The Study Of Modulating Mechanism About Axl On The Metastasis Of Hepatocellular Carcinoma And The Clinical Significance Of Axl Expression

Objective: To investigate the expression of Axl and PI3K/Akt-PAK1signalpathway in hepatocellular carcinoma and to clarify the possible role and mechanism ofthem in the tumorigenicity and metastasis process of hepatocellular carcinoma.Toinvestigate their relevance with the clinical characteristics of hepatocellular carcinoma,and provide a basis for designing future therapeutic strategy for blocking HCCmetastasis in patients.Methods: The mRNA and protein expression levels of Axl in MHCC97-H andMHCC97-L cells lines were evaluated by RT-PCR and western blot analysis and theirdiversity was clarified. We analyze the possible role and mechanism of Axl in thetumorigenicity and metastasis process of hepatocellular carcinoma by in vitro cellinvasion assays and tumorigenicity assay.(1) We silenced the expression level of Axl inMHCC97-H cells with Axl-shRNA, and examined the different expression levels of Axl.We further explore the invasion ability of MHCC97-H cells by in vitro cell invasionassays.(2)485-week-old male nude mices were randomly divided into three groups,approximately1×107MHCC97-H cells (with or without Axl shRNA interference andcontrol shRNA) were subcutaneously inoculated into the right flank of each nude mouse.When mice bearing palpable tumors (about three week), mice were sacrificed and theirtumors were isolated, weighed, and photographed. Immunohistochemical study wasused to analyse the the expression level of Axl in mice tumor tissues.(3) The key factorof phosphatidylinositol-3-kinase (PI3K)/Akt-p21-activated kinases-1(PAK1) signalingpathway was studied after Axl expression was down regulated by shRNA. After inhibiting PI3K and Akt by LY294002and Akt siRNA, we studied the associationbetween PI3K/Akt signal pathway and the invasion of MHCC97-H Cells. One hundredand thirty-seven paraffin-embedded HCC samples and forty-nine noncancerousparaffin-embedded normal liver samples were obtained from the general surgerydepartment of the second affiliated hospital of Dalian medical University from Januaryin2007to January in2012. We analyzed the expression status of Axl protein expressionin mice tumor tissues and its relationship with the invasiveness ofhepatocellular carcinoma.Results: Axl was observed higher expressed in MHCC97-H cell lines compared toMHCC97-L cell lines. The down regulation of Axl in MHCC97-H cell lines resultedin the inhibition of the invasion ability of MHCC97-H cells in both vitro and vivo.Interestingly, blocking phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway byLY294002or Akt siRNA could remarkably inhibit the p21-activated kinases-1(PAK1)activation and cell invasion. Finally, the Axl protein expression was positivelycorrelated with differentation, lymph node metastasis and clinical stage in patients withhepatocellular carcinoma patients (all P <0.01).Conclusion:(1) A higher Axl expression was found in MHCC97-H cells compared to theMHCC97-L cells. Axl played an important role in association with HCC cells invasionvia modulating the PI3K/Akt signaling pathway. The expression of Axl was not onlyshown in hepatocellular carcinoma tissue compared with noncancerous liver tissues, butalso closely was associated with differentation, lymph node metastasis and clinicalstage in patients with hepatocellular carcinoma.(2) These findings suggest that Axl can also regulate the metastasis process ofhepatocellular carcinoma and may serve as a new prognostic marker and therapeutictarget for treating hepatocellular carcinoma metastasis.