| Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and the prognosis of patients with HCC is very poor. As it is geographically biased toward the several parts of Asia and Africa, China in particular, it presents one of the major health threat in China. Although several treatments such as tumor resection, liver transplantation, transcatheter arterial chemoembolization (TAE), and local radiofrequencyablation (RFA) are now used to treat HCC, there is no overall long-term survival benefit so far. Therefore, strategies that explore new therapy for HCC are urgently needed.Recently, Li, et al. and others have reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a process termed RNA activation. This technique alters chromatin structure leading to robust and prolonged expression of the endogenous target gene. As such, RNAa has potential to be a useful tool for interrogating gene function by serving as an alternative to traditional vector-based systems and an attractive strategy to activate tumor suppressor genes for the treatment of cancer.Wilms'tumor1gene (WT1) is an important nuclear factor involved in organ development and cell growth. The role of WT1in cell biology is equally complex, and it has been shown that the repression or activation function of WT1is dependent on the cell type and on its level of expression. Moreover, WT1has been described as a tumor suppressor and as an oncogene. It was reported that plasmid-mediated transfection of WT1-KTS isoforms into hepatoma cell lines induced p53-independent apoptosis. Recently, some studies showed WT1is expressed in several human hepatocellular carcinoma (HCC) cell lines, and is also expressed in tumor tissue in42%of patients with HCC. However, the role of WT1in hepatocarcinogenesis has not been clarified.In this study, we investigate the effects of the dsRNA that specifically targets the promoter region of WT1on the growth of human hepatocellular carcinoma cells HepG2. We found that the dsRNA that specifically targets the promoter region of WT1could up-regulate WT1and induce apoptosis which was related to modulation of Bcl-2family.Part I The screening of functional small activating RNAsAim Small RNA-based strategies for the management of hepatocellular carcinoma, and screening the relatively efficient candidate small activating RNAs (saRNAs) molecules that can activate the target gene WT1.Materials and methods Based on the design rules by the literatures, we designed3pairs of small double-stranded RNA (dsRNA) for target gene WT1, and the control dsRNA is non-homologous to the whole human genome. The candidate dsRNAs were transfected into human hepatocellular carcinoma cell line HepG2. The expressions of WT1gene were evaluated at both the mRNA and protein levels using real-time PCR and Western blot analysis. The functional saRNA which defined as to increase target gene expression twice was selected.Results dsWT1-755and dsWTl-688did not activate the WT1gene significantly. Expression of WT1in dsWTl-326treated cells was significantly elevated. Compared to mock and dsControl transfections, dsWT1-329caused an over2-fold induction in both mRNA and protein level.Conclusion Screened out the functional saRNAs, dsWT1-326, it has a ability to activate the target gene WT1expression in human hepatocellular carcinoma cell line HepG2.Part II Effects and mechnism of dsWTl-326induces apoptosis in HepG2cellsAim To investigate the in vitro therapeutics of dsWT1-326on human hepatocellular carcinoma cell line HepG2and explore the molecular mechanism of cell apoptosis.Materials and methodsCell images were taken using a phase-contrast microscope at100×magnification. Cell viability was determined by the MTT assay. Colony formation was analyzed12days following transfection. Detection of apoptotic cells by flow cytometry (Annexin V and PI double-staining). The expressions of apoptosis related proteins(Bcl-2, Bak, Caspase-3and PARP) were evaluated by Western blot anlysis.Results1. dsWTl-326inhibits HepG2cell growth, and clonogenicityThe dsWT1-326and dsControl were transfected into HepG2cells at the concentration of50nM. At48h and72h following transfection, phase-contrast images of cells from the same fields were taken. Morphologically, mock and dsControl transfected cells maintained healthy growth after transfection, whereas cells transfected with WT1dsRNA gradually lost viability and the number were evidently less after72h.2.dsWT1-326inhibits HepG2cell viability and clonogenicityThe effect of dsWT1-326on proliferation and viability of HepG2cells was determined with varying concentrations of dsWTl-326and times (48-72h) by MTT assay.The effects of dsWT1-326on cell viability occurred within48h and at dsRNA concentrations as low as5nM. Inhibition of cell viability by dsWTl-326(10-50nM)was both dose-and time-dependent. Cell viability with dsRNA treatment at concentrations of2-50nM after48h ranged from87.7%to76.0%, whereas after72h ranged from83.6%to57.8%.Clonogenicity assay revealed the reduction of the number and size of colonies formed in dsWTl-326treated cells3.dsWT1-326induces significant apoptosis in HepG2cellsThe dsWT1-326mediated loss of HepG2cell viability and apoptosis were evaluated by flow-cytometric analysis of dsRNA-treated cells labeled with PI and Annexin V. We found that dsWT1-326caused a time-dependent increase in HepG2cell apoptosis. The number of early apoptotic cell at48h and72h following dsWT1-326treatment increased significantly as compared with control treatments(P<0.05), and number of late apoptotic cell at48h and72h following dsWT1-326treatment also increased significantly as compared with control treatments(P<0.01) These data also showed that dsWT1-326treatment resulted in cell necrosis in cells treated for72h, which might be a secondary event in the apoptotic process.4.The relationship of dsWTl-326treatment with the expression of apoptosis related proteinsBcl-2is known as an anti-apoptotic protein and Bak as an proapoptotic protein, so we detected their expression after50nM dsWT1-326treatment for72h. Consistent with the significantly increased HepG2apoptosis, the level of Bcl-2was decreased and level of Bak were elevated in dsWT1-326treated cells compared to mock and dsControl treated ones. Caspase-3and poly (ADP-ribose) polymerase (PARP) play central roles in apoptosis. We observed that the level of pro-caspase-3remarkably decreased in50nM dsWT1-326treated cells at72h following treatment. The cleaved caspase-3and89kDa cleaved PARP fragment were detected in dsWTl-326-treated samples. Thus the significant changes of apoptosis-related proteins caused by dsWT1-326confirmed the observed apoptosis above and the anti-tumor effect of dsWT1-326on HepG2cells. Conclusion dsWT1-326could induce apoptosis in human hepatocellular carcinoma HepG2cells. This is mediated through up-regulation of Bak, down-regulation of Bcl-2, and activation of caspase-3and PARP. The results of our study provide evidences that up-regulation of WT1by dsWT1-326may have therapeutic potential in the treatment of hepatocellular carcinoma... |
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