Membrane-cytoskeleton Organizer Ezrin Is Required In Proliferation And Metastasis In Hepatocellular Carcinoma

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. It is necessary to find new therapeutic molecular targets to ameliorate its outcome. The change of cell mobility is one of the preconditions of tumor metastasis. Cell skeleton alteration and rearrangement of F-actin was closely related to cell mobility. Ezrin is a membrane-cytoskeleton organizer that can mediate the rearrangement and the function of F-actin. In this paper, we investigated the effect of ezrin on hepatocellullar carcinoma cell growth and metastasis.Part One: Ezrin expression in HCC cell lines with different metastatic potential and HCC tumor tissue with different diferential levels.Objective: To investigate the different expression of cytoskeletal organizer ezrin and cytoskeleton protein β-actin and γ-actin in HCC cell lines with different metastatic potential and the ezrin expression in HCC tumor tissue with different diferential levels. Method: Immunofluorescence, RT-PCR and Western-blot were used to detect the gene and protein expression in hepatocelluar carcinoma cell lines with different metastatic potential. 200 points HCC tissues array were used to explore ezrin expression in HCC tumor tissue with different diferential levels. Results: Immunofluorescence demonstrated ezrin and cytoskeleton protein expressed in the cytoplasma, and co-expressed in the same cell. The expression of them in cell lines with high metastatic potential, such as SF/SMMC7721, MHCC-I and MHCC97-H was obviously higher than those with low metastatic potential as SMMC7721, Hep3B and HepG₂(X²= 13.277, P=0.010; X²=21.815, P=0.000). The mRNA and protein of ezrin and cytoskeleton protein γ-actin were over expressed in HCC cell lines with high metastatic potential. There was no β-actin expression difference in all cell lines. Tissue array showed tumor tissues usually had higher ezrin expression than normal liver and liver cirrhosis and low differential tumor tissues always had high ezrin expression(X²=36.52, P<0.05). Conclusion: Over expression of ezrin and cytoskeleton protein γ-actin was important roles in the process of hepatocellularcarcinoma metastasis.Part Two: In vitro RNA interference revealed that ezrin is necessary for the growth and invasiveness of HCC.Objectives: To investigate the effect of membrane-cytoskeleton linker ezrin on the growth and metastasis of heptocellular carcinoma cell lines. Methods: RNA interference (RNAi) was applied to down-regulate ezrin expression in 4 kinds of heptocellular carcinoma cell lines (SF/SMMC7721, MHCC97-H, MHCC-1 and HepG2). Real-time PCR and Western-blot were used to reveal the down-regulation of ezrin in gene and protein level. The day with the lowest ezrin protein level was selected as the optimal time to compare the biological behavior of cancer cell before and after RNAi treatment. The proliferation changes were detected by MTT assay and flow cytometric analysis. The invasiveness alterations were evaluated by observing morphology changes of pseudopods and transwell assay. Results: Real-time PCR and western-blot revealed that ezrin siRNA can notably down-regulate ezrin expression in both mRNA and protein level. Down-regulation of ezrin expression can distinctly decrease the proliferation rate of 4 kinds of hepatocellular cell lines (P <0.05) and the cell proportion in G2-M phase also decreased after RNAi treatment. The number of pseudopods per cell decreased after RNAi treatment (SF/SMMC7721: 20.8±3.0 vs 13.2±2.4, P<0.05; MHCC97-H: 18.4±2.7 vs 14.0±2.9, P<0.01; MHCC-1: 22.6±3.5 vsl3.3±1.9, P＜0.01; HepG2:31.0±2.9 vs 17.8±2.3, P<0.01) and the motility and invasiveness of cancer cell decreased obviously as well (SF/SMMC7721: 49.9±7.7 vs 31.9±5.2, P＜0.05; MHCC97-H: 58.5±4.2 vs 33.0±3.3,P<0.01; MHCC-1: 57.6±6.1 vs 28.3±3.4, P<0.01; HepG2: 37.3±3.0 vs 25.3±2.3, P＜0.01 ). Conclusion: Ezrin is necessary for hepatocellular carcinoma proliferation and invasion. It is probably an important factor to inhibit tumor reoccurrence and metastasis.Part Three: ezrin is crucial to the growth and metastasis of HCC testified by in vivo experiment.Objectives: MHCC-97H with high metastastic potential was chosen as study target to explore the effect of ezrin in HCC growth and metastasis by in vivo nude mice experiment. Methods: Ezrin siRNA expression plasmid was transfected into MHCC-97H by lipofectin 2000. A clone with ezrin down-regulation was selected and named 97H-ezrin(L). The proliferation and metastasis potential of cells before andafter gene transfection were observed through MTT assay, transwell assay and in vivo nude mice experiment. Results: In vitro assay showed that the proliferation and invasive ability of 97H-ezrin(L) were weaker than that of MHCC-97H(P<0.05). The tumor formation in nude mice began in 2 weeks after cell injection. MHCC-97H tumors grew rapidly than 97H-ezrin(L) tumors. Pathological analysis of the tumor showed that the number of cells in mitosis phase and pathological mitosis in 97H-ezrin(L) were lesser than that in MHCC-97H tumor. 97H-ezrin(L) also had lower lung and lymphoid node metastasis rates than control. Conclusion: these results further demonstrated that down-regulating ezrin can decrease the proliferation rate and lung and lymphoid node meastasis of MHCC-97H. Ezrin is an important molecular in HCC growth and metastasis.Part Four: SummarizationOur research firstly investigated the ezrin and actin expression in HCC cell lines and the ezrin expression in HCC tumor tissues with different differential level. Then the biological behavior of HCC cell line was compared before and after ezrin protein down-regulation by RNA interfering. The results revealed that ezrin was an important factor participating in HCC cell proliferation and invasiveness. In vivo experiment showed that tumor with ezrin down-regulation proliferated slower than control and had a lower lung and lymphoid node metastasis rate. The whole study suggests that ezrin is a key molecular to inhibit HCC growth and metastasis, and may being a new therapeutic target in HCC therapy.