Study On Directing Dicer To Cleave Target Double-strand RNA

Antisense technology is a technology that uses antisense agents to inhibit the expression of a target gene in a sequence-specific manner, and it has been widely used for functional genomics, target validation and therapeutic purposes. Three types of antisense technology can be distinguished. Firstly, the use of single stranded antisense oligonucleotides; secondly, the triggering of RNA cleavage through catalytically active oligonucleotides referred to as ribozymes; and thirdly, RNA interference induced by small interfering RNA molecules. In this study, we developed a new method that using an oligomer RNA which had high affinity to Dicer to directionally induce Dicer to process and degrade target double-strand RNA and named it DICERi. The RNA composed of the oligomer RNA and antisense RNA was named artificial small RNA(asRNA). Firstly, we selected Hela cell and MDA-MB-231 cell as experimental cells, and MALAT-1 gene, which had been reported to be involved in the genesis and development of cancer as target gene. After transfection of plasmids transcripting asRNA,we detected the expression level of MALAT-1 and cell proliferation?migration and apoptosis of Hela cell and MDA-MB-231 cell. Then, we co-transfected the plasmids transcripting asRNA and shRNA targeting Dicer mRNA into Hela cell and MDA-MB-231 cell, and the expression levels of Dicer and MALAT-1 were detected at the same time. At last, we applied the new method into EPC cell, Fathead minnow. The asRNA was targeted oncogene c-MYC, and the expression level of c-MYC and cell proliferation?migration and apoptosis were all analyzed. The main topics of this study are as followed:1. After the transfection of plasmid transcripting the corresponding asRNA into Hela cells and MDA-MB-231 cells, we used RT-PCR technology to measure the expression level of target gene MALAT-1 and the results showed that the as RNA led to a significant decrease in the expression of MALAT-1 compared with the negative control groups. The results of CCK-8 and EdU assays suggested that the facts of cell proliferation in both cells were suppressed dramaticly in the experiment groups. And the cell migrations of Hela and MDA-MB-231 cells were found reduced using wound scratch assay and Transwell assay. The results of ELISA and Hoechst staing assays indicated that the asRNA targeting MALAT-1 RNA could promote the apoptosis of both cells.2. To determine the repression effects of asRNA were truly triggered by Dicer, the plasmids transcripting sh RNA targeting Dicer and asRNA targeting MALAT-1 RNA were co-transfected into Hela cells and MDA-MB-231 cells. As expected, because of the knockdown of Dicer, the repression of target gene was released and the expression level of MALAT-1 was not affected.3. To testify that this new method could be used widely, we employed the new method in EPC cells, Fathead minnow. After the transfection of the plasmid transcripting asRNA targeting c-Myc mRNA, we tested the expression level of c-Myc and cell proliferation ?migration and apoptosis. We obtained the same results in Hela cells and MDA-MB-231 cells that the expression level of c-Myc?cell proliferation and migration were all reduced distinctly, and cell aopotosis was improved significantly.