The Effect Of Inducible Nitric Oxide Synthase Gene Transfecting On MCF-7Breast Cancer Cell

Objective:To construct the eukaryotic expression vector plasmid pYr-adshuttle-1-iNOS，and transfect it into breast cancer MCF-7to explore the effect of the iNOS gene onthe proliferation, apoptosis and the Cell cycle of MCF-7.Methods:(1)Constructing the eukaryotic expression vector plasmidpYr-adshuttle-1-iNOS.(2)Setting three groups:blank controller group,transfect withpYr-adshuttle-1-iNOS group,transfect with pYr-adshuttle-1group;transfectingpYr-adshuttle-1-iNOS and pYr-adshuttle-1into breast cancer cell MCF-7respectivelyaccording to the test group.(3)Detecting the protein expression of iNOS of each group by Western-blottiongafter all groups have been transfected for48hours.(4) Detecting the concentration of NO of each group after being transfected for0hours,24hours,48hours.(5) Analysizing the influence of transfection on proliferation of MCF-7byMTT each group after being transfected for0hours,24hours and48hours.(6) Detecting the cell apoptosis and cell cycle of each group after beingtransfected for48hours by flow cytometry.Results:(1) Established the eukaryotic expression vector plasmid pYr-adshuttle-1-iNOSsuccessfully.(2)The result of the Western-blottiong showed: the protein expression of iNOStransfected by pYr-adshuttle-1-iNOS was obviously higher than the group transfectedby pYr-adshuttle-1group and the blank group (P<0.05). At the same time there was no difference between the pYr-adshuttle-1group and the blank group(P>0.05).(3)The NO secretion was significantly higher in MCF-7transfected bypYr-adshuttle-1-iNOS than the two others groups after being transfected for24hours,48hours(P<0.05),and there was no difference between the pYr-adshuttle-1group and the the blank group (P>0.05).(4)The MTT result showed: the pYr-adshuttle-1-iNOS group had more obviousproliferation inhibition effect on MCF-7than the two other groups(P<0.05) afterbeing transfected for48hours,72hours. However, no difference occured between thepYr-adshuttle-1group and the the blank group(P>0.05).(5)The apoptosis rate of blank controll group, group transfected bypYr-adshuttle-1-iNOS, group transfected by pYr-adshuttle-1were4.8%、26.7%、6.8%by flow cytometry, showing that the high expression of iNOS gene mightincrease more cell apoptosis of MCF-7. According to the analysis of the Cell cycleby flow cytometry: the percentage of G1(the percentage of the number of cells inG1phase in all phases)of the group transfected by pYr-adshuttle-1-iNOS was higherthan the two other groups.We speculate the Cell cycle of the group transfected bypYr-adshuttle-1-iNOS might be blocked in G1/S initiatory.Conclusion:(1)the eukaryotic expression vector plasmid pYr-adshuttle-1-iNOS carrying theiNOS gene can lead to iNOS high expression in MCF-7after being transfected intoMCF-7, and then secrete high concentrations of NO persistently.(2)the high expression of iNOS cause proliferation inhibition effect in MCF-7cells.(2)the high expression of iNOS may promote cell apoptosis in MCF-7cells.(3)the iNOS high expression may cause the cell cycle of MCF-7block in G1/S.