消岩汤通过HSPA5逆转肺腺癌耐药的研究

Xiaoyan Decoction(消岩汤)Reverses Drug Resistance of Lung Adenocarcinoma through HSPA5

目的分析肺腺癌耐药细胞株A549/DDP在中药复方消岩汤作用后细胞的差异蛋白并寻找相关耐药靶点。方法通过Label-free蛋白组学筛选出两组关于耐药方面的差异蛋白,利用GO富集分析,PPI蛋白相互作用网络图的构建筛选核心蛋白,Western blot及qRT-PCR方法验证核心蛋白及基因的表达;利用CCK8法检测A549/DDP细胞增殖活力情况,利用流式细胞术检测A549/DDP细胞凋亡情况。在上调HSPA5蛋白表达的基础上,检测消岩汤对A549/DDP细胞增殖及凋亡的影响,以及A549/DDP细胞对顺铂耐药性的影响。结果在中药复方消岩汤作用后的A549/DDP细胞蛋白质谱中找到29种耐药相关差异蛋白质,其中14种上调,15种下调。通过PPI蛋白网络互做分析得出核心蛋白,选择HSPA5进行体外实验验证,结果显示与对照组相比,中药复方消岩汤可以降低A549/DDP细胞中HSPA5蛋白及基因的表达,与蛋白组学结果一致。CCK8及细胞凋亡实验显示,消岩汤可以抑制细胞增殖,促进细胞凋亡,增强耐药细胞对顺铂的敏感性;上调HSPA5蛋白表达,消岩汤抑制细胞生长及增强细胞对顺铂敏感性的能力减弱。结论HSPA5可作为肺腺癌临床治疗中的一个潜在靶点,消岩汤可以通过下调HSPA5的表达抑制肺腺癌细胞的生长,并逆转其对顺铂的耐药性。

Objective To investigate the related differential proteins in the A549/DDP lung cancer cell line after the treatment of Xiaoyan Decoction(消岩汤)and find out the drug resistance target of Xiaoyan Decoction in reversing lung adenocarcinoma.Methods Label-free proteomics was used to filter the differential proteins.Core proteins were filtered by construction of GO enrichment analysis and PPI protein interaction network.The core protein and gene expression were verified by Western blot and qRT-PCR methods.CCK8 assay was used to detect the cell proliferation and cell viability.Flow cytometry was used to detect the cell apoptosis.On the basis of up-regulated HSPA5,the effects of Xiaoyan Decoction on the proliferation and apoptosis of A549/DDP cells and cisplatin resistance were detected.Results Twenty-nine differential proteins associated with drug resistance were found using protein mass spectrometry in A549/DDP cells treated by Xiaoyan Decoction.Among them,14 proteins were over expressed and 15 proteins were down regulated.HSPA5 protein was selected for subsequent validation after the PPI protein interaction network analysis.The protein and mRNA expressions of HSPA5 in A549 cells after treatment with Xiaoyan Decoction were lower than those of the control group.These results were consistent with proteomic result.The experimental results of CCK8 and apoptosis showed that Xiaoyan Decoction could inhibit cell proliferation,promote apoptosis and enhance the sensitivity of drug-resistant cells to cisplatin.On the basis of HSPA5 upregulation,the ability of Xiaoyan Decoction to inhibit A549/DDP cell growth and enhance the sensitivity of resistant cells to cisplatin was weakened.Conclusion HSPA5 can be used as a potential target for clinical treatment of lung adenocarcinoma.Xiaoyan Decoction can inhibit the lung adenocarcinoma cell growth and reverse its resistance to cisplatin by down-regulating the expressions of HSPA5.