Mice In The Negative Regulation Of Rna Interference Mechanism Of Rna Interference In The Preliminary Study In Cancer Therapy Application

RNA interference(RNAi)is a gene silencing mechanism conserved in evolution from worm to man and has been a powerful tool for gene functional research. A lot of research groups have begun to do some researches about gene function, signal transduction pathway and the initial mechanisms of diseases. Although recently some proteins of regulating RNA silencing machinery have been identified, there is little information on the regulations of RNAi. In this work, we used esiRNA(E. coli expressed and enzyme digested siRNA) to do some exploration on negative regulation mechanism of RNAi in vitro and vivo. At the same time, we used combined esiRNA to inhibit mouse B16 melanoma cell growth in vitro and vivo.In this thesis, we firstly tested the expression inhibition of transgene of HBV surface antigen by different amounts of esiRNA in the presence or absence of mouse orthologue of eri-1. Our results revealed an unexpected decrease of inhibition effect that was associated with the introduction of higher dose of esiHBVP, compared with lower dose of esiHBVP. Blocking mouse eri-1 gene expression could rescue RNAi effect of higher dose of esiHBVP in mouse liver. We secondly used different doses of esiC-myc to inhibit B16 melanoma growth in C57BL6/6J mice. Our data showed that suppression of c-myc oncogene overexpression and anti-tumor effect in high dose esi C-myc treatment groups were less efficient than those in low dose groups. Thus, we introduced esiMERI-1 and esiADAR-1 into mice. The effectiveness of tumor growth inhibition in the high dose esiC-myc treatment groups were rescued successfully finally. We finally used single kind or combined kinds of esiRNAs to inhibit mouse B16 melanoma growth in vitro and vivo. Our result showed that the combination of different esiRNAs agents can produce additive effect to significantly minimize tumor size and tumor growth rate.In the first chapter of this thesis, we used secretary hepatitis B virus surface antigen gene as a reporter and compared its expression level in cell culture and mice challenged by different doses of siRNAs. A quicker and higher rebound of gene expression was observed in mice tail vein-injected with higher dose of siRNA, and the rebound was associated with an increase in the mRNA level of meri-1 and adar-1 genes encoding an exonuclease and RNA-specific adenosine deaminase, respectively. Down-regulation of meri-1 by RNAi enhanced the sensitivity and efficiency of siRNA inhibiting the expression of HBV surface antigen. These results indicate that RNAi machinery may be under negative regulation through the induction of a series of genes coding for destabilizing enzymes by siRNAs introduced into the cell, and also suggest that suitable amount of siRNA should be used for research or therapeutic application.In the second chapter of this thesis, we intraperitoneally injected different doses of c-myc-directed esiRNA(esiC-myc) into C57BL6/6J mice bearing B16 melanoma to investigate the inhibitory effect of esiC-myc on tumor growth. However, in high dose esiC-myc treatment groups(＞10μg/mouse/day), the tumor growth inhibition was less efficient than that of low dose treatment groups; the maximal inhibition of tumor growth was achieved in a lower dose treatment group(10pμg/mouse/day). Considering the negative regulation roles of eri-1 and adar-1 genes in RNA interference, we down regulated either/both of the two genes with c-myc gene by RNAi. Our results showed that silencing of eri-1 or adar-1 gene could rescue the tumor growth suppression in the high dose esiC-myc treatment groups obviously. The maximal inhibition of tumor growth was achieved when esiMERI-1(esiRNA of mouse eri-1 gene, 10μg/mouse/day), and esiMADAR-1(esiRNA of mouse adar-1 gene, 10μg/mouse/day)were injected together with high dose esiC-myc(30μg/mouse/day). The data strongly suggest that silencing of eri-1 and adar-1 homologues of human being should be concerned for cancer therapy by RNAi approach.In the third chapter of this thesis, based on the first and second chapter researches, we have known that high dose of esiRNA will stimulate negative regulating system of RNAi. Therefore, we adjusted the dose of esiRNA and used single kind of esiRNA or combined kinds of esiRNAs(E. coli expressed and enzyme digested siRNAs)to inhibit mouse B16 melanoma growth in vitro and vivo. Our result shown that combined different kinds of esiRNAs agents could produce additive effect to minimize tumor size. It suggested that suppression expression of both c-myc and star3 genes by RNAi could be a potent therapeutic approach for the treatment of mouse melanoma.In summary, our researches about RNAi regulation in mammalian provide necessary data for RNAi application in the future. Combination of different esiRNAs agents will be a promising therapeutic approach for human disease treatment.