| Part One:The effect and mechanism of bile acid receptor FXR on Wnt/p-catenin signal pathway in colon cancer cells.Objective:To explore the effect and mechanism of bile acid receptor FXR on Wnt/p-catenin signal pathway in colon cancer cells.Method:The expression levels of FXR and ?-catenin in human colon cancer cell lines HT-29,Caco-2 and HCT-116 were detected by Western blot.These cells were subjected to transfecting siRNA to silence FXR,followed by measurement of the transcriptional activity of ?-catenin by Dual-Luciferase(?)Reporter Assay System,and the protein level of ?-catenin by Western blot.These cells were treated with agonist of FXR GW4064 at different concentrations(1,3 and 5umol/L)for 12,24,36 and 48h respectively.Cell proliferation was quantified by MTT assay.These cells were treated with either GW4064 or vehicle,and measured the transcriptional activity of NF-?B by EMSA,then several about Wnt/?-catenin signal pathway were measured:(1)the transcriptional activity of?-catenin by Dual-Luciferase(?)Reporter Assay System,(2)the level of ?-catenin/TCF4 comlex by EMSA,(3)the total and nuclear protein expression levels of ?-catenin by Western blot;(4)the mRNA expression level of P-catenin by qRT-PCR.With the influence of GW4064,we add agonist of NF-?B signaling pathway,LPS,then repeat experiments listed above.Results:(1)HT-29 and Caco-2 cells highly expressed FXR,HCT-116 expressed less than them,the expression level of FXR and ?-catenin are different in these cell lines;(2)Wnt signal pathway in HT-29,Caco-2 and HCT-116 cells would be actived when FXR was silenced(HT-29:1.47 ± 0.14 vs 1.04 ± 0.03,Caco-2:5.18 ± 0.41 vs 3.63 ± 0.32,HCT-116:4.66 ± 0.43 vs 3.22 ± 0.26,p<0.05),without the influence of the protein level of ?-catenin;(3)The proliferation of HT-29,Caco-2 and HCT-116 cells was significantly inhibited by GW4064 in a dose and time dependent manner(HT-29 3uM 24h:0.48±0.03 vs 0.74±0.03,Caco-2 3uM 24h:0.31±0.01 vs 0.59±0.05,HCT-116 5uM 24h:0.18±0.01 vs 0.43±0.04,p<0.05);(4)GW4064 promoted the transcriptional activity of NF-?B in HT-29,Caco-2 and HCT-116 cells;(5)GW4064 promoted the activity of Wnt signal pathway in HT-29,Caco-2 and HCT-116 cells(HT-29:1.14±0.02 vs 0.87±0.04,Caco-2:3.30±0.14 vs 2.11±0.10,HCT-116:2.89±0.26 vs 1.28±0.07,p<0.05)via elevating the level of ?-catenin/TCF4 comlex,while didn't affect its mRNA and protein levels;(6)Under the influence of LPS,GW4064 also promoted the activity of Wnt signal pathway in HT-29,Caco-2 and HCT-116 cells(HT-29:1.15±0.03 vs 0.99± 0.03,Caco-2:5.24 ± 0.04 vs 3.91 ± 0.03,HCT-116:8.05 ± 0.22 vs 3.70 ± 0.05,p<0.05)via elevating the level of ?-catenin/TCF4 comlex.Conclusion:Loss of FXR promotes the transcriptional activity of ?-catenin.The agonist of FXR GW4064 significantly inhibits the proliferation of human colon cancer cells,but promotes the transcriptional activity of ?-catenin.FXR is closed with wnt pathway,and gain of the function of FXR shows different results.Part Two:The effect of probiotic on bile acid receptor FXR and its down regulatory proteins in Ulcerative Colitis related colorectal cancer animal model.Objective:To explore the the influence of bile acid receptor(FXR)by Probiotics on Ulcerative Colitis Carcinogenesis on AOM/DSS induced UC carcinogenesis mice model.Method:C57BL/6 mice were divided into 4 groups.Group 1-3 were given AOM 12.5mg/kg by intraperitoneal injection,and one week later 2.5%DSS in drinking water was fed for 5 days to establish UC carcinogenesis model.Group 1 was Saccharomyces boulardii group,which was given Saccharomyces boulardii 1.5g/kg/d by lavage every day;Group 2 was VSL#3 group,which was given VSL#3 1.5g/kg/d by lavage every day;Group 3 was served as model control group;Group 4 served as normal control.Mice were sacrificed in the end of the 12th week.Macroscopic evaluation of tumor load(TL)as well as pathologic evaluation was carried out to assess carcinogenesis severity.The mRNA expression level of Nr1h4 and Fgf15 was determined by qRT-PCR.The protein expression level of FXR,SHP/NROB2 and FGF15 was determined by immunohistochemistry.The protein level of FGF15 in serum was determined by ELISA.Results:(1)The tumor load of Saccharomyces boulardii group(n=19,TL=0.20±0.07cm),VSL#3 group(n=20,TL=0.25±0.07cm)were significantly lower than model control group(n=19,TL=0.97±0.19cm)(p<0.05).(2)In term of Nrlh4 and Fgf15 mRNA expression level in colon mucosa cells:the Nrlh4 and Fgfl5 mRNA expression in model control group was lower than blank control group(n=6,Nr1h4 0.53 ± 0.08 vs 1.10 ±0.09;Fgf15 0.80 ± 0.16 vs 1.42 ± 0.14),those in Saccharomyces boulardii group(n=6,Nr1h4 1.29 ± 0.15,Fgf15 1.87 ± 0.26)and VSL#3 group(n=6,Nr1h4 1.31± 0.21,Fgf15 2.16±0.34)were higer than model control group(p<0.05).(3)In term of FXR,SHP/NROB2 and FGF15 protein level in colon mucosa cells:the FXR expression in model control group was lower than blank control group,that in Saccharomyces boulardii group and VSL#3 group were higer than model control group;the SHP/NROB2 and FGF15 expression in model control group was lower than blank control group,that in Saccharomyces boulardii group and VSL#3 group were similar with that in model control group.(4)In term of FGF15 protein level in serum:expression of FGF15 in model control group was significantly lower than blank control group(n=6,0.51 ± 0.33 vs 2.87±0.54),those in Saccharomyces boulardii group(n=10,2.03±0.46)were higer than model control group(p<0.05),VSL#3 group(n=10,0.58± 0.21)were similar with that in model control group.Conclusion:Probiotics can inhibit the tumor formation of UC carcinogenesis induced by AOM/DSS in mice.The mechanism maybe related with that probiotics can up-regulation FXR-FGF14 axis. |
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