作者：李全营1，唐红娜1，秦长江1，翟二涛2

单位：1. 河南大学淮河医院 普通外科，河南 开封 475000；2. 中山大学附属第一医院 胃肠外科中心，广东 广州 510080

Authors: Li Quanying1, Tang Hongna1, Qin Changjiang1, Zhai Ertao2

Unit: 1.Department of General Surgery, Huaihe Hospital, Henan University, Kaifeng 475000, China；2.Department of Gastrointestinal Surgery, the First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, Guangzhou, China

摘要：

目的 探讨应激诱导磷酸化蛋白1 (STIP1)表达对于胃癌化疗抵抗的影响,并进一步探讨其潜在机制。方法 构建STIP1过表达及敲低胃癌细胞系后,应用CCK-8、单克隆形成等实验检测胃癌细胞对5-氟尿嘧啶及顺铂、奥沙利铂的敏感性;并对过表达STIP1的胃癌细胞系进行RNA测序并验证获得STIP1所调控的下游分子及信号通路;最后应用免疫荧光、免疫印迹等方法检测STIP1对胃癌细胞化疗抵抗的影响是否依赖于其下游分子。结果 STIP1过表达可显著增强胃癌细胞对于5-氟尿嘧啶及顺铂、奥沙利铂等细胞毒性药物的抵抗作用;并可促进化疗抵抗相关基因的表达;RNA表达谱测序发现STIP1可能促进重组修复相关信号通路的活化,而免疫印迹及qPCR等检测证实STIP1过表达可促进该通路相关基因的表达;免疫荧光检测进一步证实STIP1表达可影响5-氟尿嘧啶诱导的DNA损伤的重组修复能力;而应用重组修复抑制剂干预胃癌细胞后。免疫荧光检测获得STIP1表达对于5-氟尿嘧啶所诱导的胃癌细胞DNA损伤的影响主要是通过影响其同源重组修复途径。对于其下游机制的探索,通过生物信息学分析、免疫印迹、qPCR及免疫荧光,证实STIP1可调控EGR1表达进一步影响5-氟尿嘧啶所诱导的DNA损伤重组修复。结论 STIP1调控EGR1表达影响胃癌细胞DNA损伤修复,进而促进其化疗抵抗。

关键词：应激诱导磷酸化蛋白1;  早期生长反应基因1;  DNA损伤修复;  胃癌

Abstract：

Objective To investigate the effect and potential mechanism of stress-induced phospho[1]rylated protein 1 (STIP1) on chemotherapy resistance of gastric cancer. Method Gastric cancer cells with STIP1 overexpression and knockdown were constructed, and then the chemotherapy sensitivity of these cells to 5 -fluorouracil, cisplatin and oxaliplatin was detected by CCK - 8 proliferation assays and monoclonal formation assays. RNA sequence was employed to obtain the downstream molecules and signal pathways regulated by STIP1 and qPCR and western blotting were used for further validation. Finally, immunofluorescence and western blotting were used to detect whether the effect of STIP1 on chemotherapy resistance of gastric cancer cells depended on the downstream molecules. Result Overexpression of STIP1 significantly enhanced the resistance of gastric cancer cells to 5-fluorouracil, cisplatin, oxaliplatin and other cytotoxic drugs. It can also promote the expression of chemotherapy -resistance related genes. RNA sequence revealed that overexpression of STIP1 could activate the pathways related to DNA recombination and repair, immunoblotting and qPCR further confirmed that STIP1 could promote the expression of genes related to this pathway. Immunofluorescence assay further confirmed that the expression of STIP1 could affect the recombination repair ability of DNA damage induced by 5 - fluorouracil . After using recombinant repair inhibitors to intervene in gastric cancer cells, we found the effect of STIP1 on DNA damage was mainly focused on the activation of homologous recombination repair pathway. As for the exploration of its downstream mechanism, bioinformatics analysis, western blot, qPCR and immunofluorescence were employed and confirmed that STIP1 could regulate the expression of EGR1 and further affect the DNA damage recombination repair induced by 5 -fluorouracil. Conclusion STIP1 can promote chemotherapy resistance of gastric cancer by regulating the expression of EGR1 and influencing DNA repair.

Key Words: Stress -Induced Phosphoprotein 1; early growth response gene 1; DNA damage repair; Gastric cancer