Reactivation Of Silenced Hypermethylated Tumor Suppressor Genes In Colorectal Carcinomas By Hairpin SiRNA

Epigenetic silencing of tumor suppressor genes (TSGs) induced by its hypermethylated CpG islands (CGIs) is one of the critical tumoregenesis mechanisms for human tumors. This event has become the research focus of the cancer epigenomics and Human Epigenetic Projects (HEP). Colorectal carcinoma is one commonly happened human cancer. Hypermethylated CGIs of TSGs is one of the critical colorectal tumoregenesis mechanisms because it cannot only induce the epigenetic silencing of TSGs, but also related to the increased gene mutation and microsatellite instability of colorectal carcinoma. For the development of colorectal carcinoma, hypermethylated CGIs was frequently happened for many TSGs, such as DNA repair genes (hMLH1 and MGMT, et al) and cell cycle regulation genes (p16 and p53, et al). Hypermethylation of CGIs is a reversible epigenetic modification process, and its reversal can reactivate the TSGs biologic functions directly. Because hypermethylated CGIs of TSGs is always induced by the over-expression of human DNA methyltransferase 1 (hDNMT1) in human tumoregenesis, such as colorectal carcinoma, therefore, selectively inhibition of the hDNMT1 activity could decease or eliminate methylation modification of CGIs, and then reactivate the TSGs. RNA interference (RNAi) refers to post-transcriptional silencing induced by double-stranded RNA (dsRNA). After introduced cells, the exogenous dsRNA is cleaved into 19-to 23-nt RNA known as small interference RNA (siRNA) by Dicer. After the siRNA have been incorporated into a ribonuclease enzyme complex known as the RNA-induced silencing complex (RISC), the RISC was activated and then results in a sequence-specific degradation of messenger RNAs that complemented to siRNA, which induces a specific gene silencing effect. RNAi has made a great achievement in gene knockdown or knockout. According to the above researches, inhibition hDNMT1 expression by RNAi has theoretical background. In this study, the hairpin siRNA (hsiRNA913 and hsiRNA1620) were designed to target different site of hDNMT1 mRNA. According to the hairpin siRNA template oligonucleotides, two siRNA expression cassettes (SECs), which can induce the specific RNAi of hDNMT1, were amplified by PCR. These SECs were transfected into colorectal cell line HCT116 and SW480. Then SEC913 and SEC1620 were cloned into vector pSEC neo to construct the plasmids pSEC neo913 and pSEC neo1620, which were transfected into colorectal cell lines HCT116 and SW480. After transfection, the expression level of hDNMT1 was observed and analyzed at different phase on both its changing tendency and characteristic by semi-quantitative RT-PCR in which the β-actin gene was used as an internal control. The expression level and the methylation status of CpG islands of p16, MGMT and hMLH1 were analyzed by semi-quantitative RT-PCR and MSP respectively to evaluate and analyze whether the inhibition of hDNMT1 expression could reduce the hypermethylation inversion of TSGs and whether the inversion could restore the re-expression and function of these genes. The results showed that: both the SEC193 and SEC1620 which were produced in vitro and plasmids pSEC neo913 and pSEC neo1620 which expressed hairpin siRNA could decrease the expression level of hDNMT1 differently, and the differential was due to the different targeting sites of siRNA (but it has not significance difference). In addition, the effect of hairpin siRNA was much longer than that of SEC913 and SEC1620, which indicated that the vectors expressed hairpin siRNA had a long period RNAi effect. So these vectors might have more potential usage on the clinical epigenetic therapy. According to the results, the inhibition of hDNMT1 expression induced decrease or inversion on the methylation patterns of CpG islands in TSGs, such as p16, MGMT and hMLH1, and the expression level of these TSGs increased, which indicated the inversion of TSGs hypermethylation might restore or strengthen their expression. In conclusion, specific inhibition of hDNMT1 expression could inverse the hypermethylation of TSGs and this inversion could reactivate or increase the expression of TSGs. These provide a new idea on the epigenetic therapy of cancer.