基于非标记定量蛋白质组学技术分析消岩汤干预人肺腺癌细胞后蛋白表达谱特征的影响

doi:10.3877/cma.j.issn.2095-3240.2024.01.005

目的

探讨扶正祛瘀解毒法的消岩汤作用于人肺腺癌细胞后对蛋白表达谱特征的影响，并采用生物信息学富集分析差异信号通路。

方法

以消岩汤干预人肺腺癌A549细胞，提取对照组和消岩汤组细胞的总蛋白，利用非标记定量蛋白质组学技术筛选出差异表达蛋白，应用基因本体论(GO)和应用京都基因与基因组百科全书(KEGG)数据库对差异蛋白及信号通路进行富集分析，并采用Western blot对差异蛋白进行验证。

结果

与对照组比较，消岩汤组鉴别出1 217个差异表达蛋白，上调蛋白384个，下调蛋白833个。通过GO分析发现，差异表达蛋白参与了翻译、mRNA剪接、代谢等生物学过程。KEGG数据库分析显示，差异表达蛋白主要参与前20条信号通路，上调最显著的是核糖体通路，下调最显著的是RNA剪接体和MAPK通路。差异表达蛋白参与肿瘤关系密切的为MAPK通路。Western blot结果显示，与对照组比较，消岩汤组PKA、GRB2、ERK蛋白表达明显下调(t＝9.913、9.641、20.060，P均<0.05)，其结果与蛋白组学结果具有一致性。

结论

消岩汤抗肺腺癌作用可能是通过抑制RNA剪接相关的信号及MAPK通路来实现的。

关键词: 肺腺癌, 计算生物学, 蛋白质组学, 消岩汤, MAPK通路

Objective

To explore the effect of Xiaoyan Decoction, a classic prescription of strengtheing the body, removing blood stasis and detoxifying, on the protein expression profile characteristics of human lung adenocarcinoma cells, and to analyze the differentially expressed signaling pathways using bioinformatics enrichment.

Methods

Human lung adenocarcinoma A549 cells were treated with Xiaoyan Decoction, total proteins were extracted from the cells of control group and Xiaoyan Decoction, and the differentially expressed proteins were screened by label-free quantitative proteomics. The differentially expressed proteins and signaling pathways were enriched by applying Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the differentially expressed proteins were verified by Western blot.

Results

Compared with the control group, among the 1 217 differentially expressed proteins, 384 up-regulated proteins and 833 down-regulated proteins were identified in Xiaoyan Decoction group. The GO analysis revealed that the differentially expressed proteins were involved in translation, mRNA splicing, metabolism and other biological processes, and the KEGG database analysis showed that the differentially expressed proteins were mainly involved in the top 20 signaling pathways, with the most significant up-regulation of the ribosomal pathway and the most significant down-regulation of the RNA spliceosome and MAPK pathways. The differentially expressed proteins involved in the tumor were closely related to the MAPK pathway. The results of western blot showed that compared with the control group, PKA, GRB2, and ERK protein expression was significantly down-regulated in the Xiaoyan Decoction group (t=9.913, 9.641, 20.060, all P<0.05), and the results were consistent with the proteomic results.

Conclusion

The effect of Xiaoyan Decoction on lung adenocarcinoma may be realized by inhibiting RNA splicing related signaling pathway and MAPK pathway.

Key words: Adenocarcinoma of lung, Computational biology, Quantitative proteomics, Xiaoyan decoction, MAPK signaling pathway

图1 差异蛋白定量火山图注：up红色为上调基因，down绿色为下调基因，NotSig蓝色为无差异基因；X为消岩汤组；C为对照组

图2 GO显著富集分析柱图。图a为上调蛋白GO显著富集柱图；图b为下调蛋白GO显著富集柱图注：GO为基因本体论

图3 差异蛋白KEGG信号通路富集分析气泡图。图a为上调蛋白；图b为下调蛋白注：KEGG为京都基因和基因组数据库

图4 肿瘤相关MAPK通路差异蛋白表达聚类热图注：样品的聚类是纵向排列，蛋白的聚类则是横向排列，红色表示上调蛋白，蓝色表示下调蛋白，颜色强度表示蛋白质的表达水平；MAPK为丝裂原活化蛋白激酶

图5 KEGG显示MAPK通路蛋白表达差异注：深红色表示上调蛋白，深绿色表示下调蛋白，浅绿色表示无明显变化蛋白；KEGG为应用京都基因与基因组百科全书数据库；MAPK为丝裂原活化蛋白激酶

图6 消岩汤调控MAPK通路差异蛋白表达。图a为2组PKA、ERK及GRB2蛋白条带比较；图b为2组PKA、ERK及GRB2蛋白定量比较注：X为消岩汤组；C为对照组；PKA为蛋白激酶A；ERK为细胞外调节蛋白激酶；GRB2为生长因子受体结合蛋白2

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Analyzing the effect of protein expression profile characteristics after the intervention of Xiaoyan Decoction in human lung adenocarcinoma cells based on label-free quantitative proteomics technology

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Analyzing the effect of protein expression profile characteristics after the intervention of Xiaoyan Decoction in human lung adenocarcinoma cells based on label-free quantitative proteomics technology