| Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide.HCC is the second most lethal tumor in China, and there are110,000-130,000people diedin HCC each year, which accout for45%of the total HCC deaths worldwide. Despiteimprovements in tumor detection and clinical treatment strategies, the overall outcome forpatients with HCC remains very poor because of the high rate of recurrence or metastasisfollowing treatment. Therefore, it is urgent to further investigate novel indicators for theevaluation of tumor progression and the prediction of patient outcome.The Notch signaling pathway is a very conservative signal transduction system,which can regulate the cell proliferation, differentiation, apoptosis and other activities.The Notch signaling pathway is mainly composed of receptors, ligands and the DNA binding protein. The Notch receptors generated in the cytoplasm were hydrolyzed intotwo parts by the Furin-like converting enzyme in the Golgi apparatus. The two partsformed the heterodimer by the non-covalent bond, and were transported to the surface ofthe cell membrane. Once the receptors binded with the ligands, the transmembranedomain of the Notch receptor was incised by the γ-secretase, and the Notch intracellulardomain entered into the nucleus, which can start the downstream gene transcription andregulate a series of life activities. Notch1is one of the transmembrane receptors of theNotch signal pathway, and numerous studies have found that Notch1abnormallyexpressed in many tumors and associated with tumor invasion and metastasis. However,the role of Notch1in hepatocellular carcinoma is unclear.ObjectiveTo investigate the expression of Notch1in the tissues of HCC and to evaluate itsrelationship with clinicopathological features of HCC. We also tested the expression ofNotch1in different invasive ability of liver cell lines, designed and synthesized specificNotch1-siRNA to explore the role of Notch1in invasive and metastatic process ofhepatoma cell lines MHCC97-H and HepG2as well as its underlying mechanisms.MethodsWe investigated the expression of Notch1in110cases of HCC tissues and theiradjacent non-cancerous hepatic tissues by immunohistochenmical and analysed therelationship between Notch1expression and the clinicopathological characteristics. Wedetected the protein expression of Notch1in liver non-tumor cell line (HL-7702) andHCC cell lines (MHCC97-H and HepG2) by Western blot, of which capacities of invasiveand metastatic are different. The specific Notch1gene small interfering RNA (siRNA)was transfected into HCC cell lines of MHCC97-H and HepG2, using liposome, and theprotein and mRNA expressions of Notch1was tested by Western blot and real time PCR.In vitro, the invasion of the treated MHCC97-H and HepG2, which were transfected bythe Notch1-siRNA, were analyzed using Matrigel-coated Transwell cell culture chambers.Western blot and the enzyme-linked immunosorbent assay (ELISA) were used to detectthe expression and activity of MMP-2and MMP-9in each group. ResultsNotch1expression was mainly localized within the ctoplasm and at the cell membrane.In HCC tissues, Notch1staining was negative in30(27.3%) samples of HCC, whereasweak positive staining was detected in32(29.1%) samples of HCC, moderate positivestaining was detected in21(19.1%) samples of HCC, and strong positive staining wasdetected in27(24.5%) samples of HCC. The expression of Notch1differed between HCCtissues. There was no significant Notch1expression in adjacent non-cancerous hepatictissues, with only weak staining for Notch1at the cell membrance and in the cytoplasm.In the110cases of HCC, the Notch1expression was strongly correlated with AJCC TNMstage （χ~2=22.123，P<0.001）, venous invasion （χ~2=9.068，P=0.003） and tumor grade（χ~2=6.490，P=0.011）. However, there were no significant associations between Notch1expression and the gender（χ~2=0.490，P=0.484）, age （χ~2=0.188，P=0.664）, tumor size（χ~2=0.002，P=0.961）, tumor number（χ~2=2.220，P=0.136）, AFP（χ~2=1.392，P=0.238）.The protein expression of Notch1in HCC cell (MHCC97-H and HepG2) lines wasincreased compared with liver non-tumor cells (HL-7702). The Notch1-siRNA wastransiently transfected into HCC cell (MHCC97-H and HepG2), and the expression levelsof Notch1protein and mRNA were effectively downregulated, detected by Western blotand real time PCR. The Notch1-siRNA can effectively inhibit the expression of Notch1.The HepG2and MHCC97-H cells were transfected with Notch1-siRNA, and theinvasion capability of treated cells was tested by the transwell cell culture chambers. Theresults showed that the number of Notch1siRNA-transfected HCC cells (MHCC97-H andHepG2) that migrated through the Transwell was significantly less than the number of thecontrol groups. Using Western blot and ELISA, we found that the protein expressionlevels and proteolytic acticity of MMP-2and MMP-9were decreased inNotch1-siRNA-transfected HCC cells (MHCC97-H and HepG2).ConclusionNotch1may involve in the process of the invasion and metastasis of HCC.Downregulated expression of Notch1by siRNA reduced the invasiveness and the levelsand proteolytic acticity of MMP-2and MMP-9of HCC cells (MHCC97-H and HepG2), which implied that downregulated Notch1expression could decrease the invasiveness ofHCC cells via the regulation of MMP-2and MMP-9. |
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