Validating The Regulation Of MiR-21in Metabolic Enzymes Of5-Fu And The Target Gene Of MiR-21in Colon Cancer Cells

Objective:It is a very tricky question about tumor patients with drug resistance in theclinical, so understanding the drug resistance mechanism is very imminent. miR-21may be playing an important role in the mechanism of drug resistance of colon cancer.This study is exploring some new clues about the drug resistance mechanisms ofcolon cancer to5-Fu for the purpose. There will be found out some new predictivefactors for clinical individualized treatment.Method:In this study, human colon cancer cell line HT-29was used for the experiment.Then, lentivirus vector with different gene fragments were choosed transfecting intothe cells to regμlate the expression level and functions of miR-21. Using luciferasereporter assay validated that hMSH2is a potential target of miR-21. In addition, tofurther evaluate the contribution of hMSH2to biological effects of miR-21, and findout whether the expression of5-Fu metabolic enzymes can be influenced by miR-21or not, this study also used two techniques such as TaqMan probe qRT-PCR andWestern Blotting for detection from RNA and protein levels.Resμlt:1. Luciferase reporter assay showed that hMSH2is a potential target gene ofmiR-21. The group with downregμlating miR-21was found obviously enhancedluciferase activity (p<0.001); and luciferase activity in the group with upregμlatingmiR-21was decreased significantly (p<0.001). The resμlts indicated that miR-21cantarget directly at the hMSH23’-UTR.2. At the RNA levels, TaqMan probe qRT-PCR was further verified that miR-21can target at hMSH2. In addition, it was also showed that miR-21may be a negativeregμlator of TP or DPD in expression (p<0.05).3. At the protein levels, the study found that the expression trends of hMSH2、TPand DPD were consistent with the RNA level under the biological effects of miR-21,by using Western Blotting. However, the expression of TS was not obviously effected.Conclusion:hMSH2is a potential target gene of miR-21, meanwhile miR-21can partlyregμlate the expression of TP and DPD, these maybe play important roles in theprocess of colon cancer resisting5-Fu. So miR-21maybe a predictive factor todetermine whether colon cancer patients shoμld accept chemotherapy with5-Fu ornot.