Study On The Growth Of Cisplatin-resistant Human Ovarian Cancer Cell Lines SKOV3/DDP Cells By Targeting Interference In Vitro

Objective:To investigate cell growth effect on cisplatin-resistant human ovarian cancer celllines SKOV3/DDP cells by siRNA targeting Akt and go further gene therapy ofovarian cancer to provide new experimental basis.Methods:1、Using Lipofectamine2000liposome mediated method, the specificity of AktsiRNA was transfected into human ovarian cancer SKOV3/DDP cells,detecting theeffects on the growth of ovarian cancer cells after siRNA silencing Akt.2、With the transfection reagent Lipofectamine2000packages of differentconcentration of fluorescent labeled siRNA was transfected into SKOV3/DDPcells,using flow cytometry to test its transfection efficiency,determine the best siRNAtransfection concentration.3、In the screening of effective Akt siRNA fragment in the experiment,theexperiment was divided into five groups:①control②Akt siRNA-1③Akt siRNA-2④Akt siRNA-3⑤Negative Control(NC).After transfection of48h,using real-timefluorescence quantitative PCR(qRT-PCR) to detect the expression of Akt mRNA ineach group, screening effective Akt siRNA fragment.4、The effects on cell growth after Akt siRNA was transfected into ovariancancer SKOV3/DDP cells.The effective Akt siRNA fragment was transfected byLipofectamine2000into ovarian cancer SKOV3/DDP cells,experiment was dividedinto three groups:①control②Akt siRNA transfection group③Negative control(NC).5、 After transfection of48h,changes of cell cycle and apoptosis wererespectively detected by flow cytometry;CCK8detects cell proliferationactivity;Western Blot methods detect the expression levels of Akt and p-Akt protein.Results:1、With Lipofectamine2000respectively packages of50nM、80nM、100nM ofFAM-siRNA was transfected into SKOV3/DDP cells，After transfection of6h，flowcytometry results showed that transfection efficiency was more than80%,considering the toxic effects of lipo2000on cells,the optimal transfection efficiency of siRNA is50nM.2、After transfection of48h,using qRT-PCR to detect the expression of AktmRNA in each group,the results show:the expression of three fragments of siRNAgroup was obviously reduced,as compared with the control group and the negativecontrol group, the difference has statistical significance （P<0.05）,and thedown-regulation of Akt mRNA expression was the most evident in siRNA-2.Thenegative control group comparison with the control group, there was no significantdifferences（P>0.05）.This experiment selects the siRNA fragment II as effectivesiRNA fragment.3、After transfection of48h,each cell cycle was measured by flow cytometry andthe results showed that as compared with the control group and the negative controlgroup,the proportion of G0/G1phase of Akt siRNA group was evidently increased,theproportions of S phase and G2/M phase were obviously reduced,the differences wereall statistically significant（P<0.05）, as compared with the control group,the negativecontrol group had no clear difference（P>0.05）.4、After transfection of48h,each cell apoptosis was measured by flow cytometryand the results found that as compared with the control group and the negative controlgroup, early and late apoptosis rate of Akt siRNA specific group were evidentlyincreased,there was significant difference（P<0.05）, as compared with the controlgroup,the negative control group had no clear difference（P>0.05）.5、After transfection of24h,48h,72h,CCK8results showed that cell growth ofAkt siRNA transfection group was obviously limited and over time the inhibition rateincreased accordingly,the cell growth inhibition was the most obvious aftertransfection of72h.As compared the negative control group with the control group,the inhibition of cell growth had no obvious chang.6、After transfection of48h,Western Blot results showed that as compared withthe control group and the negative control group,the expression levels of Akt andp-Akt protein of the specific transfection group were obviously decreased, therewas significant differences（P<0.05）,The negative control group compared with thecontrol group， there was no significant difference. Conclusion:1. Silencing Akt gene by small interfering RNA can effectively reduce theexpression of Akt mRNA, inhibit the growth of SKOV3/DDP cells.2. Silencing Akt gene by small interfering RNA can significantly inhibited cellproliferative activity of human ovarian cancer SKOV3/DDP cells,blocking the cellcycle in G0/G1phase and reducing the expression levels of Akt protein inSKOV3/DDP cells.Inhibition mechanism may be related to knockout Akt gene ofPI3K/Akt signal pathway,blocking its activation of downstream a variety ofmolecules and promote cell apoptosis.3. Specificity silencing Akt gene by small interfering RNA can effectivelyinhibit the growth of SKOV3/DDP cells and is expected to become the new target ofgene therapy for ovarian cancer.