原花青素联合Apollon siRNA抑制肝癌HepG_2细胞增殖及凋亡

Effects on proliferation and apoptosis of HepG_2 cell lines by small interfering RNA targeted apollon combined with procyanidin

目的研究原花青素联合凋亡抑制蛋白(inhibitor of apoptosis proteins,IAPs)家族成员Apollon小干扰RNA(small interfer-ence RNA,siRNA)对人肝癌(HepG2)细胞增殖、凋亡的影响。方法设计并合成Apollon siRNA的序列,将一定浓度的人工合成的Apollon siRNA经脂质体包裹转染肝癌(HepG2)细胞,运用实时荧光定量RT-PCR检测Apollon mRNA的表达水平,蛋白质免疫印记技术检测Apollon的蛋白表达水平,并筛选出Apollon siRNA的有效序列,并进一步联合不同浓度的原花青素作用于肝癌(HepG2)细胞48 h后,采用WST-8法检测单用Apollon siRNA、单用原花青素、及Apollon siRNA联合原花青素对肝癌细胞增殖抑制作用,流式细胞术检测细胞早期凋亡率;Hoechst 33258染色观察HepG2细胞凋亡形态。结果设计的三对Apollon siRNA序列,筛选出其中有一对(NO.2)能明显下调Apollon mRNA与蛋白的表达水平,Apollon siRNA联合原花青素作用于HepG2细胞48 h后,单用原花青素的IC50为25.24 mg.L-1;Apollon siRNA与原花青素联合使用,原花青素的IC50为9.99 mg.L-1,增敏倍数为2.53,表现为协同作用。流式细胞术检测结果显示:Apollon siRNA联合原花青素能促进HepG2细胞凋亡,其早期凋亡率达25.2%;经荧光显微镜观察,Apollon siRNA组可见核高强度荧光的细胞,并见凋亡小体。结论 Apollon siRNA可增强肝癌细胞对原花青素的敏感性,作用机制可能为共同促进肝癌细胞早期凋亡有关,为临床上肝癌的治疗提供理论基础。

Objeetive To study the effect of small interfering RNA targeted on apollon for enhancing the procyanidin sensitivity of human liver cancer cells and its possible acting mechanism. Methods Chemosynthetic small interfering RNA targeted on apollon was transfect- ed to the cells using Lipofectamine TM 2000. The apollon protein expression levels were detected by western blotting. The expression of apollon mRNA was analyzed by real time fluorescent quantitative reverse transcription polymerase chain reaction. The inhibitory effects of procyanidin and small interfering RNA targeted on apollon （apollon siRNA） , used singly or in combination, on cell proliferation were de- tected by WST-8. Their inhibition rate and IC50 were calculated. The morphology of apoptotic cells was examined by fluorescence micro- scope after Hoechst 33258 staining. The percentage of apoptosis cells were determined by flow cytometry. Results Used in combination with apollon siRNA, the ICs0 of procyanidin could be lowered from 25.24 mg·L^-1 to 9.99 mg·L^-1, and the sensitivity of cells to procy- anidin increased to 2.53. The amount of apoptotic ceils increased significantly. The early apoptotic rate was 25.2%. The cells showed typical features of apoptosis, including high-intensity fluorescence, apoptotic body. Transfection with apollon siRNA （ NO. 2 ） alone or combind with matrine could down-regulate the expression of apollon mRNA and protein in ceils. Conclusions Combined use of apollon siRNA could increase the sensitivity of HepG2 cells to procyanidin. The mechanism may work together to promote early apoptosis of liver cancer cells, which provides a novel potential approach for treatment of human liver cancer.