人原发性肺腺癌转移相关分子的定量蛋白质组学研究

Quantitative Proteomic Analysis of Metastasis-Associated Proteins in Human Primary Lung Adenocarcinoma

癌细胞转移是人原发性肺腺癌(lung adenocarcinoma,AdC)死亡率高和预后差的主要原因.为了筛选潜在的肺腺癌转移相关分子标志物,依据临床诊断选取无转移的肺腺癌组织和有转移的肺腺癌组织作为研究对象,首先采用激光捕获显微切割技术(laser capture microdissection,LCM)对两组肺腺癌组织中的癌细胞进行纯化,再利用荧光差异凝胶电泳技术(two-dimensional differential in-gel electrophoresis,2D-DIGE)分离无转移肺腺癌组和有转移肺腺癌组的癌细胞总蛋白,通过Decyder软件分析两组差异表达的蛋白质点,质谱(mass spectrometry,MS)对差异表达的蛋白质点进行鉴定,Western blot验证部分差异蛋白annexin A1,annexin A2,annexin A3,B23和S100A9的表达.建立了LCM纯化的无转移和有转移的肺腺癌组织癌细胞的2D-DIGE图谱,质谱鉴定了20个非冗余差异蛋白质,其中12个蛋白质在有转移肺腺癌组中较无转移肺腺癌组表达上调,8个蛋白质在有转移肺腺癌组中表达下调.Western blot验证分析显示,差异蛋白annexin A1,annexin A2,annexin A3和S100A9的表达水平在有转移肺腺癌中较无转移肺腺癌增高,B23的表达水平在有转移肺腺癌中较无转移肺腺癌降低.免疫组化进一步证实S100A9在有转移的肺腺癌中较无转移的肺腺癌中表达上调.首次应用LCM技术联合2D-DIGE及MS技术分析鉴定出肺腺癌转移相关蛋白质,为研究肺腺癌的转移分子机制、筛选预测肺腺癌转移的分子标志物奠定了基础.

Cancer cell metastasis is the major cause of lung adenocarcinoma （AdC） with high mortality and poor prognosis. To screen metastasis-associated biomarkers of lung AdC, laser capture microdissection （LCM） was used to purify the cancer cells from primary lung AdC with （LNM AdC） and without metastasis （non-LNM AdC） according to clinical diagnosis of lymph node metastasis and distant metastasis. Then two-dimensional differential in-gel electrophoresis （2D-DIGE） was performed to isolate the total proteins of the pooled microdissected cancer cells from non-LNM AdC and LNM AdC. The differential proteins between non-LNM AdC and LNM AdC were analyzed by Decyder software and further identified by mass spectrometry （MS）. The partial differential proteins annexin A1, annexin A2, annexin A3, S100A9 and B23 were validated by Western blot. 2D-DIGE patterns of microdissected non-LNM AdC and LNM AdC were established, and 20 differential proteins in the above two tissues were identified, of which 13 proteins were up-regulated and 7 proteins were down-regulated in LNM AdC compared to non-LNM AdC. Western blot results indicated that annexin A1, annexin A2, annexin A3 and S 100A9 were significantly up-regulated in LNM AdC compared to non-LNM AdC; B23 was significantly down-regulated in LNM AdC compared to non-LNM AdC. Immunohistochemical analysis indicated S100A9 was up-regulated in LNM AdC compared with non-LNM AdC. It was the first time that metastasis-associated proteins were identified in primary adenocarcinoma by LCM coupled with 2D-DIGE and MS techniques. The findings will facilitate understanding of lung AdC metastasis and provide some direct proof for mining markers for predicting metastasis and patients＇ outcome so as to improve the diagnose and treatment of lung AdC.