d, and 32 d respectively) and nonpregnant sows We investigated the expression of GSTM2, STAT1, OPN, MUC4, ITGAV and ADAMTS1 genes. The result of RT-PCR showed that GSTM2 was upregulated in 18 d and 32 d of pregnancy. However STAT1 was upregulated in 15 and 18 respectively in pregnancy period.3. We constructed the over-expression vector of GSTM2 and then did cell transfection in ST cells. We detected the m RNA and protein level in 24 h and 48 h respectively(P<0.01). The result showed that GSTM2 over-expressed significantly. Interestingly, we found that over-expressed GSTM2 could impress phosphorylation of STAT1 protein.4. We investigated the combination of GSTM2 and STAT1 by co-immunoprecipitation technology. However, the result of western blot showed that GSTM2 combined with STAT1. Further more, the other proteins which combine with GSTM2 were detected by coomassie brilliant blue(CBB) staining and mass spectrum.5. We did cell transfection in ST cells using three pairs of si RNA of STAT1 and Lipofectamine2000. Then the STAT1 m RNA level was detected by RT-PCR. The result of RT-PCR and western blot showed that the second pair of si RNA had the most significant effect above all(P<0.05).6. The downstream genes expression of STAT1 were detected by RT-PCR. We did not find differentially expression of GSTM2. However, OPN, ITGAV, ADAMTSI and MUC4 genes were downregulate significantly(P<0.05).7. We constructed the over-expression vector of STAT1 and transfect in ST cells. The results showed that the expression of GSTM2, OPN, ITGAV, ADAMTS1 and MUC4 genes were no difference. But immune related genes IFIT1, IFIT3, MX1, OAS1 were upregulated significantly(P<0.05).To sum up, according to the sequencing data we supposition GSTM2 plays an important role in embryo implantation by adhesion molecule and inflammatory molecules signaling Pathway. Analysis of early pregnancy related genes expression in the endometrium of pig, we further speculation both GSTM2 and STAT1 have an important effect on embryo implantation. GSTM2 inhibit STAT1 phosphorylation through combination with STAT1 affect the recognition of embryo and embryo implantation.