The Effect And Mechanism Research Of Proliferation And Apoptosis In Gastric Cancer Cell For ZNF139’s Inhibited By SiRNA

Objectives: Gastric cancer is the most common malignant digestivetumor in China.The current operation-based comprehensive therapy is farfrom satisfactory due to quick proliferation and apoptosis of gastric cancer cell.Therefore, it is of vast importance to seek for a new factor who can inhibit cellproliferation and induce apoptosis of gastric cancer cell. ZNFs (zinc fingerproteins), as a cluster of transcription regulator, would control gene expressiondirectly.According to our research, ZNF139(zinc finger protein139) is inclose affinity to gastric cancer.RNAi(RNA interference)technology is a newcancer therapy that inhibits the expression, amplification and translocation ofdominant mutant oncogene and suppresses viral oncogene.This experiment,transfecting gastric cancer cell strain SGC7901with ZNF139-siRNA,is toinvestigate ZNF139’s function mechanism in controlling gastric cancer cellproliferation and apoptosis by checking gastric cancer cell viability with MTT,drawing time-dose curve of gastric cancer cell transfection, observing theapoptosis rate and cell cycle through FCM,and checking out ZNF139,CyclinD1,PCNA,Caspase-3,Bcl-2in all expression units in the way of RT-PCR.Method: Taking human gastric cancer cell strain SGC7901as object, andsynthetic plasmid mediated ZNF139-siRNA and non-specific siRNA ascarriers,the experiment includes three groups,blank control group（SGC7901）,positive experimental group（transfected ZNF139-siRNA-Ps1SGC7901） and negative control group （transfected ZNF139-siRNA-PCSGC7901）. Firstly, transfect gastric cancer cell SGC7901with different dosesof siRNA, determine the transfected cell viability through MTT and draw thetime-dose curve of gastric cancer cell to obtain the optimum dose and time forsiRNA interference.Secondly, under this optimum condition,take thetransfected ZNF139-siRNA SGC7901cell, and record cancer cell apoptosis and cycle in these three groups through FCM. Thirdly and lastly, extract thetotal mRNA of three groups,detect the mRNA expression and changing ofZNF139,CyclinD1, PCNA,Caspase-3and Bcl-2in all groups by RT-PCR,andanalyze the expression with SPSS13.0statistics approach by One-wayANOVA and Spearman Correlation,et al.Results:1Determine human gastric cancer cell SGC7901apoptosis rate and cell cyclein the three groups1.1According to FCM, the apoptosis peak occurs obviously in positiveexperimental group at24th,48th and72th hour after human gastric cancer cellSGC7901is transfected by ZNF139-siRNA-Ps1plasmid, with three apoptosisrates of4.823±0.685%,16.827±3.507%and20.107±3.305%accordingly. Noapoptosis peak appears in blank control group and negative control group.Based on the statistics analysis, statistical difference of apoptosis rate showsup three times in positive experimental group (P=0.001),in blank controlgroup and negative control group (P=0.202，P=0.904). What’s more, statisticaldifference varies with checking time in positive experimental group (24h：t=-8.826P=0.012，48h：t=-7.505P=0.016，72h：t=-9.425P=0.008).1.2Comparison of cell cycle distributionIn the first24h, there is no statistical difference of cell cycle in all three groups(F=0.388,P=0.867).After another48h, cell cycle varies between groups F=3.519,P=0.052. Inpositive experimental group, the cell number increase in Phase G0/G1is ofstatistical difference(P=0.010), whose cell ratio is about62.100±1.908%, andthe cell reduction in Phase S presents statistical difference(P=0.005), with thecell ratio of25.767±0.851%.the cell number change in Phase G2/M has nosignificant difference(P=0.811),proliferation index respectively of48.033±1.563%,37.867±2.108%,47.36±2.3447%.There is obvious statisticaldifference in Phases G0/G1and S between experimental groups and blankcontrol group(t=-7.253P=0.002，t=6.480P=0.003).After72h, cell cycle presents great difference in three groups F=5.900, P=0.013. As for positive experimental group, the cell number in Phase G0/G1increases(P=0.008), whose cell ratio is about60.100±3.365%, and the cell inPhase S reduces(P=0.006), with cell ratio of28.767±1.234%. The cell numberchange in Phase G2/M has no significant difference(P=0.841),proliferationindex respectively of48.500±1.153%,39.900±365%,47.734±1.704%.There isobvious statistical difference in Phases G0/G1and S between experimentalgroups and blank control group (t=-4.188P=0.014，t=3.483P=0.025).1.3comparison for inhibition rate of cancer cellUnder fluorescence microscope,transfection efficiency of the builtZNF139-siRNA-Ps1plasmid is around90%. It has the ability to inhibitSGC7901cell growth. Experiment shows that transfecting after48h with0.8μg/ml plasmid is the optimum condition. Its cell inhibition rate stand at22.21%，46.48%，and45.81%when declines at24h,48h and72h. TheZNF139-siRNA-PC non-specific plasmid cannot silence ZNF139expression.Its inhibition rate separate8.64%,8.42%and1.08%by the three time periodabovementioned.2The mRNA expression of ZNF139, CyclinD1, PCNA, Caspase-3and Bcl-2in three groups.2.1The mRNAs of ZNF139,CyclinD1,PCNA,Caspase-3and Bcl-2in blankcontrol group and positive experimental group cells SGC7901are allexpressed. The mRNA expression of ZNF139,CyclinD1,PCNA and Bcl-2inpositive experimental group cell SGC7901is significantly lower than gastriccancer cell SGC7901while Caspase-3expression is obviously higher thanblank control group cell(P<0.05).2.2ZNF139,CyclinD1,PCNA,Caspase-3and Bcl-2mRNA are all expressed inblank control group cell SGC7901and negative control group SGC7901cell.No significant expression difference is detected (P>0.05).3The correlation between mRNA expressions of ZNF139and CyclinD1,PCNA,Caspase-3,Bcl-2in human gastric cancer cell SGC7901of threegroups.3.1There is positive correlation between ZNF139expression and CyclinD1, PCNA,Caspase-3,Bcl-2in blank control group cell SGC7901(r=0.997P=0.048; r=0.998P=0.041;r=0.998P=0.041;r=0.997P=0.049).3.2There is positive correlation between ZNF139expression and CyclinD1,PCNA and Bcl-2(r=0.997P=0.049;r=0.997P=0.046;r=0.997P=0.045) andnegative correlation between the expression of ZNF139and Caspase-3(r=-0.998P=0.038) in positive experimental group cell SGC7901.3.3There is positive correlation between ZNF139expression and CyclinD1,PCNA,Caspase-3,Bcl-2(r=0.997P=0.045;r=0.997P=0.049;r=0.9997P=0.016;r=0.998P=0.036)in negative control group cell SGC7901.Conclusions:1The human gastric cancer cell line SGC7901experiment confirms thatthe built ZNF139-siRNA-Ps1plasmid has the ability to silence thetranscription factor ZNF139expression in SGC7901cell specifically, inhibitgrowth tumor cell, promote cell apoptosis, and block the cell in Phase G0/G1.2In transfected ZNF139-siRNA-Ps1gastric cancer cell SGC7901, of thetotal mRNA genes,the CyclinD1,PCNA and Bcl-2expression is down whileCaspase-3expression is up ZNF139expression shows the positive correlationwith CyclinD1, PCNA and Bcl-2, but negative correlation with Caspase-3,which indicates that the transcription factor ZNF139fulfills the proliferationand apoptosis of gastric cancer cell by regulating CyclinD1, PCNA, Caspase-3,Bcl-2and other associated factors.