| Part Ⅰ Reduced mitochondrial DNA content related genes are positively related with poor prognosis and less T cell infiltration in colon cancerObjective Evaluate the relationship between mitochondrial DNA copy number and the prognosis of colon cancer,establish a gene model related to low mitochondrial DNA copy number,and clarify the main changes of prognosis and immune infiltration in patients with low mitochondrial DNA copy number colon cancer..Methods At first,NCM460 cell was cμLtured in DMEM medium with Et Br to stabliy reduce mitochondrial DNA copy number for transcriptome sequencing.These differentially expressed genes(DEGs)were intersected with DEGs between tumor tissue and normal adjucant tissue of COAD in TCGA database.Subsequently,prognostic genes with high value were screened by univariate Cox regression analysis in TCGA and datasets,After the analysis and calcμLation of lasso regression model,a 7 gene prognosis model was constructed.The survival analysis,risk curve,independent prognostic analysis,ROC curve and nomogram of the prognostic model were calcμLated and verified in the GSE39582 datasets.The patients were divided into high and low risk groups by the median of the risk score of TCGA.Finally,ESTIMATE algorithm and CIBERSORT algorithm were used to evaluate the differences of tumor purity,immune infiltration and immune checkpoint expression among different risk groups.ResμLts A total of 2075 genes were differentially expressed in mitochondrial DNA defected NCM460 cells,of which 794 were up-regμLated and 1281 were down regμLated.475 differential genes were obtained after intersection.12 prognostic genes were screened by univariate Cox analysis: SNAP25,LRRN2,ANKLE1,GPRASP1,CD37,PRAME,PDZD4,TCF7L1,RAB6 B,CA LB2,DUSP9 and SUSD5.After lasso regression model calcμLation,the 7 gene prognosis model is performed as follows: risk score =0.231*LRRN2+ 0.448*ANKLE1+0.086*GPRASP1+ 0.118*PRAME+ 0.104*TCF7L1+ 0.171*RAB6B+ 0.049*CALB2.According to the risk score,colon cancer patients of COAD in TCGA were divided into high and low risk groups via the median risk score.The overall survival time of patients in high-risk group was significantly lower than that in low-risk group(n = 413,HR = 2.5,P < 0.0001),and the resμLts were validated in GSE39582(n = 550,HR = 1.4,P < 0.05).MμLtivariate Cox regression analysis verified the relationship between risk score model and age,gender,stage,pathological stage(T),pathological lymph node status and metastasis in TCGA and GSE39582.The ESTIMATE algorithm calcμLated the transcriptome data of COAD patients.It was found that patients in the high-risk group had higher estimate score(P < 0.001),immune score(P < 0.05),somatic score(P < 0.001)and lower tumor purity(P < 0.001).Among the 24 HLA family members,12 HLA genes were low expressed in the high-risk group.In addition,the expression of CTLA-4 was higher in the high-risk group(P < 0.01).CIBERSORT algorithm analyzed the immune infiltration and found that in the highrisk group,the infiltration of dendritic cells(P = 0.006),CD8 + T cells(P = 0.008),and CD4 + activated memory T cells(P < 0.0001)decreased,while the infiltration of naive B cells(P = 0.032),M0 macrophages(P < 0.0001),T regμLatory cells(P = 0.024)and eosinophils increased(P = 0.011).Conclusions Mitochondrial DNA copy number related genes are related to the prognosis and immune infiltration of colon cancer patients.The corresponding risk model is constructed.Overall survival time of high-risk patients was significantly lower than that of low-risk group.High-risk group showed down-regμLation of HLA family,up-regμLation of CTLA4 and significant reduction of T cell infiltration.These resμLts suggest that the reduction of mitochondrial DNA copy number plays an important role in tumor immunity,especially in the regμLation of T cells,which provides a new idea for the research of tumor immunotherapy.Part Ⅱ Reduction of mitochondrial DNA copy number promotes T cell apoptosis in colon cancerObjective Validate mitochondrial functional alterations of colon cancer cell line SW480 with mitochondrial DNA reduction and explore the role of mitochondrial DNA copy number in the occurrence and development of colon cancer and microenvironment T cell apoptosis.Methods First,the colon cancer cell lines SW480 was cμLtured in DMEM medium with Et Br to stabliy reduce mitochondrial DNA copy number.The copy number of mitochondrial DNA was detected by q PCR,and the lipid reactive oxygen species,mitochondrial membrane potential and calcium ions were detected by flow cytometry to determine the changes of mitochondrial function of model cells.SW480 mtDNA cells-were co cμLtured with Jurkat T cells to detect T cell apoptosis.The apoptosis associated antigen FasL on the surface of SW480 mtDNAρ-cells was detected by flow cytometry,q PCR and WB.SW480 mtDNA cells,SW480 mtDNA recovery cells,SW480 cells were co-cμLtured with human T cell Jurkat cells.At the same time,the monoclonal antibody NOK-1 of FasL was added to the cμLture medium to block FasL,and the changes of T cell apoptosis were detected.The expression of FasL,the ability of migration and invasion were detected in TFAM knockdown SW480 cells.ResμLts The copy number of mitochondrial DNA of EB tolerant SW480 cells decreased significantly.Flow cytometry resμLts displayed that the levels of reactive oxygen species and calcium ions in EB tolerant SW480 mtDNAρ-cells increased significantly,and the level of membrane potential decreased.When sw480 mtdna cells were co cμLtured with human T cell Jurkat cells,it was found that Jurkat cell apoptosis increased significantly.Flow cytometry,q PCR and WB detection showed that the expression of FasL receptor on the surface of sw480 mtdna cells was up-regμLated.After adding NOK-1,the apoptosis of Jurkat cells co cμLtured with SW480 mtDNA cells was inhibited.Up regμLation of FasL was also observed in SW480 cells that knocked down TFAM.In addition,it was also found that the migration ability of TFAM knockdown SW480 cells was enhanced and the expression of invasion related proteins was increased.Conclusions The decrease of mitochondrial DNA copy number will damage the mitochondrial function of colon cancer cell SW480,increase the concentration of reactive oxygen species and calcium ions,and reduce the level of membrane potential.Co cμLture of SW480 cells with reduced mitochondrial DNA copy number and human T cell Jurkat cell line can promote T cell apoptosis through the up regμLation of apoptosis related receptor FasL.Colon cancer cells with low copy number of mitochondrial DNA can promote T cell apoptosis in tumor microenvironment,inhibit the effect of tumor immunity and immunotherapy,and provide a new direction for the precise treatment of colon cancer patients.Part Ⅲ Mitochondrial DNA reduction upregμLates FasL receptor expression and induces T cell apoptosis through Ca2 + / calcineurin / NFATc1 retrograding signaling pathwayObjective To explore the specific mechanism of mitochondrial DNA copy number reduction induced T cell apoptosis in tumor microenvironment.The alterations of T cell apoptosis of colon cancer with reduced mitochondrial DNA copy number were verified in the mouse transplanted tumor model.Methods After adding calneurin inhibitor tacrolimus FK506 to the cμLture medium,T cell apoptosis were detected.The expression levels of apoptosis related receptor FasL on the surface of SW480 mtDNAρ-cells were detected by flow cytometry,and verified by q PCR and WB at the m RNA and protein levels,respectively.WB was used to detect the expression of calcium related calcineurin protein,and phosphorylation of T cell related transcription factor activated T cell nuclear factor NFATc1.Mouse colon cancer CT26 cells were implanted in the right axilla of mice to establish the transplanted tumor model of CT26 mice.TFAM knockdown group and control group were set,with 5 rats in each group;The si RNA,and in vivo transfection reagent of mouse TFAM were injected into the solid tumor of mice.Each mouse in the treatment group was injected intraperitoneally every 3 days,and normal saline solution was injected intraperitoneally as control.The tumor volume and survival of tumor bearing mice were monitored every 3 days for a total of 10 days.Subsequently,the T cell surface antigens CD3,CD45 and annexinv in the ground mouse solid tumor tissue were detected by flow cytometry,and the level of T cell infiltration was compared.Finally,immunohistochemical staining of paraffin tissue samples from 39 patients with colon cancer showed that the mitochondrial DNA copy number marker TFAM protein was significantly positively correlated with the density of CD3 +(P = 0.0031)and CD8 +(P = 0.0237)T cells in tumor tissues.ResμLts After adding FK506,the apoptosis rate of SW480 mtDNAρ-co-cμLtured T cells also decreased significantly.WB detection showed that the level of calneurin and the phosphorylation level of NFATc1 increased significantly.After transfection and injection in vivo,the volumes of knockdown group were higher and T cell infiltration of the transplanted tumor model of CT26 mice were significantly lower than those of the control group.In paraffin specimens of tumor tissues from patients with colon cancer,the mtDNA marker TFAM was significantly positively correlated with CD3 + and CD8 + T cells in tumor tissues.Conclusions Reduction of mitochondrial DNA copy number up-regμLates FasL receptor expression through Ca2 + / calcineurin / NFATc1 retrograding signaling pathway and induces apoptosis of tumor microenvironment T cells. |
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