锌指蛋白139对人胃癌细胞株SGC7901增殖及凋亡调控机制探讨

Mechanism for transcription factor ZNF139's inhibition of proliferation and apoptosis regulation of gastric cancer cells

目的探讨抑制锌指蛋白139(zincfinger protein 139,ZNF139)对人胃癌细胞株SGC7901增殖和凋亡的影响及相关机制。方法构建针对ZNF139的siRNA重组质粒并导入SGC7901,逆转录-聚合酶链(RT-PCR)法检测抑制效率;MTT法测定siRNA-ZNF139组、阴性对照组和空白对照组SGC7901细胞活力;流式细胞术观察转染前后细胞周期及凋亡率变化;RT-PCR和蛋白质印迹法检测Cyclin D1、PCNA、Caspase-3和Bcl-2在各组中表达情况。结果0.8μg/mL siRNA-ZNF139质粒转染SGC7901细胞24、48和72h后细胞凋亡率分别为(4.823±0.685)%、(16.827±3.507)%和(20.107±3.305)%,差异有统计学意义,F=24.59,P=0.001 3。转染48h时空白对照组、阴性对照组和siRNA-ZNF139组G0/G1期细胞比例分别为(51.800±1.552)%、(52.633±0.361)%和(62.100±1.908)%,F=25.52,P=0.001 2;转染72h时G0/G1期细胞比例分别为(51.500±1.153)%、(53.567±1.582)%和(60.100±3.365)%,F=11.97,P=0.008。0.8μg/mL siRNA-ZNF139质粒转染SGC7901细胞48h后,siRNA-ZNF139组ZNF139、Cyclin D1、PCNA和Bcl-2mRNA表达分别为0.126±0.032、0.455±0.082、0.300±0.164和0.110±0.032,明显低于空白对照组的0.613±0.119、0.740±0.098、0.523±0.109和0.311±0.133,Caspase-3mRNA表达(0.230±0.028)明显高于空白对照组(0.089±0.018),P=0.001 9;siRNA-ZNF139组ZNF139、Cyclin D1、PCNA、Bcl-2蛋白表达分别为0.524±0.044 8、0.540±0.052 5、0.535±0.046 1和0.414±0.031,明显低于空白对照组的0.861±0.047、0.750±0.033、0.886±0.058和0.821±0.030,Caspase-3蛋白表达(1.004±0.105)明显高于空白对照组(0.517±0.027),P=0.002 1。结论 ZNF139可通过调节Cyclin D1、PCNA、Caspase-3和Bcl-2基因表达而影响胃癌细胞增殖、凋亡过程。

OBJECTIVE To explore the effect and mechanism of inhibiting ZNF139 to proliferationand apoptosis of human gastric cancer cell line SGC7901.METHODS siRNA recombinant plasmid targeting ZNF139 was structured and imported into SGC7901.ZNF139 inhibition rate was tested with RT-PCR.MTT method was applied to evaluate cell viability.Apoptosis rate and cell cycle were observed through FCM.Expressions of ZNF139,Cyclin D1,PCNA,Caspase-3and Bcl-2in different group of SGC7901 were tested by RT-PCR and Western blot.RESULTS 0.8μg/mL siRNA-ZNF139 was transfected into SGC7901 cell.After 24,48,72 h,Cell apoptosis rates were（4.823±0.685）%,（16.827±3.507）%,（20.107±3.305）% respectively,and difference was found among these groups（F=24.59,P=0.001 3）.After 48 htransfection,ratios of G0/G1 phase of cells in blank group,negative group and siRNA-ZNF139 group were（51.800±1.552）%,（52.633±0.361）%,（62.100±1.908）% respectively（F=25.52,P=0.001 2）.After 72 htransfection the ratios of G0/G1 phase of cells in 3groups were（51.500±1.153）%,（53.567±1.582）%,（60.100±3.365）% respectively（F=11.97,P=0.008）.After 48 htransfection with 0.8μg/mL siRNA-ZNF139,relative expressions of ZNF139,Cyclin D1,PCNA,Bcl-2 mRNA in siRNA-ZNF139 group were 0.126±0.032,0.455±0.082,0.300±0.164,0.110±0.032 respectively,which were lower than that in blank group（0.613±0.119,0.740±0.098,0.523±0.109,0.311±0.133respectively）,whereas expression of Caspase-3mRNA was higher in siRNA-ZNF139group（0.230±0.028）than that in blank group（0.089±0.018,P=0.001 9.Relative expressions of ZNF139,Cyclin D1,PCNA,Bcl-2proteins in siRNA-ZNF139 group were 0.524±0.044 8,0.540±0.052 5,0.535±0.046 1,0.414±0.031 respectively,which were lower than that in blank group（0.861±0.047,0.750±0.033,0.886±0.058,0.821±0.030）,whereas expression of Caspase-3protein was higher in siRNA-ZNF139group（1.004±0.105）than that in blank group（0.517±0.027,P=0.002 1）.CONCLUSION ZNF139 may influence the proliferation and apoptosis regulation of gastric cancer cells by regulating Cyclin D1,PCNA,Caspase-3and Bcl-2genes.