Study About Mechanism Of The Radiation-sensitivity Difference Between Ovarian Cancer Skov3Cells And Cervical Cancer Hela Cells

Ovarian cancer is a common malignant tumor in women and a leading cause ofdeath among all gynecological malignant tumors. Radiotherapy is the main methodfor this disease. As compared with cervical carcinoma, patients with ovarian cancerhave resistant to radiotherapy, leading to therapeutic effect is not ideal in clinicaloncology. The killing effect of radiation on tumor cells is mainly by destruction ofDNA in cells. Damaged DNA activates the DNA damage response kinase [ataxiatelangiectasia mutated (ATM) or ataxia telangiectasia and Rad3related (ATR)] thatcontrols cell cycle check, retardation of cell cycle course and repairing of damagedDNA. The key enzyme related to DNA repair, DNA-activated protein kinase(DNA-PK), can determine the prognosis of cells. Successful DNA repair can restorecells into the normal cycle of life, but failure to get damaged DNA accurately repairedwill initiate an apoptotic process. Therefore, it is important to explore how the cellsresistant to radiation are responding to DNA damaging and repairing. Accordingly, thepresent work was undertaken to investigate the mechanism by which Skov3cellsderived from ovarian cancer and Hela cells derived from cervical cancer coulddifferent resist radiation. In this study, we also applied short hairpin RNA (shRNA) tointerfere in activities of checkpoint kinase1(CHK1) and DNA-PK in X-irradiatedSkov3and Hela cells in order to identify a new pathway useful for the development ofclinical treatment on ovarian cancer. The content of this experiment is as follows：1. Construction of DNA-PK shrna and CHK1shrna and the filter of effectiveinterference vector.Methods：According to the accession number of the human DNA-PKand CHK1in GenBank,hsu34994and af016582, we select CDS area, then useshrna design software to select the targets at where DNA-PK and CHK1interference RNA,which are DNA-PK1160、1888、2995、5297，CHK1140、590、617、632, design shRNA, design four shRNA vector separately,and buildcontrol vector shNC with unrelated sequence. After sequence identification and the interaction with estriction enzyme of Pst1and BamH1, shRNA vector mediatedtransfects Skov3by liposomes Lipofectimine2000,then observe the transfectionby laser scanning confocal microscopy. We test the expression of DNA-PKmRNA、CHK1mRNA in transfected cells by RT-PCR,and test the expression ofDNA-PK protein and CHK1protein by immunohistochemical method to filter theeffective shRAN vector.Results：(1)The identification of restriction endonuclease digestion of Pst1、BamH1and sequencing analysis confirm that the four shRNA vectors withDNA-PK and CHK1as theirs target are correct.(2)By laser scanning confocal microscope, the constructed shRNA caneffectively transfects the Skov3cells.According to the number of fluorescentcells,we determine the transfection efficiency is about50%to60%.(3)Through RT-PCR and immunohistochemical detection of the expressionof DNA-PK and CHK1in transfected cells,we find that in transfection grouppGPU6/GFP/Neo/DNA-PK-1888、 pGPU6/GFP/Neo/DNA-PK-5297、pGPU6/GFP/Neo/CHK1-617,the mRNA and protein expression of DNA-PK andCHK1decrease,which are effective shRNA vectors. Therefore we choosepGPU6/GFP/Neo/DNA-PK-5297、 pGPU6/GFP/Neo/CHK1-617to follow-upexperiment,which are named separately pGPU6/Neo/DNA-PK-5297、pGPU6/GFP/Neo/CHK1-617Conclusion:(1)Successfully construct four shRNA expression vectors which target at DNA-PKand CHK1gene.(2)ShRNA vector can effectively transfect Skov3cells,and silence the expressionof DNA-PK and CHK1expression,so through RT-PCR and immunohistochemicalmethod we filter the effective shRNA expression vector DAN-PKshRAN-5297,CHK1shRNA-617.2. Differences of radiation-induced apoptosis in ovarian cancer Skov3cells and Hela cell. Methods：We transfected DAN-PK shRAN-5297and CHK1shRNA-617usingthe Lipofectimine2000method, and20h later, Skov3and Hela cells wereseparatelly treated with X-ray irradiation. Irradiation was provided by a Philips deepX-ray therapy machine, the dose rate was0.287Gy/min. Before X-ray irradiation, thecells were washed with PBS. Add2ml PBS to start irradiation. After irradiation, freshcomplete medium was used immediately. When the cells were further cultured for4hor28h, that was24h or48h after cells collection, they were washed with PBS threetimes, with centrifugation using the parameter800rpm,5min for each time.5μlAnnexin V was added to stain the cells for10min, and the cells were washed withPBS for three times. Then10μl PI was added to the cell culture medium. And the cellswere treated with ice bath for30min after they were thoroughly mixed. Another washwith PBS for three times. Then we detect and analysis the cell apoptosis by flowcytometer method, and CellQuest and Modifit softwares. SPSS14software was usedfor statistical analysis, and t-test analysis was selected to find the difference of FCbetween Skov3cells and Hela cells, which was the radio of the treatment group andcontrol group.Results：(1)Adopt flow cytometry to analysis the apoptosis percentage of Skov3cells andHela cells which are separatelly treated with0、2、4、6、8、10Gy X-ray. The apoptosispercentage of Skov3cells are1.5%、2.18%、2.9%、4.67%、5.6%、12.5%，The apoptosispercentage of Hela cells are1.1%、19.79%、39.3%、56.7%、70.1%、76.2%。Therefore2Gy is chosen as X-ran irradiation dose in this study.(2)Analysis with flow cytometry demonstrated that the apoptotic response toX-radiation in Skov3cells transfected with DNA-PK shRNA-5297, CHK1shRNA-617, DNA-PK shRNA-5297and CHK1shRNA-617at4hr of post-radiationwere22.53%±2.41%（t=5.360，p<0.001）,21.53%±2.51%（t=4.837, p<0.001）,24.80%±3.20%（t=5.587, p<0.001）, respectively. Although the apoptotic responsewere higher in Skov3cells transfected with DNA-PK shRNA-5297and CHK1shRNA-617than with DNA-PK shRNA-5297, CHK1shRNA-617, but no significant. The apoptotic response at28hr of post-radiation were13.32%±3.63%（t=2.524，p=0.03）,15.30%±2.57%（t=4.518，p<0.001）,18.91%±2.81%（t=6.705，p<0.001）,respectively. The apoptotic response were significantly higher in Skov3cellstransfected with DNA-PK shRNA-5297and CHK1shRNA-617than with DNA-PKshRNA-5297, CHK1shRNA-617（t=2.981，p=0.014; t=2.323，p=0.043）.(3)Analysis with flow cytometry demonstrated that the apoptotic response toX-radiation in Hela cells transfected with DNA-PK shRNA-5297, CHK1shRNA-617,DNA-PK shRNA-5297and CHK1shRNA-617at4hr of post-radiation were22.28%±3.13%（t=1.870， p=0.091）、19.81%±3.16%（t=0.969， p=0.355）、25.99%±4.00%（t=2.82， p=0.020）, respectively. The apoptotic response wassignificantly higher in Hela cells transfected with DNA-PK shRNA-5297and CHK1shRNA-617than with CHK1shRNA-617（t=2.87，p=0.018）, although it was higherthan with DNA-PK shRNA-5297,but no significant. The apoptotic response at28hrof post-radiation were26.99%±5.28%（t=1.388，p=0.195）、26.56%±3.28%（t=1.509，p=0.162）、28.90%±3.45%（t=2.421，p=0.036）, respectively. The apoptotic responsewere higher in Hela cells transfected with DNA-PK shRNA-5297and CHK1shRNA-617than with DNA-PK shRNA-5297, CHK1shRNA-617, but no statisticalsignificance.(4)Analysis with flow cytometry demonstrated that the apoptotic response toX-radiation was significantly lower in Skov3cells than Hela cells at4hr ofpost-radiation (t=15.22, p<0.001) and at28hr (t=6.83, p<0.001). This suggested thatradiation-resistence to the same doses of X-radiation in Skov3cells is significantlylower than in Hela cells. However the apoptotic response to X-radiation wassignificantly higher in Skov3cells transfected with CHK1shRNA-617, DNA-PKshRNA-5297, CHK1shRNA-617and DNA-PK shRNA-5297than Hela cells at4hror28hr of post-radiation, in addition to no significant in cells transfected withDNA-PK shRNA-5297at28hr of post-radiation.Conclusion:(1)Skov3cells is not sensitive to irradiation.Skov3cells is less sensitive to irradiation than Hela cells at the same radiation level.Difference of sensitivity toradiation between them can be used as a cell model of ovarian and cervical cancer.(2)DNA-PK shRNA-5297-transfection and CHK1shRNA-617-transfection cansignificantly enhanced the radiation-resistence in Skov3cells.3. Study about differences of radiosensitivity between Skov3cells and Hela cells incell cycle block.Methods：The same means as method two.Results：(1)Analysis with flow cytometry demonstrated that the proportion of Skov3cellsthat entered the G2phase was19.68%±3.69%at4hr of post-radiation andsignificantly higher than cella after sham-irradiation （t=3.680， p=0.005）,12.52%±1.45%（t=7.873，p<0.001）at4hr of post-radiation. However, the proportionof Hela cells that entered the G2phase was14.09%±2.84%（t=4.826, p=0.001），11.55%±2.89%（t=2.756, p=0.020）at4hr and28hr of post-radiation, respectively. Allof the G2phase retardanted. The proportion of Skov3cells that entered the G2phasewas significantly lower than Hela at4hr of post-radiation（t=2.482, p=0.032）, butsignificantly higher than Hela at28hr of post-radiation（t=8.458, p<0.001）.(2)Analysis with flow cytometry demonstrated that the proportion of Skov3cellsand Hela cells transfected with DNA-PK shRNA-5297that entered the G2phase wassignificantly higher than cella with after DNA-PK shRNA-5297sham-irradiation at4hr and28hr of post-radiation,but no significant difference as compared with cellstransfected with shNC.(3)Analysis with flow cytometry demonstrated that the proportion of Skov3cellstransfected with CHK1shRNA-617that entered the G2phase was15.84%±2.19%,5.72%±1.60%, respectively, significantly lower than cells transfected with shNC（t=2.469, p=0.033; t=7.163, p<0.001）at4hr and28hr of post-radiation. While theproportion of Hela cells that entered the G2phase was10.42%±1.38%,9.01%±2.19%,respectively, significantly lower than cells transfected with shNC (t=4.431, p=0.001;t=3.026, p=0.013）. (4)The proportion of Skov3cells transfected with DNA-PK shRNA-5297andCHK1shRNA-617that entered the G2phase was16.14%±1.97%significantly lowerthan cells transfected with shNC（t=2.347，p=0.041）at4hr of post-radiation. Theproportion of Skov3cells that entered the G2phase was7.44%±1.99%significantlylower than cells transfected with shNC（t=4.423，p=0.001）at28hr of post-radiation,too. While the proportion of Hela cells that entered the G2phase was10.12%±1.83%,10.15%±1.47%, respectively, significantly lower than cellstransfected with shNC (t=4.431, t=6.810; p=0.001, p<0.001）at4hr and28hr ofpost-radiation. The proportion rate of Skov3cells transfected with DNA-PKshRNA-5297and CHK1shRNA-617that entered the G2phase was0.87±0.11andwas significantly higher than of Hela cell（st=2.965，p=0.014）at4hr of post-radiation,was0.64±0.17and significantly lower than of Hela cells（t=2.302，p=0.044）at4hr ofpost-radiation.Conclusion:(1)The results of flow cytometry revealed that there was G2phase arrest forSkov3cell and Hela cell when they received a2-Gy-dose X-ray radiation.(2)DNA-PK shRNA had no significant effect on the cell cycles and G2phasearrest of Skov3cell and Hela cell.(3)CHK1shRNA could relieve the G2phase arrest of Skov3cell and Hela cell,but G2phase arrest in Skov3cells transfected with CHK1shRNA was further relievedafter28hr when they had received radiation and the arrest for Hela cell was recoveredto some degree or there was no further relief. They are not consistent with the resultsof the cell apoptotic percentage. We could speculate that there were more check andrepair pathways except for CHK1check point and DNA-PK repair pathway in Skov3cell or that G2phase check point in Skov3cells might be excess or invalid, causingthat Skov3cells were non-sensitive to radiation.(4)Transfection with DNA-PK shRNA-5297and CHK1shRNA-617couldrelieve G2phase arrest in Skov3cell and Hela cell, and it was further relieved after28hr when the cells had received the radiation. The degree of G2phase arrest in Skov3 cell transfected with DNA-PK shRNA-5297and CHK1shRNA-617was higher thanthat of Hela cell at4hr of post-radiation, but lower at28hr of post-radiation.4. Discussion on ovarian cancer and cervical cancer cell radiosensitivity differencesin DNA damage response mechanism.Methods: Extract RNA from the cells,then quantificate the RNA and carryout RT-PCR. The RT-PCR products was electrophresised in1.5%agarose gel. Thenwe observed and photographied. According to DNA marker and forecasting analysisof size of PCR products,we determined what the PCR products were.Results：(1)DNA-PK shRNA-5297and CHK1shRNA-617could silence the expressionsof DNA-PK and CHK1mRNA of transfected cells effectively.(2)The expression of p53mRNA of Skov3cell and Hela cell was increased at aGy dose of X ray-radiation, but the expression of p53mRNA of cells transfected withDNA-PK shRNA-5297was close to that of the control group. The expression of p53mRNA of the group transfected with CHK1shRNA-617and group co-transfectedwith shRNA-5297and CHK1shRNA-617was close to the group which received a2-Gy-dose radiation. Thansfection with any of DNA-PK shRNA-5297and CHK1shRNA-617alone had no significant effect on the p53mRNA of Skov3cell and Helacell.(3)The expression of CDC25C mRNA of Skov3cell and Hela cell went up aftera Gy dose of X ray-radiation. The expression of CDC25C mRNA of the grouptransfected with DNA-PK shRNA-5297and group co-transfected with DNA-PKshRNA-5297and CHK1shRNA-617was close to the group which received a2-Gy-dose radiation. The expression of CDC25C mRNA of the group transfected withCHK1shRNA-617was close to normal control group. Thansfection with any ofDNA-PK shRNA-5297and CHK1shRNA-617alone had no significant effect on thep53mRNA of Skov3cell and Hela cell.(4)Transfection with CHK1shRNA-617could make the radiation-inducedexpression of CDC25C mRNA of Skov3cell and Hela cell down-regulated. The expression of CDC25C mRNA of the group transfected with DNA-PK shRNA-5297and group co-transfected with DNA-PK shRNA-5297and CHK1shRNA-617wasclose to the group which received a2-Gy-dose radiation. The expression of CDC25CmRNA of the group transfected with CHK1shRNA-617was close to normal controlgroup.(5)Transfection with CHK1shRNA-617and co-transfection with DNA-PKshRNA-5297and CHK1shRNA-617could increased the expression of CDK2mRNAof cells which received a2-Gy-dose radiation.Conclusion:Transfection of CHK1shRNA-617, DNA-PK shRNA-5297and CHK1shRNA-617decreased the expression of CDC25C and CDC25A mRNA, andincreased the expression of Cdk2mRNA in Skov3cell and Hela cell which receivedthe X-ray irradiation. DNA-PK shRNA-5297reduced the expression of p53mRNA inradiation-induced Skov3cell and Hela cell, decreases the transcription of p21in itsdownstream, enhanced the activity of Cdk2-cyclinE complexes, resulted in the reliefof cell cycle arrest and increased the apoptosis of the both cells. This conclusion wasconsistent with the previous studies.