MiR-21-5p-mediated Effects Of Galangin Against Cholangiocarcinoma And Its Mechanism

Aim Galangin is a natural flavonoid compound extracted from tubers of Alpinia oficinarum Hance,which has mutiple anti-cancer activity.However,it is not reported whether galangin has anti-cholangiocarcinoma activity.Thus,this study will explore whether the effects of galangin against intrahepatic cholangiocarcinoma cell line HCCC9810 and human extrahepatic cholangiocarcinoma cell line TFK-1 on the proliferation,migration,invasion,and apoptosis exist.And we want to clear the mechanism of galangin against CCA cells if it exists.Methods Human intrahepatic cholangiocarcinoma cell HCCC9810 and extrahepatic cholangiocarcinoma cell TFK-1 were used for research models,the IC₅₀ value of galangin against HCCC9810 and TFK-1 was obtained through the CCK-8 experiment,and the applied concentration of the subsequent experiment was determined;the effects of galangin on the proliferation ability of HCCC9810 cells were detected by Ed U assay;FACS was used to evaluate apoptosis of galangin-treated HCCC9810 and TFK-1;the effects of galangin on migration and invasion ability of HCCC9810 and TFK-1 were analyzed by Transwell assay and scratch assay;the expression level of mi R-21-5p in human cholangiocarcinoma cell HCCC9810 was detected by q RT-PCR experiment;Western blot experiments were used to examine Bcl2,Bax,MMP9,Vimentin,PTEN,AKT,P-AKT protein expression levels of galangin-treated HCCC9810and extrahepatic cholangiocarcinoma cell line TFK-1;EDU,Transwell,FACS,q RT-PCR,Western blot were used for mi R-21-5p-transfected HCCC9810/TFK-1 cells galangin-treated to detect whether mi R-21-5p mediates the anti-cholangiocarcinoma effects of galangin.Results A.（1）The activity of HCCC9810 and tfk-1 were significantly inhibited by gariangin（P<0.05）,and the calculated IC50 values were108.2umol/L and 149.7umol/L,respectively.After comprehensive consideration,150umol/L was taken as the subsequent experimental concentration.（2）Ed U experimental results showed that the average Ed U positive rate of HCCC9810treated with galangin for 24h was lower than that of the control group（P<0.05）,suggesting that galangin could significantly inhibit the proliferation of cholangiocarcinoma cells.（3）FACS results showed that the average early apoptosis rates of HCCC9810 and TFK-1 after treatment with galangin for 24hours were significantly higher than those of the control group（P<0.05）.WB results showed that the average Bax/Bcl2 in HCCC9810 and tfk-1 treated with galangin for 24h was significantly higher than that in the control group（P<0.05）,suggesting that galangin could significantly induce apoptosis of CCA cells.（4）Transwell migration assay results showed that the average number of cells passing through the polycarbonate membrane of HCCC9810 and TFK-1 treated with galangin for 24h was significantly smaller than that of the control group（P<0.05）.Transwell invasion assay showed that the average number of cells crossing the polycarbonate membrane in HCCC9810 and TFK-1 treated with galangin for 24h was significantly smaller than that in the control group（P<0.05）.WB results indicates MMP9 protein expression of HCCC9810 and TFK-1 treated by galangin for 24h were less than the control group（P<0.05）,Vimentin protein expression of HCCC9810 and TFK-1 treated by galangin for24h were less than the control group,respectively（P<0.05）,suggesting galangin inhibits CCA cells migration and invasion.B.（1）the Ed U test was conducted after transfection of HCCC9810 with100nmol/L mir-21-5p antagomir and antagomir NC for 24h.The results showed that the average Ed U positive rate of CCA cells in the mi R-21-5p antagomir group was lower than that in the control group,suggesting that the knockdown of mi R-21-5p significantly inhibited the proliferation of CCA cells（P<0.05）.Therefore,we then transfected HCCC9810 with 100nmol/L mi R-21-5p agomir to overexpression of mi R-21-5p for 24h,and then treated it with galangin for24h to explore the molecular mechanism of galangin in anti-cholangiocarcinoma effects.（2）RNA was collected for q RT-PCR,and the results showed that the expression of mi R-21-5p in the treatment group was lower than that in the control group（P<0.05）,indicating that the expression of mi R-21-5p in the treatment group was significantly lower than that in the control group（P<0.05）.The expression of mir-21-5p in the mi R-21-5p agomir+galangin group was higher than that in the agomir NC+galangin group（P<0.05）,suggesting the success of mi R-21-5p overexpression by mi R-21-5p agomir.（3）the Ed U results showed that the average Ed U positive cell rate of the mi R-21-5p agomir+galangin group was higher than that of the agomir NC+galangin group,suggesting that overexpression of mir-21-5p could partially abolish the anti-proliferation effect of galangin on CCA cells（P<0.05）.（4）FACS results showed that the average early apoptosis rates of mir-21-5p agomir+galangin in the HCCC9810 and tfk-1 groups were significantly lower than those in the control group（P<0.05）.WB results showed that the average Bax/Bcl2 of the HCCC9810 and TFK-1 mi R-21-5p agomir+galangin group was significantly lower than that of the control group（P<0.05）,suggesting that overexpression of mi R-21-5p could partially reverse the pro-apoptotic effect of galangin on CCA cells.（5）Transwell migration results showed that the average number of cells crossing the polycarbonate membrane in the mi R-21-5p agomir+galangin groups were significantly higher than that in the control group significantly（P<0.05）.Transwell invasion assay showed that the average number of cells crossing the polycarbonate membrane in the HCCC9810 and TFK-1 mi R-21-5p agomir+galangin groups was higher than that in the control group significantly（P<0.05）.The scratching experiment showed that the average migration distance of HCCC9810 mi R-21-5p agomir+galangin group was higher than that of the control group significantly（P<0.05）.WB results showed that the average expression levels of MMP9 in HCCC9810 and TFK-1 in mi R-21-5p agomir+galangin group were increased compared with the control group significantly（P<0.05）,and the average expression levels of Vimentin were increased compared with the control group significantly（P<0.05）,suggesting that overexpression of mi R-21-5p could partially abolish the anti-migration and anti-invasion function of galangin against CCA cells.C.WB results showed that the average expression level of PTEN protein in the galangin group was higher than that in the control group significantly（P<0.05）.The average expression level of p-akt in the galangin group was was lower than that in the control group significantly（P<0.05）.The average expression level of PTEN protein in the group of mi R-21-5p agomir+galangin was lower than that in the group of agomir NC+galangin significantly（P<0.05）.The average expression level of p-akt in the mi R-21-5p agomir+galangin group was significantly higher than that in the agomir NC+galangin group（P<0.05）.These results suggested that galangin could significantly increase the expression level of PTEN protein in CCA cells and significantly reduce the expression level of P-AKT,and these effects could be partially abolished by overexpression of mi R-21-5p.Conclusion（1）Galangin possess the anti-human intrahepatic cholangiocarcinoma cell HCCC9810 and extrahepatic cholangiocarcinoma cell TFK-1 effect.（2）mi R-21-5p mediates the anti-cholangiocarcinoma activity of galangin segmentally through regulation of PTEN/AKT signaling possible.