| Purpose:This study is based on colon cancer cells and the establishment of resistant cell models of colon cancer.With the aid of molecular biological and cell biological advanced technology,to investigate the effection of oxymatrine on the colon cancer cells and resistant cells and to detect the effection of oxymatrine on the TGF-?1/P38/PAI-1 signal pathways in vitro experiment.And the mechanism of EMT promoting the resistance and transfer of colon cancer cells were explored.At the same time,to clarify the molecular mechanism of reversing EMT of colon cancer by oxymatrine and explain the antitumor effects of oxymatrine to reverse the colon multi-resistant to provide new research direction for reversing multidrug resistance of colon cancer.Material and method:1.To determine the effect of oxymatrine on the role of EMT in colon cancer cells.1.1 HT-29 cells and RKO cell were experimental groups.The normal colonic epithelium FHC cells was control group,western blots were used to detect the protein expression of EMT in each group,including epithelial cell markers?E-cadherin?and mesenchymal cell markers??-SMA,FN,PAI-1?.1.2 The groups were as follow:oxymatrine+HT-29 cell group,oxymatrine+RKO cell group,HT-29 cell group,RKO cell group,and then CCK8 testing was used to evaluate cell proliferation toxicity of oxymatrine for HT-29 cell and RKO cell.1.3 The groups were as follow:oxymatrine+HT-29 cell group,oxymatrine+RKO cell group,HT-29 cell group,RKO cell group,and then the scratch test was used to evaluate effect of oxymatrine in inhibiting HT-29 and RKO cell migration ability.1.4 In order to remove cytotoxicity of oxymatrine on RKO cell and HT-29 cell,0.50 mg/mL oxymatrine was selected for 24 hours according to the results of CCK8.The expression levels of E-cadherin and?-SMA in HT-29 cells and RKO cells were detected by western blots.At the same time,ECM deposition is caused by the EMT process,and the aggregation of ECM related proteins is a key factor in tumor cell migration.FN and PAI-1 are one of the ECM related proteins and play an important role in the accumulation of ECM.We used western blots to detect the expression levels of FN and PAI-1 in HT-29 cells and RKO cells before and after the action of oxymatrine.2.The establishment of resistant cell models of colon cancer.2.1 the transgenic method was used to establish the drug resistance cell line HT-29/L-OHP of colon cancer.Design primers in accordance with the principle of primer design,using the method of scratches to inoculate of e.coli,CaCl2 method was prepared for characteristic of bacteria,converse the recombinant plasmid DNA,using containing kanamycin?kan?of AGAR plate to screen strains containing recombinant plasmid DNA,a small amount of extraction of recombinant plasmid DNA by extraction kit,and the extraction of positive plasmid was detected by PCR test after enzyme digestion and was took to invitrogen company for sequencing validation.After the successful verification,the recombinant plasmid DNA was extracted using the extraction kit.After large-scale conversion,the liposomes 2000 mediated transfection.2.2 the concentration gradient method was used to construct the HT-29/L-OHP resistant cell model of oxaliplatin in colon cancer.The beginning concentration of oxaliplatin was 5 ?mol/L?1/3 IC50?,and when the cell was growing logarithmically again,it continued to be cultured for 2 to 3 weeks,and then the cell was carried out,and the concentration of oxaliplatin was increased to 8?mol/L.And then increase the concentration of oxaliplatin in the order of 11?mol/L,13?mol/L,15?mol/L until the cells no longer grew logarithmically.2.3 The morphology of HT-29/L-OHP and HT-29/NL-OHP cells and the transfection of transgenic cells were observed by fluorescence microscope and ordinary microscope,respectively.2.4 CCK8 method was used to detect the proliferation inhibition rate of oxaliplatin for HT-29/L-OHP and HT-29/NL-OHP cells,and the drug resistance rate was calculated by IC50in the control group of HT-29.2.5 CCK8 method was used to detect the proliferation inhibition rate of 5-fluorouracil on HT-29/L-OHP and HT-29/NL-OHP cells,and the drug resistance rate was calculated by IC50in the control group of HT-29.2.6 the CCK8 method was used to detect the proliferation inhibition rate of irinotecan on both HT-29/L-OHP and HT-29/NL-OHP cells,and the drug resistance rate was calculated by IC50in the control group of HT-29.2.7 We use western blots to detect changes of epithelial markers?E-cadherin?and the markers of ectomesenchymal cells??-SMA,FN,PAI-1?in drug-resistant cell HT-29/L-OHP.HT-29 cells were as control group to confirm whether resistance occurred in the process of EMT.2.8 With colon cancer HT-29 cells as control group,Transwell invasion and migration experiments were used to detect change of cell invasion and migration in drug-resistant cell HT-29/L-OHP due to protein level changes in the process of EMT.2.9 TGF-?1signaling pathway is one of the main signaling pathways controlling EMT.With colon cancer HT-29 cells as control group,use western blot method to detect the expression of TGF-?1,Smad2,Smad4 and P38 to verify whether TGF-?1signaling pathway is activated in HT-29/L-OHP.2.10 We used the P38 blocker SB203580,then western blots were used to detect the changes of Smad2 and Smad4 protein expression levels in HT-29/L-OHP before and after adding blockers.3.Detecting effects of oxymatrine reversing EMT and drug resistance.3.1 The cell groups:oxymatrine+oxaliplatin group,oxaliplatin group,oxymatrine+5-fluorouracil group,5-fluorouracil group,oxymatrine+irinotecan group,irinotecan group.CCK8 method was used to test cell proliferation rate of each group to calculate resistance reversal rate in drug-resistant cell HT-29/L-OHP.3.2 We used HT-29 cells as the control group to detect the effect of oxymatrine on the migration of drug-resistant cell HT-29/L-OHP using transwell migration experiment.3.3 The expression levels of key proteins in TGF-?1/P38/PAI-1 signaling pathway in HT-29/L-OHP cell were detected by western blots method after different concentrations of oxymatrine were added.3.4 pEGFP-TGF-?1-shRNA recombinant plasmid was transfected into resistant cells HT-29/L-OHP by liposome 2000,and fluorescence microscopy was used to observe the transfection effect after transfection.3.5 In order to clarify the molecular mechanism of EMT,we used western blots and RT-PCR to compare the expression levels of PAI-1 protein and mRNA expression in HT-29/L-OHP cell and HT-29 cell.After recombinant plasmid pEGFP-TGF-?1-1-shRNA and pEGFP-TGF-?1-2-shRNA were transfected into resistant cells to interfere with TGF-?1 mRNA expression,the PAI-1 protein expression and mRNA expression of each group were detected by western blots and RT-PCR.3.6 After recombinant plasmid Pegfp-TGF-?1-1-shRNA and pEGFP-TGF-?1-2-shRNA were transfected into resistant cells to interfere with TGF-?1 mRNA expression,CCK8 method was used to detect changes of cell proliferation inhibition rate of oxaliplatin with or without oxymatrine.We used the P38 blocker SB203580,CCK8 method was used to detect changes of cell proliferation inhibition rate of oxaliplatin with or without oxymatrine.Results:1.To determine the effect of oxymatrine on the role of EMT in colon cancer cells.1.1 Comparing with the normal colonic epithelium cells FHC,protein expression of epithelial markers?E-cadherin?was significantly reduced or even disappear in HT-29 and RKO colon cancer cells,and protein expression of ectomesenchymal cells markers??-SMA,FN,PAI-1?was increasing.EMT change were more obvious in colon cancer cells.1.2 The inhibitory effect of oxymatrine on the proliferation inhibition of HT-29 cells and RKO cells showed time and dose dependence.1.3 After 24 hours,the scratch width of RKO cell and HT-29 cell plus oxymatrine were bigger than the cells without adding oxymatrine?P<0.05?,HT-29 cells and RKO cell migration ability was inhibited by oxymatrine.1.4 After using 0.50 mg/mL oxymatrine in the HT-29 cells and RKO cell,epithelial markers E-cadherin protein expression level was promoted,but the protein expression level of ectomesenchymal marker of?-SMA was lower?P<0.05?.1.5 Compared with the control group,the treatment of 0.50 mg/mL oxymatrine could inhibit the elevated protein expression level of FN and PAI-1?P<0.05?.2.The establishment of resistant cell models of colon cancer.2.1 RT-PCR results of pEGFP-TGF-?1 transfection proved successful pEGFP-TGF-?1transfection into HT-29 cells.2.2 Under the microscope,the drug resistant cell lines of HT-29/L-OHP and HT-29/NL-OHP appeared as a fusiform or spindle shape,cell isolation,connection between cells was reduced or even had disappear,pseudopods extended.These were completely different from the form of the parent cell HT-29 in an elliptical or irregular polygon.2.3 For two kinds of drug-resistant cell HT-29/L-OHP and HT-29/NL-OHP cells,the proliferation inhibition rate of oxaliplatin was lower than the normal colon cancer HT-29 cells.The resistance index was 11.14 and 11.64,respectively,which proved colon cancer drug-resistant cell models were successfully builded.2.4 For drug-resistant cell HT-29/L-OHP,the proliferation inhibition rate of5-fluorouraciland and irinotecan were lower than the normal colon cancer HT-29 cells.The resistance indexes were 3.5 and 7.91,respectively,which proved colon cancer multiple drug resistance model was successfully builded.2.5 Compared with colon cancer cell HT-29,the expression levels of?-SMA,FN and PAI-1protein were significantly higher than those in the control group,while the protein expression level of the epithelial marker E-cadherin was down-regulated.2.6In the transwell invasion and migration experiment,the number of HT-29/L-OHP cells in the micropore membrane was significantly higher than in the colon cancer cell HT-29 group,which was 1.9 and 2 times,respectively.2.7 Compared with the parent cell HT-29 cells,the protein expression levels of TGF-?1,Smad2 and Smad4 in the drug-resistant HT-29/L-OHP cells increased,while the P38protein of HT-29/L-OHP was also increased positively.2.8 In the drug-resistant HT-29/L-OHP group,P38 blocker SB203580 inhibits the protein expression level of Smad2 and Smad4 caused by TGF-?1.3.Detecting effects of oxymatrine reversing EMT and drug resistance.3.1 Compared with the use of oxaliplatin alone,oxaliplatin plus oxymatrine had a significant increasing in the proliferation inhibition rate of HT-29/L-OHP,and the drug resistance index was 4.84.Similarly,the drug resistance reversal index of 5-fluorouracil and irinotecan of oxymatrine was 2.41 and 4.93,respectively.3.2 Compared with drug-resistant cells HT-29/L-OHP,the number of cell in transwell membrane of drug-resistant cells HT-29/L-OHP was decreased significantly at 1.8 times by oxymatrine.3.3 The expression of E-cadherin protein was gradually enhanced with the increasing of the concentration of oxymatrine,and the expression of PAI-1 protein was gradually weakened.3.4 Recombinant plasmid pEGFP-TGF-?1-shRNA was successfully transfected into HT-29/L-OHP resistant cells and the RT-PCR showed TGF-?1 gene is obvious degradation.3.5 In RNA interference group,the outline of the cells was changed from fusiform or spindle shape into irregular polygon or rounded,which was close to the normal non-resistant colon cancer cells.3.6The protein expression level and mRNA level of PAI-1 were significantly decreased compared with that of the null transfection group and the control group?no treated HT-29/L-OHP resistant cells?.3.7 After TGF-?1 mRNA was interfered,oxymatrine had no significant change for increasing in the proliferation inhibition rate of oxaliplatin.After P38 was blocked,oxymatrine had no significant change for increasing in the proliferation inhibition rate of oxaliplatin.Conclusion:1.Oxymatrine can inhibit colon cancer cell proliferation and migration,accompanied by down-regulation of PAI-1 and FN protein expression,probably by inhibiting protein expression of PAI-1 and FN to reverse EMT change of colon cancer cells and achieve anticancer effect.2.The EMT change was caused by TGF-?1 gene and its TGF-?1/P38/Smad2 signaling pathway is one of the molecular mechanisms of drug resistance in colon cancer cells.3.Oxymatrine can reverse colon cancer drug resistance,and this effect presents the concentration dependent.And the gene transfection technology confirmed that oxymatrine reverse colon cancer drug resistance extremely likely by acting on TGF-?1/P38/PAI-1signaling pathways via reversing EMT change of colon cancer cells. |
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