Inhibition Of 293T Cells Proliferation By Blocking MiR-20 And MiR-106 Expression With Antisense RNA

microRNA (miRNA) is a well-regarded research focus in the field of life sciences in recent years. It is a class of 21-25 nt conserved, short-sequences of single-stranded small RNA that has an important role in gene expression and regulation. microRNAs are found in humans and almost all organisms. Mature miRNA can make its target mRNA degradation or inhibit the target genes expression by binding to the 3'-UTR of its target gene in the cytoplasm. The result of study confirms that as a transcriptional regulatory factor, miRNA plays an important role in gene expression of higher eukaryotes, ontogenesis, cell proliferation and differentiation, animal evolution and human diseases. miRNA participates in the balancing network of gene expression regulation and determines the fate of cells. Tumor can be induced when this balance is broken.Antisense RNA is a new technology emerging recently in molecular biology. It has become a new research focus in the area of cancer gene therapy. Antisense RNA can close the expression of certain genes to regulate the cell grow state by binding to specific mRNA. Antisense RNA may expression in cells by plasmid or virus vectors. It may also generate in induced cells. Furthermore, it can be led-in cells after artificial synthesis.miR-20 and miR-106 are members of miR-17 family. They can promote cell proliferation the same as oncogene. Therefore, the proliferation of tumor cells can be reduced by inhibiting the expression of these miRNA. At the present time, antisense RNA primarily focuses on mRNA and the antisense RNA specific to miRNA is still in initial stage. In this study, antisense RNA specific to miR-20 and miR-106 were synthesized and the 293T cells were treated with these antisense RNA. Then the effect of antisense RNA on 293T cells was studied by various methods and the value of antisense RNA in regulating cell proliferation and tumor therapy was discussed.After the 293T cells were treated with antisense RNA specific to miR-20 and miR-106, the suppression of cell proliferation by antisense RNA was studied respectively by microscopy, MTT and FACS. Compared with the control group, the treated 293T cells growth slowed down, with a large number of cells shrinkage, clustering together and apoptosis. It suggested that apoptosis can be induced by the antisense RNA.To investigate the antisense RNA-induced apoptosis, miR-20 and miR-106 expression was studied by RT-PCR and Real-time PCR. The results showed that intracellular content of miR-20 could be reduced by antisense-miR-20 and the expression of miR-106 could be inhibited by antisense-miR-106.In this study, three kinds of forecasting software were used to analyze the downstream genes regulated by miR-20 and miR-106. Then RB gene was selected for further study. The result of ELISA showed that the expression of RB protein increased significantly in cells treated with antisense-miR-106 while there was no significant changes in cells treated with antisense-miR-20. It suggested that miR-106 may regulate the expression of RB protein.In a word, the result of this study suggested that antisense RNA could inhibit proliferation of 293T cells and promote apoptosis by inhibiting the expression of miR-20 and miR-106. Moreover, the expression of anti-oncogene RB can be up-regulated by antisense-miR-106.