NCOR2/SMRT Is Involved In The Activation Of Bile Acid Nuclear Receptors To Regulate The Carcinogenesis Of Ulcerative Colitis

PART ?.FXR participates in the regulation of tumorigenicity of colon cancer cells partially in an NCOR2 dependent mannerObjective:To explore the effect and mechanism of FXR agonist in the regulation of cancer cell proliferation and migration.Methods:The expression levels of FXR and NCOR2 in common human colorectal cancer cell lines(Caco2,HT29,HCT116,SW480,SW620)and modified epithelial cell(CCD841)were screened by Western blot.The effect of FXR agonist,GW4064,on cell proliferation was detected in these cell lines by MTT assay at different concentrations.Co-immunoprecipitation was performed to measure the binding affinity of FXR and NCOR2 in vitro.After transfection of NCOR2 siRNA in the appropriate cell lines,MTT assays,Western blot,immunofluorescence staining,and qRT-PCR were used to detect the proliferation capability,?-catenin relative content,localization and relative expression of downstream genes of P-catenin as a transcription factor.Trans-well migration assays and Western blot were performed to study the migration capacity and potential mechanism in these target cell lines.Results:(1)FXR was highly expressed in Caco2 and HT29,a relatively lower level in HCT116,SW480 and CCD841,and poorly expressed in SW620.NCOR2 was highly expressed in HT29,HCT116,SW480,relatively lower expressed in Caco2 and CCD841.(2)GW4064 could significantly inhibit the proliferation capability of the above cell lines in a dose dependent manner,except SW620.(3)FXR had a strong binding affinity to NCOR2 even after FXR activation.(4)After NCOR2 RNAi silencing,the downregulation of GW4064 in cell proliferation was partially inhibited.(5)GW4064 could slightly downregulate ?-catenin expression,but the effect was not obvious in NCOR2 siRNA cell lines.(6)Further immunofluorescence staining revealed a downregulated ?-catenin expression in the nucleus after FXR activation,but the effect was not significant in NCOR2 siRNA cell lines.(7)GW4064 could inhibit the mRNA expression of ?-catenin targeted genes,but the trend was partially restricted in NCOR2 siRNA cell lines.(8)GW4064 could promote cell migration in an NCOR2 independent manner.(9)No obvious epithelial-mesenchymal transition(EMT)was observed after FXR activation.Conclusions:(1)FXR agonist,GW4064,could inhibit the proliferation capability of colorectal cancer cell lines in an NCOR2 dependent manner,partially through downregulation of the expression and transcription activity of ?-catenin.(2)GW4064 could significantly promote migration capability of colorectal cancer cells,and no obvious EMT was observed.PART II.The effect and potential mechanism of FXR activation in DSS induced colitis mice model.Objective:To explore the anti-inflammatory effect of FXR agonist,INT747,and further detect the potential mechanism in THP1 cells.Methods:Male C57BL/6 mice were divided into 3 groups.All groups were given 2.5%DSS in drinking water for 5 days except for the blank control group.The INT747 intervention group was given 5mg/(kg·d)INT747 gavage for 2 consecutive days after DSS withdrawal.All groups of mice were sacrificed on Day 7.The length of colorectum,mucosal pathological inflammation scoring and mRNA expression level of several pro-inflammatory factors were measured for inflammatory status.qRT-PCR,Western blot and immunohistochemical staining were performed to detect NCOR2 location and expression.THP1 cell line was pre-treated with 10?M INT747 for 24h and replaced with LPS containing medium for 3h before harvest The mRNA expression of TNF-?,IL-1?,IL-6 and TNF-a level in supernatant were detected.The listed experiments above were repeated in THP1 NCOR2 RNAi silencing cell line.Results:(1)The weight ratio,colorectum length,pathological inflammatory scores were slightly ameliorated in INT747 gavage group than DSS model group,but without significant difference.(2)INT747 gavage could downregulate pro-inflammatory factors expression in colorectal mucosa.(3)Western blot and immunohistochemical staining revealed NCOR2 was down-regulated in DSS model group compared with the blank control group,INT747 partially ameliorated the downregulation of NCOR2 in DSS induced colitis mucosa.(4)In THP1 cell lines,pretreatment with INT747 could ameliorate LPS induced pro-inflammatory factors(TNF-?,IL-1?)expression identified by qRT-PCR and ELISA,but not IL-6.In the NCOR2 siRNA cell line,the amelioration of INT747 on LPS induced inflammation was partially inhibited.Conclusions:(1)The FXR agonist,INT747 could ameliorate DSS induced colitis.(2)FXR activation by INT747 did not significantly influence the mRNA expression of NCOR2,but might inhibit NCOR2 degradation and promote nucleus translocation.(3)INT747 could inhibit LPS induced inflammation in THP1 cell through an NCOR2 dependent manner.