Guo Hong-Yan, Xu Ya-Ru, Li Geng-Hui, Sun Xiao-Jie, Zhao Zheng-Lin, Wu Qi, Liu Bo
Department of Biochemistry, Qiqihar Medical College.
The Clinical laboratory, the First Affiliated Hospital of Qiqihar Medical College.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2022 Jan;38(1):53-57. doi: 10.12047/j.cjap.6206.2022.010.
To investigate the effects of long-chain noncoding RNA Linc00673 overexpression on proliferation and apoptosis of gastric cancer cells and its mechanisms. The recombinant lentivirus expressing plasmid pLVX-Linc00673 and the control empty plasmid pLVX-NC were packaged and amplified in 293T cells, and the recombinant lentivirus was transfected into gastric cancer cell line MGC-803 to establish a cell line stably overexpressing Linc00673. The expression of Linc00673 gene was detected by real-time fluorescence quantitative PCR. The growth and proliferation of cells were observed by MTT assay and clone formation assay. Cell cycle and apoptosis were detected by flow cytometry. The expressions of cell cycle related regulatory genes were detected by qPCR. The expressions of key molecules in the PI3K/Akt signaling pathway and tumor proliferation related proteins were detected by Western blot. The expressions of Linc00673 in gastric cancer cell line MGC-803, BGC-823 and AGS were significantly higher than that in normal gastric mucosa cell line GES-1 (<0.05). MGC-803 cell line with stable overexpression of LINC00673 was established, and the expression level of LincC00673 was 200 times higher than that of the control empty carrier group. Overexpression of Linc00673 promoted proliferation of MGC-803 cells (<0.05) and clone formation (<0.05), inhibited cell apoptosis and affected the G1→S phase progression of cell cycle (<0.01). Overexpression of Linc00673 could affect the expressions of cell cycle regulatory gene CCNG2, P19 and CDK1 in MGC-803. Western blot showed that Linc00673 overexpression not only promoted the expressions of the key molecule pAkt in PI3K / Akt signaling pathway and its downstream target NF-κ B and Bcl-2 protein, but also up regulated the expressions of tumor related factors β-catenin and EZH2 proteins. Overexpression of Linc00673 may promote proliferation and inhibit apoptosis of MGC-803 cells through PI3K/Akt signaling pathway.
探讨长链非编码RNA Linc00673过表达对胃癌细胞增殖和凋亡的影响及其机制。将表达重组慢病毒的质粒pLVX-Linc00673和对照空质粒pLVX-NC在293T细胞中包装并扩增,然后将重组慢病毒转染至胃癌细胞系MGC-803中,以建立稳定过表达Linc00673的细胞系。通过实时荧光定量PCR检测Linc00673基因的表达。采用MTT法和克隆形成试验观察细胞的生长和增殖情况。通过流式细胞术检测细胞周期和凋亡情况。通过qPCR检测细胞周期相关调控基因的表达。通过蛋白质免疫印迹法检测PI3K/Akt信号通路中的关键分子及肿瘤增殖相关蛋白的表达。胃癌细胞系MGC-803、BGC-823和AGS中Linc00673的表达明显高于正常胃黏膜细胞系GES-1(<0.05)。成功建立了稳定过表达LINC00673的MGC-803细胞系,LincC00673的表达水平比对照空载体组高200倍。Linc00673过表达促进了MGC-803细胞的增殖(<0.05)和克隆形成(<0.05),抑制了细胞凋亡,并影响了细胞周期从G1期到S期的进程(<0.01)。Linc00673过表达可影响MGC-803中细胞周期调控基因CCNG2、P19和CDK1的表达。蛋白质免疫印迹法显示,Linc00673过表达不仅促进了PI3K/Akt信号通路中关键分子pAkt及其下游靶点NF-κB和Bcl-2蛋白的表达,还上调了肿瘤相关因子β-连环蛋白和EZH2蛋白的表达。Linc00673过表达可能通过PI3K/Akt信号通路促进MGC-803细胞的增殖并抑制其凋亡。