Effect of soluble CD40 ligand on biological behavior of THP-1 cells through long non-coding RNA linc00239 / 吉林大学学报(医学版)

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.