敲低JAK2基因表达对猪乳腺上皮细胞增殖和凋亡的影响
Effects of Knockdown with JAK2 gene Expression on Proliferation and Apoptosis of Porcine Mammary Epithelial Cells
罗杰 1杨酸 2毛同辉 3杨棋 1张依裕2
作者信息
- 1. 铜仁职业技术学院,铜仁 554300
- 2. 贵州大学动物科学学院/高原山地动物遗传育种与繁殖教育部重点实验室,贵阳 550025
- 3. 铜仁职业技术学院,铜仁 554300;铜仁市畜牧技术推广站,铜仁 554300
- 折叠
摘要
Janus激酶2(Janus kinase,JAK2)是一种激素受体偶联激酶,参与母牛(Bos taurus)乳腺上皮细胞增殖、分化、凋亡等过程,而其调控猪(Sus scrofa)乳腺上皮细胞的增殖或凋亡机制尚不清楚.为探究RNA干扰JAK2基因表达对猪乳腺上皮细胞增殖和凋亡的影响,本研究根据JAK2基因序列设计并合成4对shRNA(short hairpin RNA)干扰序列和1对阴性对照序列,构建shRNA表达载体并将其转染至猪乳腺上皮细胞中,筛选干扰效果最佳的载体,采用串联质谱标记(tandem mass tags,TMT)定量蛋白质组学技术进行蛋白组测序并筛选差异蛋白,分别采用CCK-8细胞增殖检测试剂盒、流式细胞术、qRT-PCR检测细胞增殖、凋亡、周期及相关基因的mRNA表达水平.结果显示,共筛选到差异表达蛋白699个,其中上调蛋白309个,下调蛋白390个.GO富集分析表明,差异蛋白主要富集在多肽生物合成过程和酰胺生物合成过程,以及碳水化合物衍生物结合、核糖体的结构组成等分子功能方面;KEGG富集分析可知,差异蛋白主要富集在核糖体、补体系统和丙型肝炎信号通路.干扰JAK2后,细胞增殖能力相较于对照组在不同时间节点均显著降低(P＜0.05),在72h时的OD450值极显著降低(P＜0.01),细胞凋亡率(24.0%±3.82%)显著低于对照组(33.2%±2.55%)(P＜0.05),且诱导细胞周期G1/S期阻滞;IFIT1(interferon-induced protein with tetratricopeptide repeats 1)、ISG15(interferon-stimulated gene 15)、GRB2(growth factor receptor-bound protein 2)和RACK1(receptor for activated C kinase 1)基因表达水平在沉默JAK2后极显著低于对照组(P＜0.01),RSAD2(radical S-adenosyl methionine domain containing 2)基因在沉默JAK2后极显著高于对照组(P＜0.01).综上,沉默JAK2可诱导猪乳腺上皮细胞周期G1/S期阻滞,从而抑制细胞增殖,并抑制细胞凋亡,B6E241蛋白(对应基因GRB2)可作为今后研究乳腺上皮细胞增殖相关的关键蛋白.本研究为深入探讨母猪乳腺发育和泌乳调控机制提供了基础资料.
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.
关键词
Janus激酶(JAK2)/猪乳腺上皮细胞/细胞增殖/细胞凋亡Key words
Janus kinase 2(JAK2)/Porcine mammary epithelial cells/Cell proliferation/Cell apoptosis引用本文复制引用
基金项目
贵州省生猪产业技术体系健康养殖功能实验室建设项目(GZSZCYJSTX-03)
贵州省科技支撑计划(黔科合支撑[2021]一般147号)
贵州省优秀青年科技人才培养计划黔科合平台人才([2021]5630号)
贵州省科技计划(黔科合支撑[2020]1Y038号)
贵州省千层次创新型人才培养项目(2024-[2023]-082号)
贵州省教育厅高校科技创新团队(黔教技[2023]097号)
铜仁职业技术学院畜牧兽医专业群技术技能平台项目([2022]02号)
出版年
2024
国家科技期刊平台
NSTL
万方数据