| The development of mammary gland of sows directly affects the quantity of milk,and further affects the growth and development of piglets.The number and activity level of mammary epithelial cells,which are the basis of mammary tissue growth and development,determine the lactation capacity of the mammary gland.Suppressor of cytokine signaling 1(SOCS1),a feedback inhibitor of Janus kinase signaling and activator of transcription(JAK/STAT)signaling pathways,mediates signaling of various cytokines and hormones in many cellular processes,but there is a relative lack of research on porcine mammary gland.In the present study,we isolated and cultured primary mammary epithelial cells from Collembola pigs,constructed porcine SOCS1,JAK2 and STAT3 gene sh RNA interference vectors and overexpression vectors(pc DNA3.1-),transfected mammary epithelial cells separately,screened the differential proteins among the interference groups by TMT quantitative proteomics and verified some of the differential proteins by PRM,and used RT-The m RNA expression of SOCS1,JAK2 and STAT3 genes and JAK2/STAT3 signaling pathway-related genes in the interfering and overexpressing groups were detected by RT-q PCR,and the genes corresponding to some of the differential proteins were verified at the transcriptional level.The following results were obtained:1.Successful isolation and culture of porcine mammary epithelial cells: Both tissue block culture method and trypsin digestion method could successfully isolate and culture porcine primary mammary epithelial cells,which were polygonal in shape and grew in paving stone shape against the wall,with morphology conforming to the characteristics of epithelial-like cells,positive immunofluorescence staining for cytokeratin 18,and high cell purity with normal proliferation level.2.TMT quantitative proteomic analysis and PRM validation in porcine mammary epithelial cells: TMT-LC-MS/MS was applied to screen the differential proteins among the interfering groups after SOCS1,JAK2 and STAT3 gene inhibition respectively in porcine mammary epithelial cells,and 7750 differentially expressed proteins were screened from six comparison groups,of which 7662 were quantifiable proteins.After GO enrichment analysis,differential proteins of each group were mainly enriched in nuclear chromatin,ribosome,extracellular region and extracellular matrix;SOCS1vs NC group differential proteins were mainly enriched in biological aspects such as viral defense response and immunodeficiency after EBV genetic defect response;JAK2vs NC group,STAT3 vs NC group and SOCS1 vs JAK2 group were similar and mainly enriched in peptide biosynthesis process and amide biosynthesis process;in molecular function classification,the differential proteins were mainly enriched in molecular functions such as organocyclic compound binding,nucleic acid binding,carbohydrate derivative binding,etc.KEGG enrichment showed that the hepatitis C signaling pathway was enriched in all six comparison groups,and the ribosomal signaling pathway was enriched in all five comparison groups except the JAK2 vs STAT3 group in JAK2 vs NC and STAT3 vs NC groups,the ribosomal signaling pathway was enriched in upregulated proteins,while in the SOCS1 vs NC,SOCS1 vs STAT3 and SOCS1 vs JAK2 groups,the signaling pathway was enriched in downregulated proteins;in both the JAK2 vs NC and SOCS1 vs JAK2 groups,the extracellular matrix-receptor The interaction signaling pathway was enriched in the up-regulated proteins in the SOCS1 vs NC group and the down-regulated proteins in the STAT3 vs NC group,and the necroptosis signaling pathway was enriched in the JAK2 vs NC group and the SOCS1 vs JAK2 group.Twenty differential proteins were quantified by TMT histological information assessment and PRM validation,and the overall trend of quantification results by TMT and PRM was consistent.RT-q PCR detected differential proteins corresponding to gene expression levels of only some genes with protein level expression patterns.3.Effects of SOCS1,JAK2,STAT3 gene silencing on proliferation and apoptosis of porcine mammary epithelial cells: sh RNA-SOCS1-138,sh RNA-JAK2-328 and sh RNA-STAT3-964 were successfully constructed and selected as the best interference vectors;RT-q PCR results showed that the expression levels of both JAK2 and STAT3 genes were highly significant(P<0.01)elevated after inhibition of SOCS1,and SOCS1 expression showed highly significant elevation(P<0.01)after inhibition of both JAK2 and STAT3;The expression of SOCS2,SOCS3,JAK1,PRLR and TYK2 genes were highly significantly upregulated after SOCS1 inhibition(P<0.01),and the expression of STAT2,STAT5 A,IL-6,and MCL1 genes were all highly significantly decreased after each of the three genes inhibition(P<0.01),indicating that SOCS1 gene and JAK2/STAT3 signaling pathway showed a negative feedback regulation mechanism,and the regulation of SOCS1 on JAK2/ STAT3 regulation was significantly correlated with the related genes of this signaling pathway.The cell proliferation assay indicated that the OD of SOCS1-138 cells in the interfered group was significantly higher than that of the control group at 12 h and 24h(P<0.05)and highly significantly higher at 48 h and 72h(P<0.01),while the OD of JAK2-328 group at 72 h and STAT3-964 group at 24 h,48h and 72 h were highly significantly decreased(P<0.01).The results of cell cycle and apoptosis showed that the percentage of G1 phase cells increased and the percentage of S phase cells decreased in the SOCS1-138 interference group,and the G2/G1 ratio decreased in the SOCS1-138 interference group and the apoptosis rate in the JAK2-328 group were significantly lower than that in the sh NC group(P<0.05),indicating that inhibition of SOCS1 could induce cell cycle G1/S phase block and inhibit apoptosis.4.Effects of SOCS1,JAK2,STAT3 gene overexpression on proliferation and apoptosis of porcine mammary epithelial cells: The overexpression vectors pc DNA3.1-SOCS1,pc DNA3.1-JAK2 and pc DNA3.1-STAT3 were successfully constructed;RT-q PCR results showed that after overexpression of SOCS1,the expression of intracellular JAK2 and The RT-q PCR results showed that the expression of JAK2 and STAT3 genes were down-regulated relative to the control group after overexpression of SOCS1,but did not reach a significant level.After overexpression of STAT3,SOCS2,SOCS3,JAK1,STAT2 and PIAS3 showed highly significant up-regulation(P<0.01),and PRLR and TYK2 were highly significant(P<0.01)and significantly down-regulated(P<0.05),respectively;regarding some differential protein corresponding genes expression assay,the m RNA expression levels of IFIT1,ISG15,PARP14,PARP9,GRB2,RACK1 and RSAD2 genes were all up-regulated,which were in high agreement with the expression trends of TMT quantitative protein levels.The cell proliferation results showed that the OD values of pc DNA3.1-SOCS1 cells in the overexpression group were extremely significantly lower than those in the control group at48 h and 72h(P<0.01),the pc DNA3.1-JAK2 group was significantly higher than the control group at 72h(P<0.05),and the OD values of pc DNA3.1-STAT3 group were extremely significantly higher than those in the control group at 24 h,48h and 72h(P<0.05).The cell cycle and apoptosis results showed that overexpression of SOCS1,JAK2 and STAT3 would promote cell cycle progression by blocking porcine mammary epithelial cells in G2 and S phases,and the apoptosis rate of cells in the SOCS1 overexpression group was significantly higher than that of the control group(P<0.05),while the apoptosis rate of cells in the STAT3 overexpression group was significantly lower than that of the control group(P<0.05).The apoptosis rate in the SOCS1 group was significantly higher than that in the control group(P<0.05),while that in the STAT3 group was significantly lower.This suggests that overexpression of SOCS1 inhibits the proliferation of mammary epithelial cells and promotes apoptosis.In summary,the SOCS1 gene and JAK2/STAT3 signaling pathway have negative regulatory effects on porcine mammary epithelial cells,SOCS1 gene inhibits proliferation and promotes apoptosis in porcine mammary epithelial cells,and JAK2 or STAT3 silencing induces G1/S phase block in porcine mammary epithelial cell cycle,thus inhibiting cell proliferation,and its mechanism of action is related to the regulation of related genes in the hepatitis C signaling pathway and JAK-STAT signaling pathway;The 20 differential proteins based on TMT proteomics and PRM validation and the screened genes(IFIT1,ISG15,PARP14,PARP9,GRB2,RACK1,RSAD2)could be key factors in the mechanism of SOCS1 gene and JAK2/STAT3 signaling pathway to regulate proliferation and apoptosis of porcine mammary epithelial cells. |
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