| Objective: Based on the previous work of the research group,the effects of TGF-?1 on autophagy and mitophagy of ovarian cancer cells were detected;to establish ovarian cancer cell lines with overexpression or underexpression of Lewis y antigen,to detect the effect of fucosylation modification of TGF-?1 on autophagy and mitophagy,and to explore its regulatory mechanism,so as to find a new theoretical basis for the treatment of ovarian cancer.Methods: 1.Western blot was used to detect the effect of TGF-?1 at different concentrations and time points on the expression of autophagy related proteins in ovarian cancer cells.The changes of LC3 in ovarian cancer cells treated with TGF-?1 were detected by immunofluorescence.Chloroquine(CQ),an autophagy inhibitor,was added to block the autophagy flow.The autophagy flow of ovarian cancer cells was observed by Western blot and immunofluorescence.The changes of autophagy flow of ovarian cancer cells under the action of TGF-?1 were detected after transient transfection of LC3 adenovirus.TMRM staining was used to detect the changes of mitochondrial membrane potential of ovarian cancer cells after TGF-?1 treatment.Then Western blot was used to detect the effect of TGF-?1 at different concentrations and time points on the level of mitophagy in ovarian cancer cells.The expression of PINK1,a mitophagy related protein,was detected by immunofluorescence.The apoptosis of ovarian cancer cells was detected by Annexin V/FITC flow cytometry.The effect of TGF-?1 on the apoptosis of ovarian cancer cells was detected after adding 3-methyladene(3-MA).2.Firstly,we transfected FUT1 plasmid and sh FUT1 virus respectively,constructed the ovarian cancer cell model of over expression and low expression of Lewis y antigen,detected the expression of Lewis y and TGF-?1,and verified the structural relationship between TGF-?1 and Lewis y antigen by immunocoprecipitation.We then retest the first part of the method based on these cell models.3.Western blot was used to detect the expression of TAK1 protein in ovarian cancer cells after TGF-?1 treatment.Western blot,immunofluorescence,double fluorescent LC3 adenovirus and TMRM were used to detect the changes of autophagy,autophagy flow,mitochondrial membrane potential and mitophagy caused by TGF-?1 in ovarian cancer cells after transfection of si TAK1,and the changes of related pathways.Results: 1.With the increase of TGF-?1 concentration,the expression of LC3?/I,Beclin-1,Atg5 and Atg7 increased,and the expression of p62 decreased in a concentration dependent manner.Under the action of TGF-?1 at different time points,LC3?/I,Beclin-1,Atg5 and Atg7 reached the peak of protein level at 12,24 and 48 h,while p62 was negatively correlated with time.Immunofluorescence was used to detect the change of LC3,with TGF-?1 treatment,the fluorescence of LC3 showed bright red spots,while that of untreated group showed scattered red fluorescence.After treated with CQ,the expression level of LC3 increased significantly,at the same time,the expression of Beclin-1,Atg5,Atg7 and p62 further confirmed the above results.In the TGF-?1 group,the number of small red fluorescent spots in LC3 increased more significantly.Compared with the control group,TGF-?1 significantly promoted the accumulation of autophagic bodies with yellow fluorescence signal.TMRM detection indicated that TGF-?1 induced depolarization of mitochondrial membrane potential.After treatment with different concentrations of TGF-?1,the expression levels of PINK1,BNIP3 and Parkin increased with the increase of TGF-?1 concentration,which was concentration dependent.Under the action of TGF-?1 at different time points,PINK1,BNIP3 and Parkin all reached the peak value in 24 hours;immunofluorescence detection showed that compared with the control group,the fluorescence of PINK1 was significantly enhanced and the expression was significantly increased with the treatment of TGF-?1 for 24 hours.Flow cytometry showed that with the increase of TGF-?1 concentration,the apoptosis rate of ovarian cancer cells also increased in a concentration dependent manner;3-MA treatment could alleviate the apoptosis induced by TGF-?1.2.After FUT1 gene transfection,the expression of Lewis y in the cells increased,and the expression of TGF-?1 and its Lewis y increased.At the same time,it was found that the expression of autophagy related proteins LC3 ?/I,Beclin-1,Atg5 and Atg7 increased,and the level of p62 decreased,at the same time.After transfection,the fluorescence of TMRM staining weakened,and the mitochondrial membrane potential was depolarized.After FUT1 transfection,the expression of mitophagy related proteins such as PINK1,BNIP3 and Parkin increased,and the effect of TGF-?1 on mitophagy was promoted.The results of sh FUT1 transfection were opposite.After sh FUT1 transfection,the apoptotic rate was increased,and the apoptosis induced by TGF-?1 was partially reversed.3.The expression of TAK1 protein was increased with the treatment of TGF-?1.Under the effect of TGF-?1,the expression of autophagy and mitophagy related protein and mitochondrial membrane potential depolarization were inhibited after the transfection of si TAK1,and the above phenomena were alleviated after the transfection of FUT1 gene.Under the effect of TGF-?1,there was no significant change in PI3 K,Akt and ERK1/2,but the expression of p-PI3 K,p-Akt and p-ERK1/2 was significantly down regulated.Adding MEK or PI3 K pathway inhibitors to alleviate the effect of TAK1 on autophagy and mitophagy.After TGF-?1 treatment,the expression of m TOR did not change significantly,p-m TOR was down-regulated.After FUT1 gene transfection,p-m TOR protein was down-regulated more obviously,and the down-regulation of p-m TOR protein was relieved after interfering with TAK1.Conclusion: 1.TGF-?1 can promote the development of autophagy of ovarian cancer cells in a concentration dependent manner,cause the depolarization of mitochondrial membrane potential and promote the development of mitophagy.TGF-?1 can induce apoptosis in a concentration dependent manner,and inhibition of autophagy can partially reverse the apoptosis induced by TGF-?1.2.TGF-?1 was modified with Lewis y antigen,and the Lewis y antigen modification of TGF-?1 can further promote autophagy in ovarian cancer cells,increase the depolarization of mitochondrial membrane potential and enhance the occurrence of mitophagy.Lewis y antigen modification of TGF-?1 can alleviate the apoptosis induced by TGF-?1.3.TGF-?1 can induce autophagy of ovarian cancer cells through TAK1,which leads to depolarization of membrane potential and mitophagy.Lewis y antigen modification of TGF-?1 can further promote the above effects.TGF-?1 binds to its receptor to activate TAK1,and regulates the phosphorylation of downstream m TOR protein by phosphorylation of PI3K/Akt and ERK1/2 signaling pathway,thereby affecting the expression of autophagy and mitophagy-related proteins,and thus regulating the occurrence of autophagy and mitophagy. |
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