Suppression of p53R2 gene expression with specific siRNA sensitizes HepG2 cells to doxorubicin
Suppression of p53R2 gene expression with specific siRNA sensitizes HepG2 cells to doxorubicin
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DOI：
10.1016/j.gene.2017.11.008
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发表时间：
2018-02-05
期刊：
影响因子：
3.5
通讯作者：
Yousefi, Bahman
中科院分区：
文献类型：
--
作者：
Azimi, Ako;Majidinia, Maryam;Yousefi, Bahman
前言：p53R2是一种P53诱导蛋白，它通过提供dNTPs来响应DNA损伤，从而促进DNA修复。P53R2在癌细胞和恶性肿瘤中的作用仍然存在争议。在此，我们研究了在阿霉素存在和不存在的情况下，p53R2沉默对人肝癌细胞株(HHC)(野生型p53)存活率、细胞凋亡率和细胞周期的影响。实时荧光定量聚合酶链式反应和蛋白质印迹分析检测基因沉默。用台盼蓝拒染法检测转染后细胞的生长情况。采用四甲基偶氮唑盐比色法检测阿霉素与p53R2 siRNA单独或联合作用对HepG2细胞的杀伤作用，并通过计算联合指数评价联合用药的效果。用碘化丙啶(PI)流式细胞仪分析细胞周期不同阶段的作用，DNA-组蛋白ELISA法检测细胞凋亡。结果：p53R2单独沉默对HepG2细胞的生长抑制和自发性凋亡有很强的抑制作用。P53R2 siRNA协同增强阿霉素的细胞毒作用。此外，当与阿霉素(0.4 mU M)联合使用时，细胞凋亡率显著增加(P<；0.05)。与单用阿霉素相比，p53R2联合治疗后细胞周期处于S期和G2/M期的比例较低。结论：siRNA介导的p53R2沉默在化疗中具有潜在的治疗和辅助作用。
Introduction: p53R2 is a p53-inducible protein that contributes to DNA repair by providing dNTPs in response to DNA damage. The roles of p53R2 in cancer cells and malignancies still remain controversial. Herein, we examined the effects of p53R2 silencing on HepG2 human hepatocellular carcinoma (HHC) cell line (wild-type p53) viability, apoptosis and cell cycle arrest in the presence and absence of doxorubicin.Methods: Cell transfection was performed using a liposomal approach. Gene silencing was determined by quantitative real-time PCR and western blot analysis. To evaluate the cell growth rate after transfection, trypan blue dye exclusion assay was employed. The cytotoxicity of the doxorubicin and p53R2 siRNA as single agents or in combination against HepG2 cell was analyzed by MTT assay and the drug combination effects was evaluated by calculating the combination index. The effects of treatments on different stages of cell cycle were analyzed by flow cytometry using propidium iodide (PI) and induction of apoptosis was assessed using DNA-histone ELISA.Results: We found that silencing of p53R2 alone had a strong effect on growth inhibition and spontaneous apoptosis in HepG2 cells. p53R2 siRNA synergistically enhanced the cytotoxic effect of doxorubicin. Furthermore, when used in combination with doxorubicin (0.4 mu M), a significant increase in the rate of apoptosis was observed (P < 0.05). Moreover, cell cycle at S and G2/M phases progressed at a lower rate after p53R2 combination treatment compared with dcntorubicin mono-therapy.Conclusion: These findings suggest that siRNA-mediated silencing of p53R2 has great potential as a therapeutic tool and adjuvant in chemotherapy.