The Study Of The Chemo-resistant Change About Sirna Foxo1 To The Hepg2 Cell Line

BackgroundThe primary hepatic carcinoma is one of the major reasons of the malignant tumor death in our country,second only to the lung cancer.Although the development of liver transplantation,liver cancer carcinectomy and the chemo-drug improve the clinic therapeutic effect of primary hepatic carcinoma,the 5-year survival rate still less than 50%because of the palindromia after the operation,even most of the patients have lost the operation opportunity when they get the therapy.The drug-resistant of the liver cancer patients is the major reason of treatment failure,the recovery rate and the long-term survival rate.So it is very important to find an effective method for reversing the primary hepatic carcinoma.The transcription factor FoxO1 which belongs to the Fox family is the hot spot in recently research,Fox family is named because of their homophylic conservative Fox structural domain.As a member of FoxO subfamily,FoxO1 plays a very important role in regulating the development,differentiation,metabolism of the organism,which participates in the regulation of the cell metabolism,the cell cycle,the apoptosis and the cancer genesis/inhibit,and so on.Now the relationship of the FoxO1 and the chemo-resistant is more and more important to the researchers.The chemo-resistant in the tumor therapy is the most formidable problem; especially the multi-drug resistance is the major reason in therapy failure.The mechanism of chemo-resistant is very complex,which the result of the multiple factor with complex action,including the cell signal transduction,the change of the ability of the DNA repair and expression of the MDR-related protein,and so on.Whether the drug gets the target concentration in the cancer cells is the key point which decides the cell fate.The most important mechanism is that a great number of membrane transport proteins can decrease the drug concentration in the cell by efflux of the drug, which results in the chemo-resistant.MDR1 is the major one.The proximal promoter region of the human MDR1 contained a putative FoxO-binding site,which partially overlapped with the enhancer/enhancer-binding protein beta-binding region.In the drug-resistant cancer cell strains of ovarian caner,breast cancer,endometrial cancer et al,the transcription of MDR1 gene is stimulated by FoxO1 over expression.And the FoxO 1 also can induce the cell cycle arrest and DNA repair which can increase the resistance to the anti-cancer drug.So the transcription factor FoxO1 has relationship to the chemo-resistant.Experiment aimTo study the change of expression level of FoxO1 in the HepG2 liver cancer strain,the drug-resistant HepG2 cell which is induced by 5-fu and the siRNA FoxO1 drug-resistant HepG2 cell.Observe the change of expression about the MDR1 and the chemo-resistance which followed the changes about the expression of FoxO1 among these cells.Methods1.Constructed the specific FoxO1 siRNA vector and extracted the plasmids.The specific siRNA vector of FoxO1(pSIREN-DNR-DsRed-siFoxO1)was constructed and sequenced by TaKaRa company;extracted the plasmids by the kit.2.Liver cancer HepG2 cells were cultured in 37℃,5%CO₂ saturated humidity and sterile incubator.The cells were passaged by the 0.05%parenzyme and the 0.02% EDTA and cultured 24h.The medium was added to 5μg/ml 5-fu and affects the cell 24h,then back to the normal medium.When the cells recovered the proliferating ability,after the passage,the concentration of 5-fu were increased gradually according to the cell condition.The HepG2/5-fu cell were induced by 3 month.3.Observed the cells by microscope.Detected the resistance index of the HepG2/ 5-fu cell to the 5-fu,DDP and AMD.4.Transfected the FoxO1 plasmid with marked fluorescent by liposome and silence the FoxO1.Detected the expression of FoxO1 and MDR1 in each group with RT-PCR.5.Detected the lethal effect of the anti-cancer drug to the HepG2 cells,the chemo-resistant HepG2 cells and the silence FoxO1 HepG2 cells by MTT. Observed the different level of chemo-resistant.All data were expressed as mean value±S.D,and analyzed by statistical software SPSS12.0.t test or analysis of variance was applied after equality of variances was determined.P＜0.05 was considered to be statistically significant.Results1.Induction of the chemo-resistant of the HepG2:The cells didn't have obviously change after added 5μg/ml 5-fu several hours;became a little round after 10^th h; the cells gradually died after incubated the normal medium 24^th h.Then the number of death cells increased and reached the peak at the 48-72h in normal medium.After 7d in normal medium,the cells were only left 10%from 70%～80%paved on the bottom of culture flask.The cells gradually recovered the proliferate ability,after 1 week,can get 70%～80%,then passaged to grow.After cultured the cells in medium with 10μg/ml 5-fu for 24 h,the medium changed to normal one.The cells died,at last only left 10%.The concentration of the drug increased gradually and obtained the Hepg2/5-fu resistance cell after 3 months.2.The change of the morphology and biology of the chemo-resistant cells:the morphous of the cell was observed by inverted microscope,after cultured in different concentration of 5-fu,the Hepg2 cells became larger and longer,the adherence to flask wall was firmer,the cells grew agglomerately between the cells when passaged.The population doubling time prolonged.The RI of the HepG2/5-fu to the 5-fu is 7.42(P＜0.01) and the HepG2/5-fu cell also resisted crosswise to the DDP(P＜0.05).3.The result of silence FoxO1 by the fluorescent plasmid transfection:after 24h about the plasmid transfection in liposome,the red fluor occured under the Fluorescent inverted microscope,it could last 72h,and the transfection efficiency detected by the RT-PCR was about 42%.4.The expression of the FoxO1 and MDR1 in HepG2 cells and HepG2/5-fu cells: RT-PCR analysis showed the expression of the FoxO1 and MDR1 in the chemo-resistant HepG2 cells is higher than the HepG2 cells,after the FoxO1 silence,the expression of MDR1 also decrease,while the expression of FoxO1 decreased.5.The chemo-resistant change of HepG2 cells and HepG2/5-fu cells:the chemo-resistant level of HepG2/5-fu is obviously higher than the HepG2 by MTT detection.The chemo-resistant level of HepG2/5-fu tended to decrease after silence FoxO1.Conclusion1.After the affect of the anti-cancer drug 5-fu,the expression of the FoxO1 was unregulated in the chemo-resistant cells;2.The FoxO1 siRNA could decrease the chemo-resistance of HepG2/5-fu;3.The relationship between expression of FoxO1 and MDR1 might be one of the mechanisms that the FoxO1 affects the chemo-resistance of liver cancer.