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| Effects of Knockdown with JAK2 gene Expression on Proliferation and Apoptosis of Porcine Mammary Epithelial Cells |
| LUO Jie1, YANG Suan3, MAO Tong-Hui1,2, YANG Qi1, ZHANG Yi-Yu3,* |
1 Tongren Polytechnic College, Tongren 554300, China;
2 Tongren Animal Husbandry Technology Promotion Station, Tongren 554300, China;
3 Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education/College of Animal Science, Guizhou University, Guiyang 550025, China |
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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.
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Received: 14 September 2023
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Corresponding Authors:
* zyy8yyc@163.com
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[1] 陈逸, 张一涵, 张静, 等. 2022. 大豆活性肽对体外过氧化氢诱导的奶牛乳腺上皮细胞氧化应激和炎症损伤的缓解作用[J]. 动物营养学报, 34(10): 6714-6725.
(Chen Y, Zhang Y H, Zhang J, et al.2022. Alleviation effect of soybean bioactive peptide on oxidative stress and inflammatory injury of bovine mammary epithelial cells induced by hydrogen peroxide in vitro[J].Chinese Journal of Animal Nutrition, 34(10): 6714-6725.)
[2] 樊嘉琦, 周学章. 2022. 黄芪甲苷对脂多糖刺激的奶牛乳腺上皮细胞乳蛋白及乳脂合成相关因子表达的影响[J].动物营养学报, 34(10): 6702-6713.
(Fan J Q, Zhou X Z.2022. Effects of astragaloside IV on expression of milk protein and milk fat synthesis-related factorsin lipopolysaccharide-stimulated milk bovine mammary epithelial cells[J].Chinese Journal of Animal Nutrition, 34(10): 6702-6713.)
[3] 郭丹. 2020. miR-21-5p对奶山羊乳腺上皮细胞凋亡及泌乳调控作用的研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 李广, pp. 75.
(Guo D.2020. The regulatory effect of miR-21-5p on the apoptosis and lactation of dairy goat mammary epithelial cells[D]. Thesis for M.S., Northwest A＆F University of Science and Technology, Supervisor: Li G, pp. 75.)
[4] 冉鑫. 2022. 薯蓣皂苷对LPS诱导的小鼠乳腺炎的影响及其机制[D]. 硕士学位论文, 吉林大学, 导师: 柳巨雄, pp. 80.
(Ran X.2022. Effects and its mechanisms of dioscin in LPS-induced mice mastitis[D]. Thesis for M.S., Jilin University, Supervisor: Liu J X, pp. 80.)
[5] Sigl, 李喜艳. 2013. 短期限制饲喂的泌乳期奶牛乳腺上皮细胞乳蛋白合成通路基因表达的研究[J]. 中国畜牧兽医, 40(07): 72.
(Sigl, Li X Y.2013. Expression of genes involved in the milk protein synthesis pathway in mammary epithelial cells of lactating cows fed with short-term restriction[J]. Chinese Animal Husbandry and Veterinary, 40(07): 72.)
[6] 王鹏, 刘子嶷, 刘玉芳, 等. 2023.mi R-535靶向GAB2基因通过激活PI3K/AKT信号通路促进山羊颗粒细胞增殖[J]. 中国农业科学, 56(23): 4757-4771.
(Wang P, Liu Z Y, Liu Y F, et al.2023. Mi R-535 Targets the GAB2 gene to promote goat granulosa cell proliferation through activation of the PI3K/AKT signaling pathway[J]. Scientia Agricultura Sinica, 56(23): 4757-4771.)
[7] 杨强, 郑吉松, 郎立敏, 等. 2021. 藏猪妊娠期乳腺形态和乳腺发育的标志基因表达、相关激素及信号通路的变化[J/OL]. 华南农业大学学报, 42(04): 1-12.
(Yang Q, Zhen J S, Lang L M, et al.2021. Alternations of mammary gland morphology, and marker proteinexpression, related hormones and signaling pathways of mammary glanddevelopment during gestation in Tibetan pigs[J/OL]. Journal of South China Agricultural University, 42(04): 1-12.)
[8] 张领衔, 蔡车国. 2022. 乳腺发育与乳腺干细胞的分子调控[J]. 厦门大学学报(自然科学版), 61(03): 415-428.
(Zhang L X, Cai C G.2022. Breast development and molecular regulation of breast stem cells[J]. Journal of Xiamen University (Natural Science Edition), 61(03): 415-428.)
[9] 张亚林. 2019. 不同蛋氨酸源对猪乳腺上皮细胞蛋白质合成的调控及其机制研究[D]. 四川农业大学, 导师: 方正锋, pp. 72.
(Zhang Y L.Regulation of methionine sources on protein synthesis in porcine mammary epithelial cells and tissues[D].Sichuan Agricultural University, Supervisor: Fang Z F, pp. 72.)
[10] Bomfim G F, Merighe G K F, de Oliveira S A, et al.2022. Acute and chronic effects of cortisol on milk yield, the expression of key receptors, and apoptosis of mammary epithelial cells in Saanen goats Der Pathologe[J]. Journal of Dairy Science, 105(1): 818-830.
[11] Boutinaud M, Ben Chedly M H, Delamaire E, et al.2008. Milking and feed restriction regulate transcripts of mammary epithelial cells purified from milk[J]. Journal of dairy science, 91(3): 988-998.
[12] Boutinaud M, Herve L, Quesnel H, et al.2019. Review: The cellular mechanisms underlying mammary tissue plasticity during lactation in ruminants[J]. Animals, 13(S1): s52-s64.
[13] Capuco A V, Ellis S E.2013. Comparative aspects of mammary gland development and homeostasis[J]. Annual Review of Animal Biosciences, 1: 179-202.
[14] Chakrabarti R, Celià-Terrassa T, Kumar S, et al.2018. Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche[J]. Science, 360(6396): eaan4153.
[15] Farmer C, Lapointe J, Cormier I .2017. Providing the plant extract silymarin to lactating sows: Effects on litter performance and oxidative stress in sows[J]. Animal: An International Journal of Animal Bioscience, 11(03): 405-410.
[16] Haan C, Kreis S, Margue C, et al.2006. Jaks and cytokine receptors-an intimate relationship[J]. Biochemical Pharmacology[J]. 72(11): 1538-46.
[17] Jaswal S, Anand V, Ali S A, et al.2021. TMT based deep proteome analysis of buffalo mammary epithelial cells and identification of novel protein signatures during lactogenic differentiation[J]. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, 35(6): e21621.
[18] Jena M K, Janjanam J, Naru J, et al.2015. DIGE based proteome analysis of mammary gland tissue in water buffalo (Bubalus bubalis): Lactating vis-a-vis heifer[J]. Journal of Proteomics, 119: 100-111.
[19] Jo S H, Heo W H, Son H Y, et al.2021. S100A8/A9 mediate the reprograming of normal mammary epithelial cells induced by dynamic cell-cell interactions with adjacent breast cancer cells[J]. Scientific Reports, 11(1): 1337.
[20] Li Y, Cao Y, Wang J, et al.2020. Kp-10 promotes bovine mammary epithelial cell proliferation by activating GPR54 and its downstream signaling pathways[J]. Journal of Cellular Physiology, 235(5): 4481-4493.
[21] Rodrigues D S, Lannes-Costa P S, Santos G S, et al.2022. Antimicrobial resistance, biofilm production and invasion of mammary epithelial cells by Enterococcus faecalis and Enterococcus mundtii strains isolated from bovine subclinical mastitis in Brazil[J]. Letters in Applied Microbiology, 75(2): 184-194.
[22] Shillingford J M, Miyoshi K, Robinson G W, et al.2002. JAK2 is an essential tyrosine kinase involved in pregnancy-mediated development of mammary secretory epithelium[J]. Molecular Endocrinology, 16(3): 563-570.
[23] Tong J, Sun Y, Wang Z, et al.2022. Evaluation of biological mechanisms of artemisinin on bovine mammary epithelial cells by integration of network pharmacology and TMT-based quantitative proteomics[J]. Frontiers in Pharmacology, 13: 968149.
[24] Wagner K U, Krempler A, Triplett A A, et al.2004. Impaired alveologenesis and maintenance of secretory mammary epithelial cells in JAK2 conditional knockout mice[J]. Molecular and Cellular Biology, 24(12): 5510-20.
[25] Wang B, Shi L, Men J, et al.2020. Controlled synchronization of prolactin/STAT5 and AKT1/mTOR in bovine mammary epithelial cells[J]. In Vitro Cellular and Developmental Biology-Animal, 56(3): 243-252.
[26] Wang X, Chen H, Bühler K, et al.2022. Proteomics analysis reveals promotion effect of 1α,25-dihydroxyvitamin D3 on mammary gland development and lactation of primiparous sows during gestation[J]. Journal of Proteomics, 268: 104716.
[27] Wu Z, Heng J, Tian M, et al.2020. Amino acid transportation, sensing and signal transduction in the mammary gland: Key molecular signalling pathways in the regulation of milk synthesis[J]. Nutrition Research Reviews, 33(2):287-297.
[28] Zhang J, Ye J, Yuan C, et al.2018. Exogenous H2S exerts biphasic effects on porcine mammary epithelial cells proliferation through PI3K/Akt-mTOR signaling pathway[J]. Journal of Cellular Physiology, 233(10): 7071-7081.
[29] Zhao H, Chen S, Hu K, et al.2021. 5-HTP decreases goat mammary epithelial cells apoptosis through MAPK/ERK/Bcl-3 pathway[J]. Gene, 769: 145240. |
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