转录组测序结合蛋白组学技术筛选肝癌转移基因  

Identification of genes related to hepatocellular carcinoma metastasis by a combined transcriptomics and proteomics approach

作　　者：秦荔荣[1] 周怡[2] 邓小芳[3] 李洪涛[2] 臧宁[2] 何敏[3,4] 

机构地区：[1]广西医科大学第一附属医院消化内科,广西壮族自治区南宁市530021 [2]广西医科大学医学科学实验中心,广西壮族自治区南宁市530021 [3]广西医科大学公共卫生学院,广西壮族自治区南宁市530021 [4]区域性高发肿瘤早期防治研究教育部重点实验室,广西壮族自治区南宁市530021

出　　处：《世界华人消化杂志》2015年第13期2050-2057,共8页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;Nos.81260445;30960332;区域性高发肿瘤早期防治研究教育部重点实验室研究基金资助项目;Nos.GK2013-13-A-01-02;GK2014-ZZ04~~

摘　　要：目的:通过高通量转录组测序和相对定量血清蛋白组学技术筛选肝癌转移相关的关键基因.方法:利用Ion Proton.测序仪比较人肝癌细胞株(Smmc-7721)和正常人肝细胞株(L-02)的差异表达基因,对差异表达基因进行聚类分析、GO功能注释和富集分析,获得肝癌细胞转移相关基因;收集10例肝细胞癌(hepatocellular carcinoma,HCC)患者血清及匹配10例正常人血清,通过相对和绝对定量的同位素标记(isobaric tags for relative and absolute quantitation,iTRAQ)联合基质辅助激光解吸电离串联飞行时间质谱(ma t r ixassisted laser desorption/ionization tandemtime of flight mass spectrometry,MALDITOF/MS)检测肝癌与正常对照组血清差异表达蛋白;将两组学结果进行交集分析,确定肝癌转移关键基因,并在76例HCC和癌旁组织中进行免疫组织化学验证.结果:对肝癌细胞及正常肝细胞进行转录组测序分析,共得到肝癌细胞差异表达基因618个,生物信息学分析差异表达基因主要聚集在转移相关、转录因子相关、氧化还原过程等14个分子功能,其中转移相关功能基因所占比例最大,达15.05%(93/618);血清蛋白组学分析共得到69个肝癌血清差异表达蛋白,其中在肝癌组上调33个,下调36个;将转录组测序筛选的与转移功能相关基因与蛋白质组学进行交集分析,发现有3个共同交集的差异因子,其中差异最明显的是肝癌中表达上调的热休克蛋白90 AA1(heat shock protein 90 AA1,HSP90AA1);免疫组织化学验证结果显示,HSP90α表达强阳性在门脉转移和无门脉转移HCC组织中的比例分别为66.7%(16/24)和25%(13/52)(P<0.005),HSP90α在有门脉转移的肝癌组织表达明显增高.结论:利用转录组结合血清蛋白组策略,发现HSP90AA1可能是在肝癌转移重要基因.AIM： To screening key genes related to hepatocellular carcinoma （HCC） metastasis by high-throughput transcriptomics sequencing and serum proteomics. METHODS： Differentially expressed genes between liver cancer cells Smmc-7721 and normal liver cells L-02 were analyzed by Ion ProtonTM high-throughput sequencing. Bioinformatics methods were used to perform GO annotation, clustering and enrichment analysis. Ten serum samples from HCC patients and 10 normal serum samples were recruited to detect the differential protein expression by isobaric tags for relative and absolute quantitation （iTRAQ） and matrix- assisted laser desorption/ionization tandem time of flight mass spectrometry （MALDI- TOF/MS）. The transcriptomics data and serum proteomics data were analyzed together to screen key genes related to HCC metastasis. Then, a screened key gene was verified by immunohistochemistry in 76 HCC and adjacent tissues. RESULTS： A total of 618 differentially expressed genes （DEGs） in liver cancer cells were identified by transcriptome sequencing,and the gene functions were enriched in 14 terms, including metastasis process, transcription and REDOX process, among which metastasis process owned the most DEGs [15.05% （93/618）]. The proteomics data showed that a total of 69 differentially expressed proteins in HCC were detected, including 33 up-regulated and 36 down-regulated ones. Combination analysis found three common factors in transcriptomics and proteomics, among which heat shock protein 90 AA1 （HSP90AA1） was up-regulated in HCC and presented the most significant ratio. According to the immunohistochemical results, the strongly positive rates of HSP90α in HCC with portal vein metastasis and without were 66.7% （16/24） and 25% （13/52）, respectively （P 〈 0.005）. HSP90α was overexpressed in HCC with portal vein metastasis.CONCLUSION： Transcriptomics and proteomics analysis revealed that HSP90AA1 might be a key gene related to HCC metastasis.

关 键 词：肝细胞癌 血清蛋白组学 转录组测序 HSP90AA1 转移 

分 类 号：R735.7[医药卫生—肿瘤]