Analysis Of The Internal Ribosomal Entry Site Of P-gp MRNA And Research On The Relationship Between The IRES And MDR

Multi-drug resistance (MDR) is one of the major causes of chemotherapy failure in current clinical cancer therapy. Previous studies have showed the abnormal expression of some drug resistant factors related to the mechanism of MDR. Such as P-glycoprotein (P-gp), MDR related protein 1 (MRP1) and breast cancer resistance protein (BCRP), and P-gp is one of the most important resistance-associated proteins. Although the structure and function of these drug resistant factors have been widely reported, the reason about their abnormal expression in MDR cells is still poorly understood. In this paper, the abnormal expression was analyzed at the translation level, and it's expected that a new gene sequence IRES (internal ribosomal entry site) which may regulate the expression of MDR related protein could be found finally.In this paper, we chose human breast cancer cell line MCF-7/W (wild type) and MCF-7/MDR (drug resistance type) as material. The drug resistance property of the MDR cell line was identified by SEM (scanning electron microscope) and drug sensitivity test; and the expression of MDR related proteins were studied by immunocytochemistry assay and western blot. The results showed only the expression of P-gp significantly increased in MDR cells versus wild type cells, while the other MDR related proteins had no significant variety in expression. Therefore, these data together indicate the possibility to use P-gp mRNA to detect the IRES activity.The red and green fluorescent protein genes were used as reporter genes, the EMCV (encephalomyocarditis virus) 5'-UTR (untranslated region) was used as positive IRES to establish the dicistronic expression plasmid to detect IRES activity of P-gp mRNA 5'-UTR. The expression efficiency of the reporter genes was analyzed by fluorescence microscopy, fluorescence spectrophotometer and Western Blot. Results showed that additional bases at the end of EMCV IRES significantly affected the IRES activity of EMCV; EGFP reporter gene may contain IRES element itself, so it isn't suitable as the downstream reporter gene of IRES sequence; EGFP can be used as upstream reporter gene and DsRed can be used as downstream reporter gene for IRES activity analysis. Finally we got the best plasmid pCI-EGFP- MDR1DsRed for activity analysis of P-gp mRNA 5'-UTR. The indicated plasmid was then transfected into 293T and MCF-7 cells. According to the results, the 5'-UTR of P-gp mRNA showed IRES activity exactly, but the activity was much weaker than EMCV.Then the cells thansfected with plasmid pCI-EGFP-MDR1DsRed were treated with rapamycin. As a result, the cap-dependent translation had not been inhibited and the cap-independent translation also had not been activated. It may be caused by the possibility that EGFP contains IRES activity itself and the poor IRES activity of P-gp mRNA 5'-UTR.In this paper, we proposed and demonstrated that there exists IRES activity of P-gp mRNA 5'-UTR for the first time. But the relationship between the IRES activity of P-gp mRNA 5'-UTR and the upregulation of P-gp under the presure of chemotherapy drugs remains to be further studied.