Effect Of Mitochondrial Transcription Factor A On Autophagy Of Tumor Cells Under Starvation And Its Mechanism

Background: In recent years,due to the increase of environmental pollution and aging population,the incidence and fatality rate of tumor are getting higher and higher,and the treatment methods for tumor are also emerging in an endless number,but their therapeutic effects vary due to different tumor types and their own characteristics.And single therapy often has a tumor adaptive response,so combination therapy emerged.In the basic research stage,it was shown that "starvation therapy" can significantly reduce the proliferation of tumor cells by restricting the nutrient intake of tumor cells,and limit the tumor deterioration and metastasis in the transformation model.However,starvation therapy also activates an intracellular autophagy response,Through the decomposition of substrates,cells can obtain more materials and provide protection for tumor cells.Therefore,how to limit the protective autophagy induced by starvation therapy is of great concern.Under starvation stress,mitochondrial functional homeostasis changes and is associated with autophagy.transcription factor A(TFAM),which maintains the stability of Mitochondrial genome and regulates its expression,is upregulated under starvation conditions,and is positively correlated with autophagy.Whether the application of starvation therapy can inhibit autophagy response by restricting the expression of TFAM and improve the inhibition effect of starvation conditions on tumor is worth exploring.Therefore,in this paper,the effect of TFAM on the autophagy process of tumor cells under starvation condition was investigated,and the effect of TFAM deficiency on the proliferation of tumor cells under starvation condition was detected,which not only broadened our understanding of mitochondria regulation of autophagy response,but also provided experimental theoretical basis for the development of tumor therapy targeting mitochondria.Purposes: To investigate the effect of mitochondrial transcription factor A on autophagy of tumor cells under starvation condition and its mechanism.Methods: U2 OS cells,He La cells and MCF7 cells were studied.Transcriptome sequencing and database were used to screen out genes related to mitochondrial and autophagy up-regulation differences under starvation stress.q RT-PCR was used to verify the changes in m RNA levels of differential genes.The expressions of TFAM and LC3II/I in U2 OS cells,He La cells and MCF7 cells under starvation conditions were detected by q RT-PCR and Western Blot.The number of autophagosomes in the cytoplasm before and after starvation was detected by immunofluorescence.The stable cell line was established,and the LC3II/I changes after sh-TFAM and starvation combined with sh-TFAM were detected by Western Blot.The number of autophagosomes in the cytoplasm after starvation combined with sh-TFAM was detected by immunofluorescence technique.The changes of mitochondrial function before and after starvation were detected by immunofluorescence.The changes of mitochondrial function were detected by immunofluorescence after starvation combined with sh-TFAM.The changes of autophagy related genes LC3II/I,ATG101,ATG13,P62,Beclin1,FIP200,UVRAG and ULK1 in starvation combined with sh-TFAM cell lines were detected by Western Blot.CCK8 assay was used to detect the changes of proliferation ability of tumor cells in sh-TFAM group under starvation condition,and to explore the effect of mitochondrial transcription factor A on autophagy of tumor cells under starvation condition and its mechanism.Results: The expressions of TFAM and LC3II/I in U2 OS cells,He La cells and MCF7 cells were up-regulated after EBSS treatment for 3h,6h and 9h,and the number of autophagosomes in the cytoplasm increased gradually.Down-regulation of TFAM inhibited LC3II/I expression and decreased autophagosome.After 6h of EBSS treatment,the content of ATP,mitochondrial membrane potential and mitochondrial derived reactive oxygen species were significantly decreased compared with the untreated group,and the copy number of mitochondrial DNA increased with the extension of starvation time.Starvation for 6h combined with sh-TFAM also inhibited LC3II/I expression and autophagosome formation.Compared with NC+EBSS group,ATP content,mitochondrial DNA copy number content and mitochondrial membrane potential were significantly decreased in sh-TFAM+EBSS group,while mitochondrial derived reactive oxygen species increased with the extension of starvation time.The protein levels of ATG13,ATG101 and P62/SQSTM1 in sh-TFAM+EBSS group were decreased compared with NC+EBSS group.Cells treated with actinomycin(CHX)in control group and TFAM down-regulated group showed decreased intracellular P62 protein level 3,6 and 9 h after TFAM down-regulated group.Cells treated with CHX + MG132 or Baf A1 for 6 h showed decreased intracellular P62 protein level after TFAM down-regulated group compared with control group.P62/SQSTM1 m RNA expression was up-regulated.After the treatment of actinomycin D on the cells of control group and TFAM knockdown group,the m RNA level of P62/SQSTM1 in TFAM group decreased significantly after 3h,6h and 9h downregulation.Inhibited TFAM expression and intermittently treated with EBSS,the proliferation rate of tumor cells was significantly reduced.Conclusion: Under starvation condition,TFAM is involved in the regulation of autophagy response of tumor cells.Once TFAM is deficient,it not only affects the initiation stage of autophagy reaction,but also interferes with mitochondrial functional homeostasis,causing DNA damage of cells and slowing down the proliferation rate of tumor cells.