稳定敲低Notch3基因H1299细胞系的建立及其对糖代谢调节的作用  

Establishment and effect of stable knockdown Notch3 gene in H1299 cells on glucose metabolism regulation

作　　者：韩芳竹 肖金阳 刘尨彧 李春艳 宋晓宇 HAN Fang-zhu;XIAO Jin-yang;LIU Meng-yu;LI Chun-yan;SONG Xiao-yu(Institute of Translational Medicine,China Medical University,Shenyang 110112,China;Department of Biological Science and Biotechnology,China Medical University,Shenyang 110112,China;Science Experimental Center,China Medical University,Shenyang 110112,China)

机构地区：[1]中国医科大学转化医学研究院,辽宁沈阳110112 [2]中国医科大学生物科学与生物技术系,辽宁沈阳110112 [3]中国医科大学科学实验中心,辽宁沈阳110112

出　　处：《解剖科学进展》2024年第6期661-663,667,共4页Progress of Anatomical Sciences

基　　金：辽宁省教育厅基金(LK201617)。

摘　　要：目的通过RNA干扰技术敲低非小细胞肺癌H1299细胞的Notch3基因表达,初步探讨敲低Notch3对H1299细胞糖代谢的影响。方法利用表达Notch3 shRNA的慢病毒包装质粒和嘌呤霉素(puromycin)抗生素筛选、构建Notch3敲低的H1299细胞系,通过Western blot确认敲减细胞系的建立;CCK-8检测细胞增殖,验证敲减对细胞功能的影响;糖酵解压力测试试剂和Seahorse能量仪检测细胞外酸化率;Western blot检测糖酵解关键酶己糖激酶(HK2)、丙酮酸激酶M2(PKM2)蛋白表达;JC-1染色和流式细胞仪检测线粒体膜电位。结果基因敲减后,Notch3蛋白表达水平降低,抑制细胞增殖,表明成功建立Notch3敲减细胞系。Notch3敲减降低线粒体膜电位,提高HK2和PKM2的表达水平和细胞外酸化率。结论Notch3稳定敲低细胞系构建成功,Notch3敲低抑制H1299细胞的增殖,促进细胞的糖酵解。Objective To investigate the effect of Notch3 knockdown on glucose metabolism in H1299 cells of non-small cell lung cancer(NSCLC)by RNA interference.Methods Lentiviral plasmid expressing Notch3 shRNA and puromycin were used to screen and construct H1299 cell lines with Notch3 knockdown.Western blot was used to confirm the establishment of knockdown cell lines.Cell proliferation was detected by CCK-8 to verify the effect of knockdown on cell function.The extracellular acidification rate was detected by glycolysis pressure test reagent and Seahorse energy instrument.Western blot was performed to detect the protein expression of the key enzyme hexokinase2(HK2)and pyruvate kinase M2(PKM2).Mitochondrial membrane potential was detected by JC-1 staining and flow cytometry.Results After gene knockout,the expression of Notch3 was decreased,which cell proliferation was inhibited,indicating that the Notch3 knockout cell line was successfully established.Notch3 knockdown decreased mitochondrial membrane potential,increased the expressions of HK2 and PKM2 and the rate of extracellular acid acidification.Conclusion The stable knock down cell line of Notch3 was successfully constructed,which provided a powerful tool for studying the regulatory effect of Notch3 on cell function.Notch3 knockdown inhibited the proliferation of H1299 cells and promoted glycolysis.

关 键 词：非小细胞肺癌 H1299细胞 NOTCH3 糖酵解 

分 类 号：R734.2[医药卫生—肿瘤]