| BackgroundHepatocellular carcinoma (HCC) is the worldwide common malignancy of theliver, the ffth most frequently diagnosed cancer, and the second leading cause ofcancer deaths in China and Asia. It is an insidious disease without eliciting pain, thediagnosis of which is usually made at a late stage, thus excluding curative treatmentsand leaving patients with few therapeutic options. The high mortality of HCC ismainly attributed to invasion pattern and intrahepatic and/or extrahepatic metastasis,but its exact mechanism remains unclear. Therefore, a thorough understanding of theunderlying mechanisms of tumor metastasis is vital for the effective prevention andtherapeutics in the field of liver cancer research.Silent Information Regulator2(Sir2) was found to be a nicotinamide adenosinedinucleotide (NAD+)-dependent histone deacetylase, isolated initially from yeasts.The sirtuins comprise a small family with seven members, respectively namedSIRT1-SIRT7, which sharing extensive homologies with Sir2. As Sir2possess anestablished capacity to prolong yeast lifespan, the researchers focus more attention onthe mammalian sirtuin family implicated in the regulation of metabolism, aging,inflammation and cancer progression. SIRT1remains the most extensively studiedmember, which has been found to deacetylate histone and nonhistone proteins.Emerging evidence demonstrates that abnormal expression of SIRT1is closely linkedto cancer through deacetylating oncogenes or tumor suppressor genes. Recently,correlation between SIRT1expression and tumor metastasis has also arousedwidespread concern in several types of cancer. Different study showed that SIRT1serve different functions in different cellular environments. For example, SIRT1canpromote metastasis in prostate cancer through regulation of EMT. In addition, anotherstudy showed that SIRT1is up-regulated in the majority of human hepatocellularcarcinoma cell lines and tissues. Functional studies revealed that gene silencing ofSIRT1can inhibit proliferation of HCC cells and induce cell apoptosis, which suggestthat up-regulated SIRT1may be involved in HCC carcinogenesis and progression.EMT (epithelial–mesenchymal transition) was known as the acquisition ofmesenchymal phenotype by epithelial cells, which is a key process that is required ina variety of human epithelia tumor. More recently, EMT is associated with livercancer migration and metastasis. We speculated that EMT-associated transcriptionfactors might be one of the target genes of SIRT1in HCC. However, it is not clear that whether SIRT1is involved in HCC carcinogenesis and metastasis throughtargeting EMT, and whether therapeutic intervention on SIRT1expression couldeffectively reverse the metastatic phenotype of HCC cells. So it is still need to beexplored that roles and mechanisms of SIRT1in HCC carcinogenesis and metastasis.AimsTo explore the regulatory role and the underlying mechanisms SIRT1in HCCinvasion and metastasis, with the aim of better elucidating the mechanisms of HCCmetastasis and laying a foundation for formulating novel therapeutic target fortreatment of HCC.Methods1. A total of99HCC tissues and corresponding non-tumor tissues, as well asclinicopathological data were obtained. Expression of SIRT1in liver cancerand normal liver tissue was detected by immunohistochemistry. Meanwhilethe relationship between nuclear SIRT1expression and clinicopathologicalcharacteristics was analyzed.2. The human HCC cell lines SMMC-7721, Hep-G2, Hep-3B and Huh7weremaintained and cultured in our lab. Expression of SIRT1was also detected byRT-PCR and Western Blotting analysis.3. SIRT1(Ad-SIRT1) and SIRT1siRNA (sh-SIRT1) were transferred into cellsmediated by adenovirus carrying green fluorescent protein to up-regulate anddown-regulate SIRT1. The transfection efficiency and the expression ofSIRT1protein were evaluated48h after transfection.4. Observe the effects of SIRT1on biological behavior of HCC cells in vitro:SIRT1activator resveratrol and Ad-SIRT1were used to activate SIRT1, andSIRT1inhibitor nicotinamide and sh-SIRT1were used to suppress SIRT1.Then wound healing test and transwell migration and invasion assays wereemployed for investigating the metastatic properties.5. For further in vivo detection, we establish a liver cancer metastasis model innude mice to validate the role of SIRT1on liver cancer metastasis ability.6. To explore EMT is the potential mechanism underlying hepatic metastasis bySIRT1, we perform RT-PCR and immunofluorescence techniques to detectEMT-associated genes expression after altering the activity of SIRT1. Results1. Percentage of positive SIRT1expression was significantly higher in livercancer tissues than that of normal liver tissues (P<0.001). High SIRT1expression was strongly correlated with the tumor size (P=0.043), tumornumber (P=0.031) and TNM stage (P=0.038), but it was not correlated withother factors. Patients with high SIRT1expression had significantly pooreroverall survival (P=0.035).2. SIRT1was expressed in all the HCC cell lines, and we choose SMMC-7721and Hep-G2to apply the experiment.3. The transfection efficiency of Ad-SIRT1was30%,87%,95%, and thetransfection efficiency of sh-SIRT1was30%,55%,78%at10,50,100MOIrespectively. Ad-SIRT1up-regulated the expression of SIRT1protein, whilesh-SIRT1down-regulated SIRT1after transfection for48h.4. There were statistically significant correlations of the SIRT1level with bothcell migration and invasion potential.5. In the in vivo studies, metastasis potential of up-regulated SIRT1HCC cells innude mice was significantly increased.6. SIRT1could induce EMT and regulate the mRNA level expression ofEMT-related genes.Conclusions1. SIRT1was up-regulated in HCC cell lines and tissues. Overexpression ofSIRT1increased potential of cell migration and invasion, whereas inhibition ofSIRT1reduced cell migration and invasion in HCC cells. These indicated thatSIRT1plays an important role in HCC metastasis. It can be expected tobecome a new prognosis reference of HCC.2. SIRT1is involved in HCC invasion and metastasis mediates inducing EMT.Both of them are likely to be a new potential therapeutic target for treatment ofHCC. |
PDF Full Text Request