TFAM Inhibits Colitis And Colitis-associated Colorectal Tumorigenesis By Regulating Mitochondrial Activity

Background:Chronic inflammation is an important factor leading to tumorigenesis.Colitis-associated colorectal cancer（CAC）is one type of colorectal cancer developed from inflammatory bowel diseases（IBD）such as ulcerative colitis and Crohn’s disease,with a younger average age of onset and a higher grade of malignance than sporadic colorectal cancer.Because unable to effectively block the progression from inflammation to cancer by rapidly restoring intestinal epithelial barrier,the diagnosis and treatment of CAC is not satisfactory right now.Mitochondria is a pivotal cellular sensor for environmental change and plays a key role in maintaining intestinal epithelial homeostasis.Mitochondria also take part in inflammatory response and tumorigenesis through a variety of pathways.As an important regulator of mitochondrial function,mitochondrial transcription factor A（TFAM）is involved in the transcription and replication of mitochondrial DNA（mtDNA）,it also plays a critical role in regulating the mitochondrial activity.Therefore,it is conducive to have a better understanding of mitochondrial dysfunction in the inflammation-cancer transformation in the intestinal epithelia through the study of the function and mechanism of TFAM in colitis and colitis-associated colorectal tumorigenesis.Methods:Normal human colorectal tissues,IBD tissues and CAC tissues were collected.DSS was used to induce colitis in mice,AOM combined with DSS were used to establish CAC mouse model.Immunohistochemistry and western blot were used to determine the expression of target gene in tissues.We generated mice disrupted TFAM specially in intestinal epithelia(TFAM^ΔIEC)through villin-Cre⁺mice crossing with TFAM ^flox/floxmice.The body weight,stool consistency,hematochezia,colon length,q RT-PCR analysis of proinflammatory cytokines expression in colonic mucosa,HE staining and histological score,survival time under lethal dose of DSS,were all used to evaluate the symptoms of DSS-induced colitis in mice.The number and volume of intestinal neoplasia at 1 month and3 months,tumor pathological grade,mice survival rate were used to evaluate the influence on AOM/DSS induced CAC tumorigenesis.Lentiviral transfection was used to interference and overexpression TFAM in normal human colon mucosal epithelial cell lines FHC and NCM460.Mitochondrial functions were determined by mitochondrial respiratory chain complex activity,oxygen consumption rate and mitochondrial ATP.Cell proliferation were detected by Ed U and cell cycle.Results:Compared with human normal colorectal tissues,the expression of TFAM were significantly decreased in active IBD tissues and no significant difference in inactive IBD tissues,however,the expression of TFAM was significantly increased in human CAC.Similarly,compared with normal colorectal tissues in mice,the expression of TFAM were significantly downregulated in colitis tissues and upregulated in CAC tissues in mice model.TFAM^ΔIECand WT mice did not show obvious inflammatory symptoms under water feeding,however,in DSS-induced colitis,TFAM^ΔIECmice had much more body weight lost,softer stool,heavier hematochezia,shorter colon length than WT mice.TFAM^ΔIECmice also have higher percent of crypt damage,much more and deeper inflammatory cell infiltration,higher histological score,higher mucosal expression of proinflammatory cytokines（IL-1β,IL-6,IL-11,TNF-α）than WT mice.TFAM^ΔIECmice also have less survival time than WT mice under continuous drinking of lethal dose DSS.In AOM/DSS induced CAC model,TFAM^ΔIECmice presented earlier colorectal tumorigenesis,larger tumor volume,higher proportion of high grade dysplasia and lower survival rate than WT mice.In vitro,knockdown of TFAM significantly decreased the mitochondrial respiratory chain complex activity,oxygen consumption rate and mitochondrial ATP than negative control in normal human colon epithelial cell lines FHC and NCM460,while overexpression of TFAM significantly increased above indicators.Moreover,knockdown of TFAM in FHC and NCM460significantly inhibited cell proliferation,decreased the proportion of cells in the S phase of cell cycle and blocked in G1 phase than control,while overexpression of TFAM significantly promoted cell proliferation,increased the proportion of cells in the S phase of cell cycle and decreased in G1 phase than control.However,oligomycin,an inhibitor of ATP synthesis,significantly inhibited cell proliferation and decreased the proportion of cells in the S phase of cell cycle and blocked in G1 phase in FHC and NCM460 with TFAM overexpression.Conclusion:The expression of TFAM was significantly downregulated in active colitis and upregulated in CAC.Knockout of TFAM specially in intestinal epithelia aggravated experimental colitis and promoted CAC tumorigenesis in mice.TFAM promoted intestinal epithelia proliferation and accelerated cell cycle through enhancing mitochondrial activity.