TXNDC5蛋白在胃癌细胞及组织中相互作用蛋白的免疫共沉淀验证

Verification of the interaction protein of TXNDC5 protein in gastric cancer by immunocoprecipitation

目的 我们前期通过串联亲和偶联蛋白质组学技术成功地筛选出胃癌细胞SGC7901中与TXNDC5相互作用的蛋白质,在这些蛋白质中除去功能意义较为明确的蛋白,TXNIP、PRDX2、PDCD4和ADIPOR1可能在肿瘤发生发展中具有一定作用,但其具体分子作用机制尚不清楚。本研究进一步验证在胃癌组织和细胞中TXNDC5蛋白与上述蛋白的关键相互作用。方法 以pcDNA3.1为基础构建pcDNA3.1-TXNDC5真核表达载体,并以此载体瞬时转染人胃癌SGC7901细胞,RT-PCR法及Western blotting法检测TXNDC5蛋白表达。采用免疫共沉淀(Co-immunoprecipitation,Co-IP)法验证TXNDC5高表达细胞系中其与TXNIP、PRDX2、PDCD4、ADIPOR1的相互作用。取人胃腺癌新鲜手术标本提取总蛋白,采用Western blotting法检测TXNDC5蛋白表达,选取确认为TXNDC5高表达的胃癌组织总蛋白采用Co-IP验证TXNDC5与TXNIP、PRDX2、PDCD4、ADIPOR1的相互作用。结果 通过Co-IP法验证,TXNDC5高表达胃癌细胞系中,TXNDC5与TXNIP、PRDX2、PDCD4存在相互作用;TXNDC5高表达胃癌组织中,TXNDC5与TXNIP、PDCD4存在相互作用。结论 本研究通过Co-IP技术成功地验证了胃癌细胞和组织中TXNDC5与TXNIP、PDCD4存在相互作用,提示TXNDC5在胃癌中的促癌分子机制可能通过与TXNIP和PDCD4相互作用影响其相关信号通路有关。

Objective To screen the proteins interacting with TXNDC5 in gastric cancer cell line SGC7901 by tandem affinity coupled proteomics( TAP),and to remove the proteins with clear functional significance.TXNIP,PRDX2,PDCD4 and ADIPOR1 may play roles in the occurrence and development of tumor,but their specific molecular mechanism was not clear.This research aimed to further verify the key interaction between TXNDC5 and the above proteins in the gastric cancer tissues and cells.Methods pcDNA3.1-TXNDC5 eukaryotic expression vector was transiently transfected into human gastric cancer SGC7901 cells.The expression of TXNDC5 protein was detected by RTPCR and Western blotting.Co-immunoprecipitation(Co-IP) was used to verify the interaction between TXNDC5 and TXNIP,PRDX2,PDCD4,ADIPOR1.The total protein was extracted from fresh surgical specimens of human gastric adenocarcinoma,and the expression of TXNDC5 protein was detected by Western blotting.The interaction between TXNDC5 and TXNIP,PRDX2,PDCD4,ADIPOR1 was verified by Co-IP.Results Co-IP results showed that TXNDC5 interacted with TXNIP,PRDX2 and PDCD4 in gastric cancer cell lines with high expression of TXNDC5,and TXNDC5 interacted with TXNIP and PDCD4 in gastric cancer tissues with high expression of TXNDC5.Conclusion This study successfully verified the interaction between TXNDC5 and TXNIP,PDCD4 in gastric cancer cells and tissues through Co-IP technology,and proposed that the cancer promoting molecular mechanism of TXNDC5 in gastric cancer may be related to its related signal pathways through the interaction with TXNIP or PDCD4.