应用氨基酸稳定同位素标记-质谱技术构建肺癌细胞14—3—3sigma相互作用的分子网络

Analyzed the molecular interaction network of tumor suppressor gene 14-3-3 sigma in lung cancer cell based on stable isotope labeling by amino acids in cell culture technology

目的筛选14—3—3sigma在非小细胞肺癌细胞中的功能相关蛋白，构建相互作用的分子网络。方法构建稳定过表达14—3—3sigma蛋白的非小细胞肺癌细胞株（PG细胞，大细胞肺癌），采用四甲基偶氮唑蓝（MTT）方法检测PG细胞的生长速度。利用稳定同位素标记氨基酸（stableisotopelabelingbyaminoacidsincellculture，SILAC）技术，联合线性离子阱回旋共振质谱仪（LC-LTQ—n’）鉴定由14-3-3sigma过表达引起的差异表达蛋白，将质谱鉴定为表达〉2倍或〈0．5倍的蛋白作为差异蛋白。通过检索人类蛋白参考数据库（Humanproteinreferencedatabase，HPRD）和蛋白质交互作用网络数据库（Kyotoencyclopediaofgenesandgenomes，KEGG）构建分子网络。结果显示C4克隆中外源14—3—3sigma表达量最高（命名为PGSC4），后续实验用PGSCA细胞进行。转染14—3-3sigma后，PG细胞生长速度明显减慢。建立了包含147个蛋白差异蛋白的数据库。构建的分子网络中含有26个蛋白，其中参与了多个细胞活动（细胞周期调控、细胞分化、凋亡等）的重要激酶——酪蛋白激酶Ⅱ0t亚基（caseinkinaseIIsubunitalpha，CSNK2A1）是表达升高最明显的蛋白质，与DNA损伤调节机制相关的转录调节因子MENl（Menin）则是表达降低幅度最大的蛋白质。结论14．3—3sigma可以抑制非小细胞肺癌PG细胞的生长速度，与细胞周期、DNA损伤修复等机制相关的蛋白质发生了变化，并构成了相互作用的分子网络。

Objective To analysis the molecular interaction network of 14-3-3 sigma in non small cell lung cancer （NSCLC） cells. Methods Established stable over-expressed 14-3-3 sigma protein PG cells, MTT assay was used to assess the growth rate of PG cells. Though stable isotope labeling by amino acids in cell culture （SILAC） and Mass spectrometry （MS） technology, to identify difference expressed proteins caused by over expressed 14-3-3 sigma. The protein expressed 〉 2 or 〈 0. 5 times was termed as the differential protein. By searching Human protein reference database （HPRD） and Kyoto encyclopedia of genes and genomes （KEGG） , established the molecular interaction network of tumor suppressor gene 14-3-3 sigma. Results The growth rate of over-expressed 14-3-3 sigma PG cell was obviously slower down compared to vector PG cells. A database including 147 differential protein was established. And a molecular interaction network of 14-3-3 sigma containing 26 protein was constructed. In this network, the expression of CSNK2A1 （casein kinase Ⅱ subunit alpha）, involved in numerous cellular processes, such as cell cycle progression, apoptosis and transcription, was the most significantly increased. A DNA repair protein, MEN1 （Menin） which functions as a transcriptional regulator was the most significantly decreased. Conclusion After stable transfected with 14-3-3 sigma gene, growth rate of PG ceils was inhibited, the proteins associated with cell cycle, DNA damage repair mechanisms were significantly changed, and constructed the molecular interaction network.