| STAT5(Signal transducer and activator 5)is encoded by STAT5a and STAT5b homologous genes,which are a class of intracellular transcription factors,functioned in mammary gland development and lactation response to PRL in vivo.Studies showed that cell specific abundance of STAT5a and STAT5b contributed to the proliferation and differentiation of mammary epithelial cells,but the mechanism was not clear.At present,the study of STAT5 gene function are mainly conducted in human and mouse,but its role in the development and lactation of buffalo mammary gland has not been reported.In this study,we firstly analyzed the expression pattern of STAT5a and STAT5b in buffalo,and studied their function in mammary gland epithelial cell proliferation and milk protein systhesis by regulating genes expression.Secondly,the differently expressed genes of mammary epithelial cells response to PRL were analyzed by transcriptomics and the interaction of STAT5a and STAT5b with their target genes was analyzed by ChIP-PCR.Finally,quantitative proteomics were used to study the differential expression proteins in different developmental stages of mammary gland,and the important differentially expressed proteins were validated.The results laid a foundation for further exploring mammary gland development and the physiological mechanism of lactation in buffalo.The study results are as the following:Part Ⅰ Cloning and analysis of STAT5a and STAT5b and their promoters of buffaloThe coding sequence(CDS),5’ and 3’ untranslated region(UTR)and promoter sequences of buffalo STAT5a and STAT5b genes were cloned and analyzed.The results showed that the full-length CDS of buffalo STAT5a and STAT5b was 2385bp and 2364bp,encoded 794 and 787 amino acids respectively.Sequence similarity and phylogenetic analysis showed that the CDS of STAT5a gene shared 95%similarities with that of STAT5b gene,and were highly-conserved in different species.The length of buffalo STAT5a 5’UTR and 3’UTR was 700bp and 1358bp,while the length of buffalo STAT5b 5’UTR and 3’UTR was 1500bp and 174bp.Two promoter fragments(PI and P2)of STAT5a were cloned,whose length was 500bp and 700bp respectively.Three promoter fragments(P3,P4 and P5)of STAT5b were cloned,whose length were 500bp,800bp and 1500bp respectively.Among them,hypennethylation sites were only located in P2.293T and mammary epithelial cells were transfected with buffalo luciferase vectors which contained different promoter fragments,and the promoter activity was analyzed based on the measurement of luciferase activity.The results showed that the ratio of luciferase in P1～P5 plasmid transfected groups was significantly higher than that of in untransfected group(P<0.05).The ratio of luciferase in P3 plasmid was significantly higher than that of in other groups with 5 mg/mL PRL treatment in buffalo mammary epithelial cells(P<0.05).Part Ⅱ The expression pattern of STAT5a and STAT5b gene in buffalo tissuesTo understand the expression pattern of STAT5 in different buffalo tissues,the morphological structure of buffalo mammary gland was firstly analyzed by HE staining and transmission electron microscopy.The expression of STAT5a and STAT5b genes in buffalo tissues was detected by immunohistochemistry and real-time quantitative PCR methods.The results showed that alveolar was intact in mammary gland tissues during lactation period.More organelles,including mitochondrion,endoplasmic reticulum and ribosome along with the secretion of particles and lipid droplets were observed.Immunohistochemical results showed that STAT5a and STAT5b genes mainly expressed in mammary epithelial cells.QRT-PCR showed that the expression of STAT5a and STAT5b in lactation groups was significantly lower than that of in non-lactation groups(P<0.05).Meanwhile,STAT5a and STAT5b expressed in all tissues detected,and the expression levels were significantly different(P<0.05).The expression of STAT5a gene had the highest expression in mammary gland,while STAT5b gene highest in brain.Part Ⅲ The effects of STAT5 gene expression on proliferation of buffalo mammary epithelial cells and milk protein gene expression.To investigate the effect of STAT5 gene expression on the proliferation and milk protein systhesis,firstly the pSicoR-GFP-shRNA lentiviral expression vectors of buffalo STATSa and STATSb gene were constructed and analyzed by virus packing,and then infected buffalo mammary epithelial cells.Cell cycle and expression of milk protein gene expression were detected.Flow cytometry results showed that the percentage of G1 phase cells increased significantly with the increase of MOI value(P<0.05),The expression of Csnlsl,Csnls2,Csn3,aLA and βLG were detected by QRT-PCRt.The results showed that compared with the control group,the expression of Csnlsl,Csnls2,Csn3,aLA and PLG were significantly decreased(P<0.05)with the inhibition of STAT5a and STAT5b.Secondly,the mammary gland specific expression vector of buffalo STAT5a gene was constructed,and transfected into the mammary epithelial cells by liposome method.QRT-PCR results showed that the expression of Csnlsl,Csnls2,Csn3 and βLG were significantly increased(P<0.05),while the expression of aLA gene was not significantly different from that of in untransfected group(P>0.05).Part Ⅳ The analysis of differentially expressed genes in buffalo mammary epithelial cells response to PRL induction.In order to investigate the effect of PRL on the gene expression of buffalo mammary epiththelial cells,the differentially expressed genes were screened by RNA-seq,the targeted genes of STATSa and STAT5b were validated by ChIP-PCR.The molecular mechanism of PRL regulating STATSa and STATSb gene expression to promote cell growth was discussed.The results of transcriptomics analysis showed that 925 genes were up-regulated and 176 genes were down-regulated after PRL treatment.The differentially expressed genes can be localized to 271 signaling pathways,in which the expression of the genes that enriched in the signal transduction and tumor was higher,including JAK-STAT5 signaling pathways induced by PRL.QRT-PCR results showed that the expression of STAT5a and STAT5b was significantly up-regulated with PRL treatment in mammary cells(P<0.05),while the expression of other genes tested was consistent with the results of transcriptome.ChIP-PCR showed that the SOCS2 gene bands could be amplified by using DNA template extracted from STAT5a and STAT5b antibody enriched PRL treated mammary epithelial cells,the control group had no band.It indicated that the SOCS2 gene was downstream target gene of STAT5a and STATSb,PRL induction promoted interaction of STAT5a and STAT5b with SOCS2.Part Ⅴ Quantitative proteomics analysis of buffalo mammary gland tissuesDifferentially expressed proteins of non-lactation and lactation buffalo mammary gland were analyzed by TMT-LC-MS/MS The results showed that 309 proteins were up-regulated and 421 proteins were down-regulated in lactation mammary gland tissues.The up-regulated proteins were mainly located in mitochondria and their intima,down-regulated protein located in the extracellular matrix,and were mainly functioned in oxidative phosphorylation,extracellular matrix receptor interaction pathway.Real-time quantitative PCR results showed that the mRNA of CYP27A1,DNAJC12,KRT18,HDAC1 and FASN genes expression were basically consistent with that of TMT quantitative proteome.The above results demonstrated that STAT5a and STAT5b expressed in different stages of buffalo mammary gland,and expression of STAT5 gene affected mammary epithelial cell proliferation and milk protein synthesis.PRL could induce the expression of STAT5a and STAT5b to bind and target SOCS2 gene,thus positively promoting cell growth.The highly abundant proteins through mitochondria and oxidative phosphorylation pathway in lactation period changed sigificantly,and functioned in the physiological process of buffalo lactation. |
