CAJ | 학술논문

目的筛选14-3-3 sigma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14-3-3 sigma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(stableisotope labeling by amino acids in cell culture,SILAC)技术,联合线性离子阱回旋共振质谱仪(LC-LTQ-FT)鉴定由14-3-3 sigma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(Human protein reference database,HPRD)和蛋白质交互作用网络数据库(Kyoto encyclopedia of genes and genomes,KEGG)构建分子网络.结果显示C4克隆中外源14-3-3 sigma表达量最高(命名为PGSC4),后续实验用PGSC4细胞进行.转染14-3-3 sigma 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Ⅱα亚基(casein kinaseⅡsubunit alpha,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MEN1 (Menin)则是表达降低幅度最大的蛋白质.结论 14-3-3 sigma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.
목적 사선14-3-3 sigma재비소세포폐암세포중적공능상관단백,구건상호작용적분자망락.방법 구건은정과표체14-3-3 sigma단백적비소세포폐암세포주(PG세포,대세포폐암),채용사갑기우담서람(MTT)방법검측PG세포적생장속도.이용은정동위소표기안기산(stableisotope labeling by amino acids in cell culture,SILAC)기술,연합선성리자정회선공진질보의(LC-LTQ-FT)감정유14-3-3 sigma과표체인기적차이표체단백,장질보감정위표체＞2배혹＜0.5배적단백작위차이단백.통과검색인류단백삼고수거고(Human protein reference database,HPRD)화단백질교호작용망락수거고(Kyoto encyclopedia of genes and genomes,KEGG)구건분자망락.결과 현시C4극륭중외원14-3-3 sigma표체량최고(명명위PGSC4),후속실험용PGSC4세포진행.전염14-3-3 sigma 후,PG세포생장속도명현감만.건립료포함147개단백차이단백적수거고.구건적분자망락중함유26개단백,기중삼여료다개세포활동(세포주기조공、세포분화、조망등)적중요격매——락단백격매Ⅱα아기(casein kinaseⅡsubunit alpha,CSNK2A1)시표체승고최명현적단백질,여DNA손상조절궤제상관적전록조절인자MEN1 (Menin)칙시표체강저폭도최대적단백질.결론 14-3-3 sigma가이억제비소세포폐암PG세포적생장속도,여세포주기、DNA손상수복등궤제상관적단백질발생료변화,병구성료상호작용적분자망락.
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 cells was inhibited,the proteins associated with cell cycle,DNA damage repair mechanisms were significantly changed,and constructed the molecular interaction network.