The Membrane-cytoskeleton Organizer Ezrin Is Necessary For HGF/c-Met Mediated Invasiveness And Metastasis In Hepatocellular Carcinoma Cell

Hepatocellular carcinoma (HCC) is a malignant tumor with high relapse and metastasis. Despite recent improvement in long-term survival, the prognosis of HCC is still poor. As we all known, metastasis is one of the characteristics of malignant tumor. To investigate the mechanism of metastasis is helpful for illustrating biological activities of malignant tumor; it is also useful for finding new therapeutic molecular targets and ameliorating the poor outcome of malignant tumor.Hepatocyte growth factor/scatter factor-Met signaling has been implicated in tumor growth, invasion, and metastasis. Activation of hepatocyte growth factor and its receptor Met may enhance tumor growth, invasion, and metastasis, while block Met activation can suppress tumor metastasis. The membrane-cytoskeleton organizer ezrin has been reported to be an effecter of hepatocyte growth factor. As a membrane-cytoskeleton organizer, ezrin can transfer signaling from membrane to the effecter—cytoskeleton F-actin, and mediate migration and morphogenesis.In our previous study, hepatocelluar carcinoma cell lines with different metastasis potential and 200 points HCC tissues array were used to explore ezrin expression in HCC. It came that over expression of ezrin was found in low differential tumor tissues and HCC cell lines with high metastasis potential. Small interference RNA (siRNA) sequences directed against ezrin can downregulate expression levels of ezrin, they also suppress tumor growth, invasion, and metastasis as well. But the mechanism involved in this process is not well understood. In this study, by means of adenovirus mediated RNA interference (RNAi) we try to explore the mechanism by which ezrin influence tumor invasion and metastasis.Part One: The construction and application of adenovirus vectors carrying short-hairpin RNA against ezrin Objective To establish adenovirus vectors carrying short-hairpin RNA (shRNA) against ezrin. By means of these constructions, which may result in a steady-going downregulation of ezrin, we can investigate the crucial role of ezrin on metastasis of heptocellular carcinoma cells, and we may also explore the mechanism involved in this process as well.Methods Short-hairpin RNA which designed according to effective small-interfering RNA was cloned into a shuttle vector to establish recombined adenovirus constructs. After transfecting 293 packaging cell lines, reconstructed adenovirus containing short-hairpin RNA against ezrin (ezrin-shRNA/Ad) were harvested. Human heptocellular carcinoma cells: MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 10, 50 and 100, and ezrin expression was determined by Western blotting. Next, we asked how long the RNAi effect continues after cell passage. MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 100, and ezrin expression was determined at 3-day intervals for up to 9 days after cell passage.Results Human heptocellular carcinoma cells MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 10, 50 and 100. Ezrin expression was determined on day 3 after infection by Western blot. The expression of ezrin protein was dramatically suppressed by ezrin-shRNA/Ad at m.o.i. 50 and 100; Control Ad virus infected cells showed no effect on ezrin expression. Ezrin expression was still strongly suppressed even undergoing cell passage. On MHCC97-H, ezrin expression was still suppressed after a second passage, while a strong suppression was still observed after a forth passage on SF/SMMC7721 cell lines.Conclusion Reconstructed adenovirus containing short-hairpin RNA against ezrin can downregulate ezrin expression. Ezrin expression was still strongly suppressed even after cell passage.Part two: The effect of Adenovirus mediated RNAi on the invasiveness and metastasis of hepatocelluar carcinoma cell linesObjective To explore the effect of Adenovirus mediated ezrin suppression on growth, migration, invasiveness and metastasis of heptocellular carcinoma cells, and to study the mechanism involved in this process as well.Methods Human heptocellular carcinoma cells MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 50, the proliferation were determined by MTT assay. Scanning electron microscopy (SEM) and Matrigel Invasion Assay (Transwell assay) was used to study cellular morphology and invasion. Cell-cell interaction and cell -matrix adhesion was estimated by a Calcein AM labeled fluorescence OD. Immunofluorescence was used to observe expression and location of ezrin, E-cadherin and CD44 protein. RT-PCR was performed for ezrin, actin, E-cadherin, CD44 andβ-catenin expression. MHCC97-H infected with ezrin-shRNA/Ad was injected into nude mice, MHCC97-H as control, the proliferation and metastasis were observed in vivo nude mice experiment.Results In vitro: After infected with reconstructed adenovirus ezrin-shRNA/Ad, the proliferation rate of MHCC97-H and SF/SMMC7721 were distinctly decreased. Proliferation curve show the proliferation suppression of SF/SMMC7721 occurred at the day4 after infection, while the proliferation suppression of MHCC97-H appeared only at the second day. Scanning electron microscopy performed with MHCC97-H and SF/SMMC7721 revealed rather rounded cellular morphology with reduced pseudopods number after infection with ezrin-shRNA/Ad. The decreased invasion were also confirmed by Matrigel Invasion Assay, compared to control Ad virus, MHCC97-H and SF/SMMC7721 infected with ezrin-shRNA/Ad resulted in significant reduction in the number of Matrigel invading cells (number of Matrigel invading MHCC97-H cells :ezrin-shRNAl/Ad 44.00±7.06; ezrin-shRNA2/Ad 35.50±3.70; control Ad virus 56.50±7.01; uninfected 63.80±6.26). Enhanced cell-cell interaction and reduced cell -matrix adhesion were observed with MHCC97-H and SF/SMMC7721 cells after ezrin-shRNA/Ad infection, as indicated by Calcein AM labeled fluorescence OD. Immunofluorescence showed weak ezrin expression, concomitant with increased E-cadherin expression, mainly located on membrane, and decreased expression of CD44, after ezrin-shRNA/Ad infection. RT-PCR was performed for ezrin, actin, E-cadherin, CD44 andβ-catenin expression. Expression of E-cadherin andβ-catenin showed a moderate upregulation in response to ezrin-shRNA/Ad treatment, while CD44 andγ- actin was found to be downregulated. Ezrin-shRNA/Ad treatment seemed to have no effect onβ-actin expression. In vivo: The tumor formation in nude mice began in 2 weeks after cell injection. Tumors of MHCC-97H infected with ezrin-shRNA/Ad grew rapidly than tumors of control MHCC-97H. Infected MHCC97-H also had lower lung and lymphoid node metastasis rates than control (Infected MHCC97-H of 50% and 30.0% vs MHCC97-H of 77.8% and 55.6%).Conclusion Cell proliferation and invasiveness, as well as metastasis potential of HCC cell lines were depressed by ezrin-shRNA/Ad mediated RNA interference, which indicated ezrin is involved in hepatocellular carcinoma proliferation, invasion and metastasis.Part three: The role of Ezrin played in HGF/met cell signalingObjective To explore the mechanism that ezrin involved in HGF mediated tumorgrowth, invasion, and metastasis.Methods Human heptocellular carcinoma cells SMMC77211, SF/SMMC7721 andSF/SMMC7721 infected with ezrin-shRNA/Ad reconstruction were treated with orwithout HGF stimulation. Western-blot was performed for phosphorylated ezrin.Cellular migration was determined by Wound healing assay. Scanning electronmicroscopy (SEM) was used to study cellular morphology. RT-PCR was performedfor E-cadherin and CD44 expression.Results Ezrin is a substrate for HGF/met cell signaling. Upon HGF treatment,phosphorylated ezrin appeared in 30 minutes. Knocking down of ezrin byezrin-shRNA/ Ad mediated RNAi, impairs motility and morphogenesis response toHGF, as measured by the slower wound healing and less pseudopods number. HGFshows no effect on migration and morphogenesis of ezrin-shRNA/Ad infected celllines. Phosphorylated p44/42 MAP kinase and phosphorylated Akt disappeared afterinfection. Phosphorylated p44/42 MAP kinase reappeared upon exposure of cells toHGF.Conclusion Ezrin is an effecter of HGF mediated migration and morphogenesis inhepatocellular carcinoma cell lines. SummarizationIn this study, we have successfully obtained reconstructed adenovirus whichcontaining short-hairpin RNA against ezrin (ezrin-shRNA/Ad). Upon treated withezrin-shRNA/Ad, the expression levels of ezrin protein was dramaticallydownregulated and ezrin expression was still strongly suppressed even after cellpassage.Cell proliferation and invasiveness of hepatocellular carcinoma (HCC) cell linesMHCC97-H and SF/SMMC7721 were depressed by ezrin-shRNA/Ad mediated RNAinterference, which indicated ezrin is involved in HCC proliferation and invasion. Theexpression and location of actin, E-cadherin, CD44 may be involved in this process aswell. In vivo nude mice experiment confirmed that ezrin played an important role onHCC growth and metastasis.Upon HGF treatment, phosphorylated ezrin appeared. Knocking down of ezrin byezrin-shRNA/ Ad mediated RNAi, impairs cellular motility and morphogenesisresponse to HGF, which indicated ezrin is an effecter of HGF mediated migration andmorphogenesis in hepatocellular carcinoma cell lines.The whole study suggests that ezrin play an important role on HCC growth, migrationinvasion and metastasis, and may become a new therapeutic target in HCC therapy.