PDCD4对肝癌细胞生物学功能的影响机制及其在化疗敏感性中的作用

Effect of PDCD4 on the biological function of hepatocellular carcinoma cells and the sensitivity of chemotherapy

目的探讨程序性细胞死亡-4(PDCD4)对肝癌细胞生物学功能的影响机制及其在化疗敏感性中的作用。方法应用q RT-PCR和Western Blot检测肝癌细胞HepG2、SMMC-7721、MHCC97H和正常的肝细胞L02中PDCD4的水平。采用肝癌细胞Hep G2为研究对象,将转染PDCD4过表达载体(p Ds Red2-N1-PDCD4)的HepG2细胞设置为PDCD4过表达组;将60μmol/L替莫唑胺作用24 h的HepG2细胞设置为替莫唑胺组;将正常肝癌细胞Hep G2(不加替莫唑胺)设置为对照组;将转染pDs Red2-N1-PDCD4载体后经60μmol/L替莫唑胺作用24 h的Hep G2细胞设置为联合组。采用噻唑蓝(MTT)法检测各组细胞的存活率,流式细胞仪检测细胞凋亡情况,Western Blot检测细胞中Bax、Bcl-2、MMP-9表达水平。结果 PDCD4在肝癌细胞HepG2、SMMC-7721、MHCC97H中的表达水平均低于正常的肝细胞L02(P﹤0.01),肝癌细胞HepG2中的PDCD4表达水平最低;PDCD4过表达组、替莫唑胺组、联合组的肝癌细胞存活率均明显低于对照组(P﹤0.01);PDCD4过表达组、替莫唑胺组、联合组的肝癌细胞凋亡率均明显高于对照组(P﹤0.01);PDCD4过表达组、替莫唑胺组、联合组肝癌细胞中Bax的表达水平均明显高于对照组(P﹤0.01);Bcl-2、MMP-9的表达水平均明显低于对照组(P﹤0.01);联合组肝癌细胞中Bax的表达水平明显高于PDCD4过表达组和替莫唑胺组(P﹤0.01)。结论 PDCD4在肝癌细胞中低表达,PDCD4可抑制肝癌细胞的增殖,促进肝癌细胞的凋亡,增加肝癌细胞的化疗敏感性,其作用机制可能与Bax、Bcl-2、MMP-9有关。

Objective To investigate the mechanism of programmed cell death-4 （PDCD4） on the biological function of hepatocellular carcinoma （HCC） cells and the sensitivity of chemotherapy. Method The level of PDCD4 in HcpG2, SMMC-7721, MHCC97H and normal L02 cells were detected using qRT-PCR and Western Blot assay. HepG2 were trans-fected with PDCD4 overexpression vector （pDsRed2-N I-PDCD4） to establish the PDCD4 overexpression group; another group of HepG2 cells were treated with 60 pmol/L temozolomide （TMZ） for 24 h as the TMZ group; while the control group was normal HepG2 cells without TMZ： HepG2 cells transfected with pDsRed2-NI-PDCD4 vector after 24 h treat-ment of 60μmol/L TMZ were the combined group. The survival rate of cells in each group was evaluated by MTT, and cell apoptosis was detected by flow cytometry, while expression levels of MMP-9, Bax, Bcl-2 by Western Blot. Result The expression level of PDCD4 in HepG2, SMMC-7721, and MHCC97H cells were significantly lower than that in nor- real hepatic cells L02 （P〈0.01）, and the lowest level of PDCD4 was observed in HepG2. Compared with the control group, the survival rate of the PDCD4 over-expression group, TMZ group, and the combined group were significantly lower （P〈0.01）; And the apoptosis rate in the three groups were higher than that of the control group （P〈0.01）; PDCD4 overexpression group, TMZ group and combined group had higher expression level of Bax （P〈0.01）, but lower expres- sion level of Bcl-2 and MMP-9 than that of the control group （P〈0.01）; While higher expression level of Bax was observed in the combined group than in PDCD4 overexpression group and TMZ group （P〈0.01）. Conclusion The expression of PDCD4 in hepatocellular carcinoma cells is low, while PDCD4 may inhibit the proliferation of HCC cells, while protnote apoptosis and increase the sensitivity to chemotherapy of HCC cells, of which the mechanism may be related to expression ofBax, Bcl-2 and MMP-9 protein.