Effect Of Butyric Acid On Epithelial-mesenchymal Transformation Of Colorectal Cancer Cells

Objective:Colorectal cancer is one of the most common malignant tumors in the world,and its mortality rate is on the rise.The main reason for the high mortality of colorectal cancer is that colorectal cancer is prone to metastasis,and epithelial-interstitial transformation is considered to be the primary link of colorectal cancer metastasis.Butyric acid is a kind of short-chain fatty acid produced by insoluble dietary fiber fermentation in the intestinal tract.Studies have shown that butyric acid can affect the process of epithelial-interstitial transformation.Therefore,the purpose of this study is to observe the effect of butyric acid on epithelial-mesenchymal transformation of colorectal cancer cells was observed in vitro and in vivo.And explore the possible mechanism of its role.Methods:Mouse colorectal cancer cell line CT26,human colorectal cancer cell line HT29and human colorectal cancer cell line SW620 were inoculated into 96-well plate and treated with 1.25 mM,2.5 mM,5 mM and 10 mM sodium butyrate(NaB),respectively.Another control group was set up.CCK8 assay was used to determine the inhibitory effect of different concentrations of NaB on the proliferation of different colorectal cancer cells.Wound healing test and transwell chamber migration test were used to detect the effect of NaB on the migration ability of three colorectal cells.Matrigel invasion test was used to detect the effect of NaB on cell invasiveness.The effect of NaB on the adhesion between cells and extracellular matrix was detected by cell-matrix adhesion assay.The effects of NaB on transforming growth factor?(TGF-?),E-cadherin,Vimentin mRNA and protein were detected by RT-PCR and Western-blot,respectively.Under aseptic condition,the liver metastasis model of in situ colorectal cancer was established by injecting 0.2 mL(cell concentration 2×10~6 cells/mL),into the cecal serous layer of mice.The mice were randomly divided into 4 groups with 15 mice in each group,as follows:high dose butyric acid group(glycerol tributate 3g/kg),middle dose butyric acid group(glycerol tributate 1 g/kg),low dose butyric acid group(glycerol tributate 0.3 g/kg)and saline group.Weigh them once a week.Each group was given intragastrically,starting from the 3rd day after operation,once a day.At the end of the experiment,the mice were killed and the tumor weight and the number of liver metastases were recorded.The expression of TGF-?,E-Cadherin,N-Cadherin,Vimentin,matrix metalloproteinase-2(MMP-2)and matrix metalloproteinase-9(MMP-9)mRNA were detected by real-time quantitative PCR(RT-PCR).The expression of TGF-?,E-Cadherin,N-Cadherin and Vimentin protein was detected by Western-blot.The expression of MMP-2 and MMP-9protein was detected by immunohistochemistry.Results:The results of CCK8 assay showed that the proliferation of CT26,HT29 and SW620 cells was significantly inhibited by different concentrations of butyric acid(F=281.899,13.009,116.209,respectively;P<0.001),and showed a dose-effect relationship with the increase of intervention concentration.The results of wound healing test and Transwell chamber migration test showed that butyric acid inhibited the migration of three colorectal cancer cell lines in a dose-dependent manner with the increase of intervention concentration(F=2.028~640.901,P<0.001).Matrigel invasion assay showed that butyric acid could inhibit the invasiveness of three colorectal cancer cell lines(F=1145.075,195.888,390.464,respectively;P<0.05),and the higher the intervention concentration,the more obvious the inhibitory effect on colorectal cancer cells.The results of cell-matrix adhesion test showed that butyric acid decreased the adhesion of three colorectal cancer cell lines to extracellular matrix,and with the increase of butyric acid intervention concentration,he more obvious the inhibitory effect on the adhesion of colorectal cancer cells(F=8.079,6.171,14.874,respectively;P<0.005).Different concentrations of butyric acid could inhibit the expression of TGF-?and Vimentin protein in CT26,HT29 and SW620 cells,while the mRNA and protein expression of E-Cadherin were significantly up-regulated(F=4.041~451.611,P<0.05).In vivo,butyric acid significantly inhibited the liver metastasis rate of colorectal cancer in mice.The liver metastasis rate of colorectal cancer in the control group was 30.77%,and the liver metastasis rate of colorectal cancer in the low,middle and high dose groups was7.14%,0.00%and 14.29%,respectively,which was lower than that in the control group.The results of RT-PCR test and Western-blot test showed that butyric acid could decrease the expression of TGF-?,Vimentin,N-Cadherin,MMP-2,MMP-9 mRNA and protein,and promote the expression of mRNA and protein in E-Cadherin(F=1.593~862.335,P<0.05).Conclusion:1.Butyric acid inhibited the proliferation,migration,invasion and adhesion of CT26,HT29 and SW620 cells.2.Butyric acid can inhibit the liver metastasis of colorectal cancer in situ in mice.3.Butyric acid can inhibit the expression of epithelial-interstitial transformation related proteins TGF-?,Vimentin,N-Cadherin,MMP-2,MMP-9,promote the expression of E-Cadherin,and inhibit the epithelial-mesenchymal transformation of colorectal cancer.4.Butyric acid can inhibit tumor metastasis by inhibiting epithelial-mesenchymal transformation of colorectal cancer cells.