摘要
目的:探讨CD40配体(CD40L)通过长链非编码RNA(lncRNA)linc00239对人单核细胞白血病THP-1细胞生物学行为的影响,阐明其可能的作用机制.方法:构建linc00239过表达载体(pcDNA-linc00239)和干扰载体(sh-linc00239),转染至THP-1 细胞,采用实时荧光定量PCR(RT-qPCR)法检测转染效率.THP-1细胞分为对照组、空载体(vector)组、pcDNA-linc00239组、sh-linc00239 组、vector+CD40L 组、pcDNA-linc00239+CD40L 组 和 sh-linc00239+CD40L 组.RT-qPCR法检测各组细胞中linc00239表达水平,CCK-8法检测各组细胞增殖活性,流式细胞术检测各组不同细胞周期细胞百分率和细胞凋亡率,RT-qPCR法和Western blotting法检测各组细胞中B细胞淋巴瘤2(Bcl-2)和Bcl-2关联X蛋白(Bax)mRNA和蛋白表达水平,Western blotting法检测各组细胞中蛋白激酶B(AKT)和磷酸化 AKT(p-AKT)蛋白表达水平并计算 p-AKT/AKT 比值.结果:与vector组比较,pcDNA-linc00239组细胞增殖活性和G2 期细胞百分率明显升高(P<0.05或P<0.01),细胞中linc00239、Bcl-2 mRNA及蛋白表达水平和p-AKT/AKT比值明显升高(P<0.05或P<0.01),G1 期细胞百分率、细胞凋亡率和细胞中Bax mRNA及蛋白表达水平明显降低(P<0.05);与vector组比较,sh-linc00239组和vector+CD40L组细胞增殖活性和G2期细胞百分率明显降低(P<0.05或P<0.01),细胞中linc00239、Bcl-2 mRNA和蛋白表达水平及p-AKT/AKT比值明显降低(P<0.05或P<0.01),G1 期细胞百分率、细胞凋亡率和细胞中Bax mRNA及蛋白表达水平明显升高(P<0.05或P<0.01).与pcDNA-linc00239组比较,pcDNA-linc00239+CD40L组细胞增殖活性和G2期细胞百分率明显降低(P<0.05或P<0.01),细胞中linc00239、Bcl-2 mRNA和蛋白表达水平及p-AKT/AKT比值明显降低(P<0.05或P<0.01),G1 期细胞百分率、细胞凋亡率和细胞中Bax mRNA及蛋白表达水平明显升高(P<0.05或P<0.01);与sh-linc00239组比较,sh-linc00239+ CD40L组细胞增殖活性和 G2 期细胞百分率明显降低(P<0.05 或 P<0.01),细胞中linc00239、Bcl-2 mRNA和蛋白表达水平及p-AKT/AKT比值明显降低(P<0.05或P<0.01),G1 期细胞百分率、细胞凋亡率和细胞中Bax mRNA及蛋白表达水平明显升高(P<0.05或P<0.01).结论:CD40L可通过linc00239抑制THP-1细胞增殖和细胞周期进展,并诱导细胞凋亡.
Abstract
Objective:To discuss the effect of CD40 ligand(CD40L)on the biological behavior of the human monocytic leukemia THP-1 cells through long non-coding RNA(lncRNA)linc00239,and to clarify its potential mechanism.Methods:The linc00239 over-expression vector(pcDNA-linc00239)and interference vector(sh-linc00239)were constructed and transfected into the THP-1 cells.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the transfection efficiency.The THP-1 cells were divided into control group,vector group,pcDNA-linc00239 group,sh-linc00239 group,vector+CD40L group,pcDNA-linc00239+CD40L group,and sh-linc00239+CD40L group.RT-qPCR method was used to detect the expression levels of linc00239 in the cells in various groups;CCK-8 assay was used to detect the proliferation activities of the cells in various groups;flow cytometry was used to detect the percentages of the cells at different cell cycles and the apoptotic rates of the cells in various groups;RT-qPCR and Western blotting methods were used to to detect the expression levels of B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)mRNA and proteins in the cells in various groups;Western blotting method was used to detect the expression levels of protein kinase B(AKT)and phosphorylated AKT(p-AKT)proteins in the cells in various groups,and the ratio of p-AKT/AKT was calculated.Results:Compared with vector group,the proliferation activity of the cells and the percentage of the cells at G2 phase in pcDNA-linc00239 group were significantly increased(P<0.05 or P<0.01),the expression levels of linc00239,Bcl-2 mRNA and protein,and the ratio of p-AKT/AKT were significantly increased(P<0.05 or P<0.01),the percentage of the cells at G1 phase,apoptotic rate,and expression levels of Bax mRNA and protein in the cells were significantly decreased(P<0.05);compared with vector group,the proliferation activity of the cells and percentage of the cells at G2 phase,expression levels of linc00239,Bcl-2 mRNA and protein,and ratio of p-AKT/AKT in the cells in sh-linc00239 group and vector+CD40L group were significantly decreased(P<0.05 or P<0.01),while the percentage of the cells at G1 phase,apoptotic rate,and the expression levels of Bax mRNA and protein in the cells were significantly increased(P<0.05 or P<0.01);compared with pcDNA-linc00239 group,the proliferation activity of the cells and percentage of cells at G2 phase in pcDNA-linc00239+CD40L group were significantly decreased(P<0.05 or P<0.01),the expression levels of linc00239,Bcl-2 mRNA and protein,and ratio of p-AKT/AKT were significantly decreased(P<0.05 or P<0.01),while the percentage of cells at G1 phase,apoptotic rate,and the expression levels of Bax mRNA and protein were significantly increased(P<0.05 or P<0.01);compared with sh-linc00239 group,the proliferation activity of the cells and percentage of cells at G2 phase in sh-linc00239+CD40L group were significantly decreased(P<0.05 or P<0.01),the expression levels of linc00239,Bcl-2 mRNA and protein,and ratio of p-AKT/AKT were significantly decreased(P<0.05 or P<0.01),and the percentage of the cells at G1 phase,apoptotic rate,and expression levels of Bax mRNA and protein were significantly increased(P<0.05 or P<0.01).Conclusion:CD40L can inhibit the proliferation and cell cycle progression of the THP-1 cells through linc00239 and induce the apoptosis.
基金项目
贵州省卫健委科学技术基金项目(gzwjkj2020-1-082)
维普
万方数据