| Objective Aberrant Wnt/β-catenin signaling pathway was associated with a majority of tumorgenesis,whether Wnt/β-catenin signaling pathway play a role anti-tumor chemotherapy drugs resistances remain unknown.Through the study of Beclin 1-mediated autophagy and Gemcitabine resistance in human osteosarcoma cell line MG63,to investigate the relationship between Wnt signaling pathway and Gemcitabine resistance through overexpression of Beclin 1 gene in human osteosarcoma cell line MG63.Methods 1.Cell Culture Human osteosarcoma cell line MG63 was cultured in DMEM with 10% FBS at 37℃ in an atmosphere containing 5% CO2.Cells were subcultured at approximately 90% of cell density,adding 1m L 0.125% trypsin and 0.02%EDTA digestion for 3-5 minutes,complete medium to terminate the reaction,the centrifugal supernatant(1000 rpm),and 1/3 cells were cultured,the other cells with DMSO liquid nitrogen preservation reserve.2.Confocal Microscopy To visualize the induction of autophagy,connecting the Ds Red-LC3-GFP to the retroviral vector,Gag-pol and VSVG together with the helper plasmid were transfected into 293 T cell lines.After screening by puromycin,to establish a stable Ds Red-LC3-GFP cell line expressing target cells.293 T cell lines cultured in serum free medium condition for 3 days as a positive control to visualize the induction of autophagy.While Ds Red was constitutively expressed in all the situations,the percentage of autophagy-positive cells were characterized by GFP negative cells under a confocal fluorescence microscope.3.Western Blotting 1×106 cells were washed twice with PBS,and incubated with pyrolysed solution containing 0.1mol/L PMSF.Cell lysates were centrifuged at 13,000 rpm for 20 min.According to the molecular weight of target protein,maked up 10% separated gel and 4% concentrated gel.Equal amounts of the protein were suspended,resolved by SDS-PAGE and transferred onto a 0.45μm pore diameter NC membrane.After blocking with 5% non-fat dry milk in TBS for 1 h at room temperature,membrane were incubated with primary antibody(anti-Beclin 1or anti-LC3B)at room temperature for 10 minutes and then 4℃ overnight.The next day membrane was taken out and incubated at room temperature for 30 minutes,washed with TBST for 5 times,each time for 3 minutes.After washed with TBS with 20% Tween 20 5 times,the NC membrane was treated with goat anti-rabbit Ig G antibody for 40 min at room temperature.ECL wasadded to the membrane,then reaction 3-5 minutes,film exposure,developing 2min,fixing.Specific bands were detected by an enhanced chemiluminescence system.4.PCR detect the expression of Caspase 3、Caspase 7、BCL-2、cyclin D1、c-FLIP Total cellular RNA was extracted using TRIZOL reagent and c DNA was synthesized according to manufacturer’s instructions(Promega,USA).The PCR program include initial denaturation for 10 min at 95℃,followed by 40 cycles of denaturation for 15 s at 95℃,annealing/extension for 1min at 60℃.The levels of indicated molecules were measured by calculating the threshold cycle(Ct)of target gene after normalization against the Ct value of GAPDH dependent on the delta delta cycles to threshold(ΔΔCT)method.The primers were designed by Primer premier 5.0 software and synthesized by Sangon Biotech(Shanghai,China).5.PCR detect the expression of ATG4,Beclin 1,LAMP1,ATG5,ATG12 Total cellular RNA was extracted using TRIZOL reagent and c DNA was synthesized according to manufacturer’s instructions(Promega,USA).The PCR program include initial denaturation for 10 min at 95℃,followed by 40 cycles of denaturation for 15 s at 95℃,annealing/extension for 1min at 60℃.The levels of indicated molecules were measured by calculating the threshold cycle(Ct)of target gene after normalization against the Ct value of GAPDH dependent on the delta delta cycles to threshold(ΔΔCT)method.The primers were designed by Primer premier 5.0 software and synthesized by Sangon Biotech(Shanghai,China).6.Flow Cytometry To determine cell apoptosis,1×106 MG63 cells were washed,digested and resuspended in 100μL binding buffer.5μL FITC-conjugated Annexin V antibody was added and incubated for 20 min at room temperature.20 minutes before flow cytometry analysis,5μL PI was added to visualize DNA.Cell apoptosis was detected by flow cytometry.7.ELISA Technical Analysis 10μL cell supernatant by 0.05mol/L Na HCO3 diluted to 100μL with ELISA,at room temperature oscillated 1.5 hours.PBST washing 3 times.Sealed closed liquid(PBST plus 1% non-fat dry milk)350μL/hole,at room temperature for 1 hours,washed 3 times with PBST.The original pepsinogen antibody was diluted to 100μL with sealed closed liquid,at room temperature oscillated 1 hour,washed 3 times with PBST.HRP marker closed sealed liquid dilution of goat anti-mouse 100μL/hole,ELISA plate at room temperature oscillated for 1 hours,washed 3 times with PBST.Adding OPD color liquid 100μL/hole,light response about 10-30 minutes.1mol/L H2SO4 100μL/hole terminated reaction,the absorbance measured at the wavelength of 450 nm.Statistical Analysis.All statistical analyses were performed using SPSS software version 16.0.Data were presented as mean± standard error.One-way or two-way variance analysis was performed to determine statistical significance for multiple comparisons.p<0.05 was considered to indicate a statistically significant difference.Results 1.Beclin 1 gene over-expression induce autophagy in human osteosarcoma cell line MG63.Western blotting detections showed over-expressed Beclin1 gene in 293 T cell line.Beclin-1 gene over-expression is associated with enhanced autophagy with associated dampening green fluorescence observed in human osteosarcoma cell line MG63.Western blot analysis was performed to detect the LC3 B,a specific marker of the steady-state levels of autophagosomes,conversion and progression of autophagy.Consistent with fluorescence microscope displayed above,increased conversion of LC3 B was detected after Beclin-1 gene over-expression.2.Beclin 1 gene over-expression induce autophagy in human osteosarcoma cell line MG63.As expected,Beclin1 overexpression significantly reduced the expression of pro-apoptotic caspase3(p<0.001)and caspase7(p<0.001)gene expression in gemcitabine-treated cells compared with untransfected cells at 50 μg/ml gemcitabine.The results demonstrated that overexpression of Beclin1 was associated with reduced gemcitabine-induced apoptosis.3.Activation of wnt/β-catenin signaling pathway attenuates autophagy.Beclin1 expression was significantly increased following inhibition of the Wnt/β-catenin signaling pathway by XAV939,indicating that altered Beclin1 expression may be involved in Wnt/β-catenin signaling pathway-attenuated autophagy.4.Activation of wnt/β-catenin signaling pathway inhibit autophagy and reduces resistance of Beclin1 gene over-expressed MG63 cellstogemcitabine-induced apoptosis.The results demonstrated that the Wnt/β-catenin signaling pathway may inhibit autophagy and increase gemcitabine-induced apoptosis in the MG63 cell line by down-regulating the expression of.Beclin1.Conclusion Beclin1 gene over-expression could induce autophagy and reduce Gemcitabineinduced apoptosis of MG63 osteosarcoma cells.Beclin1 over-expression can reduce the apoptosis induced by Gemcitabine,but the activation of the Wnt signaling pathway can reverse inhibition.Our results provide a new insight of anti-tumor chemotherapy drugs resistance.Aberrant Wnt/β-Catenin signaling may enhance Beclin1-mediated autophagy,thus preventing chemotherapy drug-induced cell apoptosis.Our findings provide a potential evidence for enhancing the sensitivity of tumors to apoptosis induced by chemotherapy drugs through modulating aberrant Wnt/β-Catenin signaling related autophagy and autophagy protein Beclin1. |
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