DcR3表达在肝癌细胞凋亡和增殖中的作用研究

Study of the value of DcR3 expression in the proliferation and anti-apoptosis of hepatoma cells

目的通过体外试验探讨诱骗受体3(DcR3)对肝癌细胞凋亡和增殖的影响。方法构建DcR3慢病毒载体和慢病毒非特异性载体,转染HepG2细胞株后,用荧光显微镜观察绿色荧光蛋白(GFP)判断转染效率。采用蛋白质免疫印迹(Western blot)检测DcR3蛋白表达水平;并绘制生长曲线检测细胞增殖情况;采用荧光定量PCR (qPCR)方法检测细胞凋亡指标半胱天冬酶-3 (caspase 3)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)转录水平变化。结果检测3株肝癌细胞HepG2、MHCC-LM3和MHCC-97H的DcR3蛋白表达水平,发现细胞株HepG2较MHCC-LM3、MHCC-97H中的DcR3蛋白表达水平明显低,差异具有统计学意义(P<0.05)。上调HepG2细胞株的DcR3的mRNA水平后,细胞凋亡指标caspase 3和Bax转录水平明显下降,抗凋亡基因Bcl-2转录水平升高,差异有统计学意义(P<0.05)。当培养到第4天时,LV-DcR3组增殖速度[(39.45±3.61)]与LV-NC组[(25.98±5.34)]比较,差异具有统计学意义(P=0.022);当培养到第6天时,LV-DcR3组增殖速度[(65.84±6.16)]较LV-NC组[(33.34±4.55)],差异有统计学意义(P=0.002)。结论DcR3具有促进肝癌细胞增殖和抗凋亡作用,其机制可能与上调抗癌基因Bcl-2的表达有关。

Objective To analyze the value of decoy receptor 3(DcR3)expression in the proliferation and anti-apoptosis of hepatoma cells by vitro testing.Methods The DcR3 recombinant lentivirus vectors and DcR3 lentiviral non-specific vector were synthesized and transfected to HepG2 cells.The GFP green fluorescent protein were observed by fluorescence microscopy to determine the transfection efficiency.The DcR3 protein levels were analyzed by Western blot.The growth inhibition was examined by the draw growth curve,and qPCR assay was used to detect the changes of transcription levels of caspase 3,Bax and bcl-2.Results The expression levels of DcR3 in HepG2,MHCC-LM3 and MHCC-97 H were detected.The expression of DcR3 in HepG2 cells was significantly lower than that in liver cancer cell line MHCC-LM3 and MHCC-97 H,and which had significant difference(P<0.05).After up-regulating the DcR3 of HepG2 cell line.Compared with LV-DcR3 group,the transcriptional levels of caspase 3 and Bax in LV-DcR3 group were down-regulated significantly,whereas the expression level of Bcl-2 was up-regulated significantly,the difference was statistically significant(P<0.05).When cultured to the 4 th day,the proliferation rate of LV-DcR3 group [(39.45±3.61)]was significantly different from that of LV-NC group [(25.98±5.34)],the difference was statistically significant(P=0.022).When cultured to the 6 th day,the proliferation rate of LV-DcR3 group[(65.84±6.16)]was significantly different from that of the LV-NC group[(33.34±4.55)],the difference was statistically significant(P=0.002).Conclusion DcR3 promotes the proliferation and anti-apoptosis of liver cancer cells through up-regulation of the expression of the anti-oncogene Bcl-2.