Expression Of RUNX3 In Human Colon Cancer Cell Lines Hct-116 Resistant To 5-fluorouracil And Analysis Of The Drug Resistance Mechanism

This study aimed to establish a human colon cancer cell line HCT-116/5-FU,resistant to 5-fluorouracil(5-FU)and investigate the relationship between Runt-related transcription factor 3(RUNX3)and drug resistance of colorectal cancer.Establishment of human colon cancer drug-resistant cell line by low concentration gradient increment combined with high-dose intermittent impact method HCT-116/5-FU,CCK-8 method was used to determine the50% inhibitory concentration of 5-FU on parent strain HCT-116 and drugresistant strain HCT-116/5-FU,draw the cell growth curve,and calculate the cell doubling time.Knockdown of RUNX3 expression in HCT-116 cells by si RNA technique divided into RUNX3 knockout group(siRUNX3-1 group),(si-RUNX3-2 group),and negative control group(siNC group).The knockdown efficiency of RUNX3 was verified by q RTPCR and western blot at the m RNA and protein levels.A stable human colon cancer drug-resistant cell line HCT-116/5-FU was successfully constructed.The doubling time of HCT-116/5-FU drugresistant cell line was longer than that of HCT-116 parent cell line(P<0.001).The TD of drug-resistant cell population was 1.38 times higher than that of parent cells,(P=0.002).and the cell morphology of drugresistant cell line changed.The expression level of RUNX3 m RNA in drug resistant cell line HCT-116/5-FU was significantly lower than that in parent cell line HCT-116(p=0.048).The expression level of P-gp,MRP1 and LRPm RNA in drug resistant cell line HCT-116/5-FU was significantly higher than that in parent cell line HCT-116(p=0.008,p=0.001,p=0.001).The relative expression of RUNX3 protein in drug-resistant cell line HCT-116/5-FU was significantly lower than that in parent cell line HCT-116(p<0.001).The relative expression of P-gp,MRP1 and LRP protein in drug-resistant cell line HCT-116/5-FU was significantly higher than that in parent cell line(all p<0.001).The HCT-116 cell model with low expression of RUNX3 was successfully constructed.There was no significant difference in the expression of RUNX3 m RNA between siRUNX3-1 group and si-NC group(P =0.064),but the expression of RUNX3 m RNA in si-RUNX3-2 group was significantly lower than that in si-NC group(P=0.034),and the expression of RUNX3 protein in siRUNX3-1 group and si-RUNX3-2 group was lower than that in si-NC group(all p < 0.001),And the knocking efficiency of si-RUNX3-2 group is higher.si-RUNX3-2 group was selected to complete the follow-up test.The IC50 of si-RUNX3 group was significantly higher than that of si-NC group(P<0.001).Down-regulation of RUNX3 expression could reduce the sensitivity of HCT-116 cells to 5-FU.The relative expressions of P-gp,MRP1 and LRP proteins in si-RUNX3 group were significantly higher than those in si-NC group(all p<0.001).HCT-116/5-Fu cells showed stable drug resistance,and the expression level of RUNX3 in the drug-resistant strains was lower than that in the parent strains.The expressions of P-gp,MRP1 and LRP in the si-RUNX3 group were higher than that in the si-NC group.Meanwhile,the loss of RUNX3 expression reduced the sensitivity of HCT-116 cells to 5-Fu.The loss of RUNX3 expression may increase the resistance of cells by upregulating the expression of P-gp,MRP1,LRP and other drug-resistant proteins.