KL1在人非小细胞肺癌A549细胞中的作用及其相关机制研究

目的:研究人非小细胞肺癌A549细胞中Klotho基因表达的KL1蛋白的相关功能及其机制?方法:分别用表达KL1蛋白和表达KL蛋白(Klotho基因表达的另一种同源蛋白)的质粒转染非小细胞肺癌细胞株A549后,通过Western blot验证转染结果;MTT法检测细胞的生长情况;平板细胞克隆形成实验检测细胞增殖活性;流式细胞术检测细胞凋亡;Western blot检测细胞转染后对碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)信号通路的中下游靶位点蛋白ERK1/2磷酸化水平的影响?结果:转染48 h后细胞显著表达外源性KL(P < 0.01)?KL1蛋白(P < 0.01)?KL1基因过表达后可以抑制A549细胞生长(P < 0.01);同时显著抑制细胞增殖(P < 0.01),促进细胞凋亡(P < 0.01);KL1过表达能够明显降低bFGF通路中ERK1/2的磷酸化水平(P < 0.01);过表达KL1与过表达KL比较,两者在影响细胞生长?增殖?凋亡和对bFGF信号通路的激活中并无明显统计学差异(P > 0.05)?结论:KL1与KL蛋白均能够抑制肿瘤细胞生长增殖,促进肿瘤细胞凋亡,作用效果相当?KL1能够抑制bFGF信号转导通路,可能是KL中发挥抑制肿瘤作用的主要功能片段?

Effects and related mechanism of KL1 in human non-small cell lung cancer A549 cells

Objective:To study the effects and possible mechanism of action of KL1 protein in human non-small cell lung cancer cells A549. Methods:Western blot was used to detect the overexpression of KL or KL1 in A549 cells transfected with pcDNA3.1-MYC-KL1 or pcDNA3.1-MYC-KL. MTT assay,colony-forming assay and flow-cytometry analysis were designed to determine the change of cell growth,proliferation and apoptosis of A549 cells mediated by KL or KL1, respectively. Effects on the phosphorylation of ERK1/2 in the middle and lower target site of the basic fibroblast growth factor(bFGF) signaling pathway were detected by Western blot after transfection. Results:After transfected 48 h,exogenous KL or KL1 was overexpressed significantly in A549 cells (both P < 0.01). Compared with the control group,the cell growth was inhibited in A549 cells after transfected with KL1 (P < 0.01),the proliferation was remarkably inhibited (P < 0.01)and the apoptosis was promoted significantly(P < 0.01). KL1 overexpression in A549 cells was associated with reduced bFGF-induced phosphorylation of ERK1/2 (P < 0.01). There was no signifcant difference between overexpressed KL and KL1 in cell growth,proliferation and activation of bFGF signal pathway (P > 0.05). Conclusion:The KL and KL1 have a similar signifcant ability to inhibit the growth,proliferation and promote the apoptosis of A549 cells. This may be due to the same ability to inhibit bFGF pathway. Thus,KL1 may be the main functional fragment of the KL protein.