Abstract Janus kinase 2 (JAK2), a hormone receptor-coupled kinase, is involved in the proliferation, differentiation and apoptosis of cow (Bos taurus) mammary epithelial cells. However, the regulation mechanism of JAK2 on the proliferation or apoptosis of porcine (Sus scrofa) mammary epithelial cells is still unclear. To study the effect of RNA interference with JAK2 gene expression on the proliferation and apoptosis of porcine mammary epithelial cells, in this study, 4 pairs of shRNA (short hairpin RNA) interference sequences and 1 pair of negative control sequences designed and synthesized according to the JAK2 gene sequence were transfected into porcine mammary epithelial cells, and the vectors with the best interference effect were screened. After successfully interfering with JAK2, the mammary epithelial cells were subjected to quantitative proteomics by using TMT (tandem mass tags) technology. Proteome sequencing and screening of differential proteins were performed, and the mRNA expression levels of cell proliferation, apoptosis, cycle and related genes were detected by CCK-8 cell proliferation detection kit, flow cytometry and qRT-PCR, respectively. The results showed that 699 differentially expressed proteins were screened, including 309 up-regulated proteins and 390 down-regulated proteins. GO enrichment analysis showed that the differential proteins were mainly enriched in the peptide biosynthesis process and amide biosynthesis process, as well as enriched in the molecular functions such as carbohydrate derivatives binding and the structural composition of ribosomes; KEGG enrichment analysis showed that the differential proteins were mainly enriched in the ribosomes, the complement system, and the hepatitis C signaling pathway. After interfering with JAK2, the cell proliferation ability was shown to be reduced at different time nodes compared with the control group (P<0.05), with extremely significant reduction in the OD450 value at 72 h (P<0.01), and the apoptosis rate (24.0%±3.82%) was significantly lower than that of the shNC group (33.2%±2.55%)(P<0.05), and the blockage of the cell cycle in the G1/S phase was induced. IFIT1 (interferon-induced protein with tetratricopeptide repeats 1)、ISG15 (interferon-stimulated gene 15)、GRB2 (growth factor receptor-bound protein 2) and RACK1 (receptor for activated C kinase 1) gene expression levels were extremely significant lower than those in the control group after silencing JAK2 (P<0.01), and RSAD2 (radical S-adenosyl methionine domain containing 2) gene was extremely significant higher than that in the control group after silencing JAK2 (P<0.01). In conclusion, silencing JAK2 induced G1/S phase block of porcine mammary epithelial cell cycle, which inhibited cell proliferation and apoptosis, and B6E241 protein (encoded by the gene GRB2) could be served as a key protein related to mammary epithelial cell proliferation for future studies. This study provides basic information for further study of the regulation mechanism of mammary gland development and lactation in sows.
LUO Jie,YANG Suan,MAO Tong-Hui, et al. Effects of Knockdown with JAK2 gene Expression on Proliferation and Apoptosis of Porcine Mammary Epithelial Cells[J]. 农业生物技术学报, 2024, 32(8): 1857-1868.
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