抑制葡萄糖调节蛋白78表达逆转人卵巢癌细胞顺铂耐药机制的分析

Inhibition of GRP78 expression reverses cisplatin resistance in human ovarian cancer

目的探讨抑制葡萄糖调节蛋白78（GRP78）表达对卵巢癌细胞顺铂敏感性的影响及其在卵巢癌顺铂耐药中的作用和机制。方法构建特异性靶向GRP78的siRNA表达质粒，MTT法检测细胞生存率，逆转录多聚酶链链式反应和免疫印迹方法检测内质网应激相关、凋亡相关基因蛋白表达，流式细胞术检测细胞凋亡率。结果顺铂（6mg／L）作用于SKOV3细胞诱导GRP78、CHOP和cleaved—caspase4表达均增高；作用于SKOV3／DDP细胞可诱导GRP78表达明显增高，但CHOP和cleaved—caspase4表达无明显差异。抑制GRP78表达与顺铂联合作用能明显增加SKOV3／DDP细胞cleaved—caspase4和3表达，增加细胞凋亡率。抑制SKOV3／DDP细胞GRP78表达，能降低顺铂诱导的p-Akt和p-mTOR表达上调，增加XBPlmRNA剪切表达和CHOPmRNA表达。结论抑制GRP78表达能与顺铂协同作用增加细胞凋亡的发生而逆转SKOV3／DDP细胞对顺铂的耐药性，其机制可能是通过影响Akt／mTOR信号通路的活性、CHOP表达水平和caspases活性导致的。

Objective To explore the effects of GRP78 suppression on the sensitivity to cisplatin and elucidate the role and mechanism of GRP78 in ovarian cancer cisplatin resistance. Methods The GRP78 siRNA expression plasmid was constructed to suppress GRP78 expression. Cell viability was detected by methyl thiazolyl tetrazolium （MTF） assay. Endoplasmic reticulum stress-related apoptosis related protein expressions were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. And cell apoptosis was detected by flow cytometry. Results The expressions of GRP78, CHOP and cleaved-caspase 4 were induced significantly by cisplatin （6 rag/L） in SKOV3 cells. And the expression of GRP78 was induced significantly by cisplatin in SKOV3/DDP cells. But the expressions of CHOP and cleaved-caspase 4 showed no significant difference. Inhibition of GRP78 expression and cisplatin combined treatment significantly increased the expressions of cleaved-caspase 4 and eleaved-easpase 3 in SKOV3/DDP cells. Inhibition of GRP78 expression reduced the cisplatin-indueed up-regulations of p-Akt and p-mTOR and induced XBP1 mRNA shear expression and CHOP mRNA expression. Conclusion Inhibition of GRP78 expression reverses cisplatin resistance in SKOV3/DDP cells. The mechanism may be through the activity of Akt/mTOR signaling pathway, CHOP expression levels and caspase activity.