Up-regulation Of Tumor Suppressive Genes By DsRNAs And Its Therapeutic Effects On Bladder Cancer

Very recent studies have reported that chemically synthesized small duplex RNAs complementary to the promoters of target genes can up-regulate gene expression in different cancer cell lines and have named this phenomenon as RNA-induced gene activation (RNAa) and such dsRNA molecules as small activating RNAs (saRNAs). Following mechanistic studies on RNAa indicated that epigenetics and antisense transcripts are associated with RNAa. However, it's still confused about the prevalence, exact mechanisms and design rules of RNAa, and there are also some disputations on the mechanisms of RNAa to be solved.This paper focused on discussing the prevalence of RNAa, identifying its exact mechanisms, improving its design rules, and evaluating its therapeutic potential. The main investigations and results are as follows:1) Several dsRNAs were designed to target the promoters of p21, par-4, KAI1 and NKX3-1 respectively. The functional saRNAs, dsP21-306 and dsPar4-435, were picked out according to their abilities to induce the target gene expression to more than two times detected by PCR and Western blot. It was suggested that the efficacy of functional saRNAs was dependent on both the target site and the genetic background of target cells.2) We synthesized several dsRNAs whose target sites were adjacent to the target site of the functional saRNAs and then compared these dsRNAs with the functional saRNAs in regulation of target genes. It was suggested that one or two new functional saRNAs can be obtained by shifting the original target sites to one or two bases nearby along the gene promoters. Meanwhile, all the functional saRNAs including the former ones were analyzed and found that the bases in 3'ends of their sense strands had regularity which might be important to their functions.3) We discussed the relationship between RNAa and the epigenetic changes in par-4 gene promoter by ChIP, and found that H3K9 di-methylation decreased and H3K4 di-methylation increased in the chromatin around the target site after dsPar4-435 transfection, but H3K9 acetylation didn't have evident changes. These results suggested that although the trends of the epigenetic changes were consistent with RNAa, the concrete changes might be different in different genes. Then we detected transcripts existing in par-4 gene promoter in several cells. The transcripts were mainly antisense RNAs and increased when par-4 mRNA was up-regulated by dsPar4-435. The exact mechanisms still need to be investigated in the future.4) We evaluated the in vitro therapeutics of functional saRNAs on bladder cancer cells by MTT, flow cytometry and Western blot. Both dsP21-306 and dsPar-435 could induce apoptosis in T24 cells effectively and dsP21-306 could also induce G1-phase cell cycle arrest in these cells. Therefore, they could inhibit the growth of bladder cancer cells, suggesting that up-regulation of TSGs expression by saRNAs have therapeutic potential and promise for bladder cancer.In conclusion, RNAa phenomenon occurs in some genes, and its mechanisms are associated with epigenetics and antisense transcripts. Meanwhile, the in vitro therapeutics of functional saRNAs on bladder cancer cells was effective and provided evidence and basis for the future clinical application in the treatment of cancer or other diseases. However, there are still a lot of problems need to be investigated and improved, including the exact mechanisms, design rules and in vivo therapeutic experiments of RNAa.