NIT1 Suppresses Tumour Proliferation By Activating The TGF?1-Smad2/3 Signalling Pathway In Colorectal Cancer

BACKGROUND AND OBJECTIVE:Colorectal cancer(CRC)is one of the most common digestive tract malignant tumours.Although the diagnosis and treatment of colorectal cancer have made significant progress in the last ten years,the incidence and mortality will also remain high long term.Therefore,it is of great significance to investigate the detailed molecular mechanism of the occurrence and development of colorectal cancer in depth.In Drosophila melanogaster and Caenorhabditis elegans,NitFhit protein is a fusion protein which plays an important role in regulation of cell proliferation and apoptosis.It is generally known that FHIT is a clear tumour suppressor gene that suppresses cell growth and promotes apoptosis.Loss of FHIT is associated with many human malignancies.So we speculate that NIT1 might be a potential tumour suppressor candidate.However,little is known about the specific role of NIT1 in tumour development and progression.The role of NIT1 in colorectal cancer progression is not yet reported.In this study,we aim to investigate the potential role of NIT1 in the development and progression of colorectal cancer.We mainly study the expression of NITI in colorectal cancer,analyze the relationship between NIT1 expression levels and the clinicopatho logical characteristics of tumour tissue samples,investigate the functional roles and molecular mechanisms of NIT1,and eplore its upstream regulation mechanisms.METHODS:1.The public databases(Oncomine and GEO)were used to detect NIT1 expression in CRC;2.qRT-PCR,western blot and IHC assays were used to detect the expression of NIT1 in fresh surgically resected CRC tissues and paraffin-embedded CRC tissues.Then,the relationship between the expression levels of NIT1 and the clinical pathology parameters were explored;3.The stable NIT1 expressed cell lines and NIT1 repressed cell lines were established to evaluate the possible role of NIT1 in CRC proliferation by CCK8 cell proliferation assays,plate colony formation assays and nude mouse xenograft model;4.Gene Set Enrichment Analysis(GSEA)was used to reveal the enrichment of the cell cycle-related signalling pathway and the apoptosis-related signalling pathway when NIT1 was down-regulated or up-regulated.Flow cytometry assays and western blot assays were used to detect the cell cycle distribution and apoptosis;5.The public databases(BioGRID,HPRD,MINT,IntAct and PrePPI)were used to analyze interacting proteins.Co-IP assays were used to further verify the interaction between NIT1 and SARA.Western blot was used to confirm the activation of the TGF?1-Smad2/3 signalling pathway;6.The luciferase reporter assays,ChIP assays and western blot assays were performed to examine the transcriptional regulation of NIT1.qRT-PCR was used to analyze the relevance of the expression ofNIT1 and SMAD3.RESULTS:1.Down-regulation of NIT1 is correlated with CRC progression and poor prognosis.The low expression level of NIT1 protein is significantly correlated with poor differentiation(p<0.001)and more serosal invasion(p<0.001).The result of survival analysis indicates that patients with low NITl expression levels have a poor prognosis;2.The results of CCK8 cell proliferation assays and plate colony formation assays reveal that overexpression of NITl significantly restrains the cell proliferation(p<0.001;p<0.05)and knockdown of NITl yields opposite effects(p<0.001;p<0.01).Nude mouse xenograft model reveals that knockdown of NIT1 promotes tumour growth in vivo(p<0.01);3.NIT1 induces cell cycle arrest and caspase3-dependent apoptosis;4.NIT1 recruits Smad2/3 and then activates the TGFP-Smad2/3 pathway by interacting with SARA and SMAD2/3 in CRC;5.The results of the luciferase reporter assays,ChIP assays and western blot assays show that SMAD3 directly binds to the promoter regions(1678 to 1690 bp)of NITI and then enhances the transcription of NITl.Spearman correlation analyses demonstrate that NIT1 and SMAD3 are positively correlated in expression(r=0.852;p<0.0001).CONCLUSIONS:In summary,NIT1 activates the TGF?-Smad2/3 signalling pathway,which plays a key role in the suppression of CRC proliferation by interacting with SARA.In addition,the induction of TGF?1 or overexpression of SMAD3 increased the expression levels of NITl.Therefore,there was a positive feedback loop involving the regulation of CRC cell proliferation between NIT1 and activation of the TGF?-Smad signalling pathway.The increased expression of NIT1 leads to the continuous activation of the TGF?-Smad signalling pathway and then suppresses the cell proliferation of colorectal cancer.This outcome helps us to further comprehend the potential molecular mechanisms of CRC proliferation.