天津医药

天津医药 ›› 2020, Vol. 48 ›› Issue (4): 241-247.doi: 10.11958/20193827

• 细胞与分子生物学 •    下一篇

抑制 BHK21细胞 STYK1/NOK基因的转录组分析 #br#

高婧雅 1，张骊 2，谢炎 2，郭庆军 2，田大治 2，李俊杰 2，蒋文涛 2△，刘力 3△
  

  1. 1天津医科大学一中心临床学院（邮编 300192）；2天津市第一中心医院移植外科；3北京协和医学院中国医学科学院基础医学研究所微生物系
  • 收稿日期:2019-12-19 修回日期:2020-03-05 出版日期:2020-04-15 发布日期:2020-06-23
  • 作者简介:高婧雅（1993），女，硕士在读，主要从事肝癌、细胞通路及凋亡研究
  • 基金资助:
    国家自然科学基金资助项目（81870444）；天津市科技计划项目（17ZXMFSY00040）；天津市自然科学基金（19JCQNJC10300）；天
    津市第一中心医院科技基金（    CL201801）；中国医学科学院医学与健康科技创新工程（2017-I2M-3-007）

Transcriptome sequencing and analysis of the inhibited STYK1/NOK gene in BHK21 cells #br#

GAO Jing-ya1, ZHANG Li2, XIE Yan2, GUO Qing-jun2, TIAN Da-zhi2, LI Jun-jie2, JIANG Wen-tao2△, LIU Li3△ #br#   

  1. 1 Tianjin Medical University First Center Clinical College, Tianjin 300192, China; 2 Department of Liver Transplantation,
    Tianjin First Center Hospital; 3 Department of Microbiology, Institute of Basic Medical Sciences, Chinese Academy of
    Medical Sciences and School of Basic Medicine, Peking Union Medical College
  • Received:2019-12-19 Revised:2020-03-05 Published:2020-04-15 Online:2020-06-23

PDF (PC)

3635

摘要： 目的 转录组分析抑制丝氨酸-苏氨酸-酪氨酸激酶 1（STYK1/NOK）后仓鼠肾正常细胞 BHK21的基因表
达，并分析其可能作用的信号通路。方法 BHK21细胞分为空载体组（转染 6 µg空载体 pBs/U6）、低转染浓度组（2
µg si-STYK1/NOK质粒+4 µg空载体 pBs/U6）和高转染浓度组（6 µg si-STYK1/NOK质粒）。转染 48 h后，Western blot
检测 STYK1/NOK蛋白表达，基于检测结果，选取空载体组和高转染浓度组提取总 RNA进行转录组测序。将测序所
得的序列进行过滤比对，获得差异表达基因后，应用 R软件进行生物功能（GO）分析和 KEGG信号通路分析。应用
STRING蛋白质互作数据库中的互作关系进行差异基因蛋白互作网络的分析。采用 qRT-PCR验证关键差异表达基
因亚甲基四氢叶酸脱氢酶 2（Mthfd2）、转录激活因子 5（Atf5），ⅡA分泌型磷脂酶 A2（Pla2g2a）、6-磷酸果糖-2-激酶/
果糖-2,6-双磷酸酶 3（Pfkfb3）的表达。CCK-8法检测空载体组和高转染浓度组细胞增殖情况。结果 Western blot
结果表明高转染浓度组明显抑制 STYK1/NOK蛋白表达。高转染浓度组与空载体组相比，共发现差异表达基因 44
个，包括 19个上调基因和 25个下调基因。GO富集分析发现差异表达基因主要影响生物调控、细胞进程、代谢调控、
细胞生物过程、细胞、细胞部分、胞外区、配体和催化活性。KEGG通路分析发现差异表达基因主要集中作用于免疫
系统、癌症、细胞周期、信号转导和氨基酸代谢等方面。qRT-PCR结果表明，高转染浓度组Mthfd2、Atf5的相对表达量
显著上调（P＜0.05），Pfkfb3、Pla2g2a的相对表达量显著下调（P＜0.05），与测序结果相一致。细胞增殖实验表明高转
染浓度组细胞增殖明显高于空载体组（P＜0.01）。结论 抑制 BHK21细胞 STYK1/NOK表达后，致使 BHK21细胞原
癌基因表达增强，抑癌基因表达减少，促进细胞增殖。

关键词: 转录组, 基因表达谱, 丝氨酸-苏氨酸-酪氨酸激酶 1, BHK21细胞, 差异表达基因

Abstract: Objective To analyze the gene expression of hamster kidney normal BHK21 cells after inhibiting serine
threonine tyrosine kinase 1 (STYK1/NOK) by transcriptome analysis, and analyze its possible signaling pathways. Methods
BHK21 cells were divided into empty vector group (transfected with 6 µg empty vector pBs/U6), low concentration
transfection group (2 µg si-STYK1/NOK plasmid + 4 µg empty vector pBs/U6) and high concentration transfection group ( 6 µg si-STYK1/NOK plasmid). Western blot assay was used to detect STYK1 / NOK protein expression in 48 hours after
transfection. Based on the detection results, the empty RNA group and high transfection concentration group were selected to extract total RNA for the transcriptome sequencing. The sequences obtained by sequencing were filtered and compared to obtain differentially expressed genes. After obtaining the differentially expressed genes, R software was used to analyze the biological functions of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The interactions in the STRING protein interaction database were used to analyze the differential gene-protein interaction networks. QRT-PCR was used to verify the key differentially expressed genes methylenetetrahydrofolate dehydrogenase 2 (Mthfd2), activating
transcription factor 5 (Atf5), phospholipase A2 group Ⅱ A (Pla2g2a) and 6-phosphofructo-2-kinase/fructose-2, 6-
biphosphatase 3 (Pfkfb3). CCK-8 method was used to detect cell proliferation in empty vector group and high transfection
concentration group. Results Western blot results showed that the expression of STYK1/NOK protein was significantly
inhibited in the high-concentration transfection group. Analysis of the transcriptome sequencing results revealed a total of 44
differentially expressed genes, including 19 up-regulated genes and 25 down-regulated genes in the empty vector group and the high-concentration transfection group. GO enrichment analysis found that the differentially expressed genes mainly
affected biological regulation, cellular processes, metabolic regulation, cellular biological processes, cells, cellular parts,
extracellular regions, ligands and catalytic activity. KEGG pathway analysis found that the differentially expressed genes
were mainly concentrated in the immune system, cancer, cell cycle, signal transduction and amino acid metabolism. The
qRT-PCR results showed that the relative expression levels of Mthfd2 and Atf5 were significantly increased in the high
concentration transfection group (P＜0.05), and the relative expression levels of Pfkfb3 and Pla2g2a were significantly
decreased (P＜0.05), which was consistent with the sequencing results. Cell proliferation experiments showed that the cell
proliferation was significantly higher in the high-concentration transfection group than that in the empty vector group (P＜
0.01). Conclusion The inhibition of STYK1 / NOK expression can result in the enhanced expression of proto-oncogenes,
the promoted expression of tumor suppressor genes and the decreased cell proliferation in BHK21 cells.

Key words: transcriptome, gene expression profiling, serine threonine tyrosine kinase 1, BHK21 cell, differentially
expressed genes

中图分类号: