Effects Of Lentivirus-mediated RNAi Inhibiting PIK3CA On Apoptosis And Proliferation Of Ovarian Epithelial Cancer Cell Line SKOV3

Objective: A lentiviral vector of RNAi targeting human PIK3CA gene(P110α)was constructed and the lentiviral vector was transfected into human ovarian cancer cell line SKOV3. The inhibitory effect of PIK3CA was detected by Real-time PCR and the changes of proliferation of SKOV3 cells contransfected with lentivial vector were examined by MTT. FCM was employed to investigate the changes of apoptosis and cell cycle of SKOV3 cells. The aim of the present study was to explore the prospects of lentivirus-mediated silencing PIK3CA application in gene therapy for ovarian cancer in order to provide a new treatment for ovarian cancer.Methods: 1 The RNAi sequences targeting PIK3CA gene were designed as follows: four best siRNA sequences were selected according to the basic design principles and the efficiency prediction formula of RNAi, and a negative control sequence was chosen. The designed siRNA sequences encoded into frameworks of shRNA were synthesized for DNA fragments of five targets.2 The pGCL-GFP vector was digested by Hpa I and Xho I.3 The DNA oligos were cloned into the digested pGC-L-G- FP vector and the linking clones were transformated into E.coli competence cell. The masculine clones were identified by PCR and then sequenced.4 The plasmids of masculine clones pGCL-GFP-PIK3CA were extracted from E.coli.5 The pEGFP-C1 vector was digested by XhoⅠand SacⅡ. The PIK3CA fragments extracted from plasmids containing PIK3CA by PCR were digested by XhoⅠand SacⅡ.6 The digested products of PIK3CA were linked with digested pEGFP-C1. The plasmids of masculine clones were identified by PCR and sequenced.7 The fusion protein pEGFP-C1-PIK3CA was transform- ated into 293T cells with Lipofectamine 2000. Then the expression of fusion protein GFP/RFP was detected by fluores- cence microscope after 36-48 hours so as to investigate the expression of PIK3CA gene.8 Different concentration of pGCL-GFP-PIK3CA and pE- GFP-C1-PIK3CA plamids were transformated into 293T cells. The expression of PIK3CA gene was determined by Western Blot for the selection of the most effective target for RNA interference.9 The plamids of pGCL-GFP-PIK3CA, pHelper 1.0(gag/pol element)and Helper 2.0(VSVG element)were extracted and transformated into 293T cells. The supernatants of cells full of lentivirus were collected 48 hours later. The supernatants were concentrated and the high-titer concentrated solutions of lentivirus were gained. The fluorescent expressions were observed four days later and the titres of virus were detected in 293T cells with the method of hole-by-hole dilution titer.10 SKOV3 cells were transformated by virus soluntion, five holes one group. Three days later, the expressions of GFP were observed by fluorescence microscope. Five days later, the cells were collected and the expressions of PIK3CA mRNA were detected by Real-time PCR.11 The 96-well plate were inoculated with three groups cells, five holes one group. The growth of the cells in three groups was observed in the first three days by MTT.12 The 6-well plate were inoculated with three groups cells, five holes one group. The cells were collected five days later and the cell cycle and cell apoptosis was detected by FCM.Results: 1 Four RNAi targets and a negative control target were selected. The virus frameworks were constructed and formed into double strands DNA oligo. The DNA oligo was conformed by 12% PAGE gel eletrophoresis.2 The pGCL-GFP vector was digested by Hpa I and Xho I conformed by Agarse gel eletrophoresis.3 DNA oligos cloned into the digested pGCL-GFP vector were transformated into E.coli competence cell and identified by PCR. The masculine clones psc1-1, psc2-1, psc3-1, psc4-1, pscNC-1 were checked out and sequenced right by ABI3730 implement in Invitrogen company. 4 The plamid of masculine clone pGCL-GFP-PIK3CA was extracted.5 Agarse gel eletrophoresis showed the digested pEGFP- C1 vector was right. The agarse gel eletrophoresis displayed the extracted PIK3CA and digested PIK3CA fragments were right.6 The digested PIK3CA fragments were linked to digested pEGFP-C1 succesfully. The masculine clones were checked out and sequenced right.7 The fusion protein pEGFP-C1-PIK3CA was transforma- ted into 293T cells. The expressions of PIK3CA by fluorescence microscope were right.8 The plamids of pGCL-GFP-PIK3CA and pEGFP-C1- PIK3CA were transformated into 293T cells. The 3#(psc340)target was effective for inhibiting PIK3CA by Western Blot.9 The plamids of pGCL-GFP-PIK3CA, pHelper 1.0 (gag/pol element)and Helper 2.0(VSVG element)were transformated into 293T cells. The virus was gained and the determined titre was 2×108 TU/ml.10 The effect of inhibiting PIK3CA of SKOV3 cells transformated by lentivirus was detected by Real-time PCR.The relative content of PIK3CA gene in KD group was 0.3303, which less than in CON(1.1087)or NC(1.1087)group(P<0.01). It appeared no difference about relative content between CON and NC group(P>0.05). There were no pollutions, primer dimerides or non-specificity amplifications because there were no mixed hump or abnormal broaden main maximum. It indicated that PIK3CA was inhibited stably and long by RNAi.11 The growth state was detected by MTTThere was no difference between CON and NC group(P>0.05). But the KD group growed slowly than CON and NC group obviously(P<0.01). Inhibiting PIK3CA gene can step down the growth of SKOV3 cells. The SKOV3 cells were inhibited because of inhibiting PIK3CA.12 The changes of cell cycle and cell apoptosis by FCMThe apoptosis rate in KD group was 3.877% larger than CON or NC group obviously(P<0.05). The numbers of G2/M phase in KD group were less than that in CON or NC group. The numbers of S phase in KD group were more than that in CON and NC group obviously(P<0.05). There was no difference in cell cycle and cell apoptosis between CON and NC group(P>0.05). The numbers of G2/M phase were reduced and S phase were increased by inhibiting PIK3CA in SKOV3. It was possible that inhibiting PIK3CA can make cells maintain in S phase.Conclusions: 1 Lentivirus may be an ideal vector carring siRNA in ovarian cancer cell.2 The expressions of PIK3CA mRNA and protein were inhibited long and obviously after RNAi lentivirus transforma- ted into ovarian cancer cell.3 PIK3CA can regulate the growth and proliferation of ovarian cancer cell line SKOV3 obviously. 4 Inhibited PIK3CA can increase the rate of apoptosis in ovarian cancer cell line SKOV3 and reduce the number of cells in G2/M phase and increase the number of cells in S phase. It is possible that inhibited PIK3CA can make cells maintain in S phase.