Expresssion And Significance Of NHERF1andβ-catenin In Cutaneous Malignant Melanoma Tissues And Cell Lines

Cutaneous malignant melanoma is a highly lethal tumor, originating from melanocyte and resistant to medical intervention of any kind in advanced disease. Initiatives have concentrated on the mechanism and the therapy of melanoma for decades. Na+/H+exchanger regulatory factor-1(NHERF1) is a scaffolding protein, playing an important role in regulating of ion transport, and stabilizing the transmenbrane protein involved in epithelial microvilli structure. It is also a regulatory protein in signaling and trafficking of more than30kinds of proteins. In recent years, it has been found that NHERF1has an emerging role in various tumors. NHERF1gene mutations or loss of heterozygosity can lead to increased degree of malignancy of the tumor cells. In normal tissue, NHERF1is usually cyto-membrane expressed demonstrated by immunohistochemistry and plays a tumor suppressor role, on the other hand, negative expression in cyto-membrane or overexpression in the cytoplasm or nucleus correlate with increased degree of malignancy. Although a growing number of studies have shown that NHERF1plays an important role in oncogensis, its role in anti-tumorigenesis is still poorly understood. Further studies, in addition to the continuate exploring of its tumor suppressor mechanism, willbe focused on its role in the tumor biological behavior and the clinical correlation.Expression of NHERF1and β-catenin in cutaneous malignant melanoma and melanocytic nevus tissuesTo detect the expression of NHERF1and β-catenin in cutaneous malignant melanoma and melanocytic nevus. The expression of NHERF1and β-catenin in47cutaneous malignant melanomas and37melanocytic nevi were examined with immunohistochemistry. The cytoplasm moderate/strong positive expression rate of both NHERF1and β-catenin were gradually increased from melanocytic nevus group, melanoma in situ group to invasive melanoma group. Correlation analysis with Spearman’s rho test for NHERF1and β-catenin expression showed no significant correlation between the expression in melanoma tissue samples and melanocytic nevus tissue samples. The cytoplasm moderate/strong positive expression rate of both NHERF1and β-catenin were gradually increased with the increase of the degree of malignancy of lesions from melanocytic nevus group, melanoma in situ group to invasive melanoma group.Expresssion and significance of NHERF1and β-catenin in malignant melanoma cell lines A375, M14and MV3To investigate the expression of NHERF1and3-catenin in human melanoma cell lines A375, M14and MV3, as well as their significance. The human melanoma cell lines A375, M14, MV3and human melanocytes were cultured. Immunocellulochemistry, Real-time PCR and Western blotting were performed to detect the expression of NHERF1and β-catenin in these cells respectively; the proliferation rates of these cell lines were measured by the MTT method. In the immunocytochemistry, NHERF1and β-catenin were showed both membranous staining in the melanocytes; while they were absent in the membranous staining in the melanoma cell lines A375, M14and MV3, but presented cytoplasmic and/or nuclear staining with different intensity. Among the3melanoma cell lines, the MV3cells showed partly weak cytoplasmic staining of NHERF1, with the lowest level of the relative quantity of mRNA and absence of immunoblotting NHERF1protein expression. But, its proliferation rate was the highest by MTT assay, much higher than that of the other two cells; while the A375cells showed intensive cytoplasmic and nuclear staining of NHERF1, with the highest expression of the relative quantity of mRNA and intensive immunoblotting NHERF1protein expression. But, their proliferation rate were much lower than that of the MV3cells. Meanwhile, the MV3cells showed intensive cytoplasmic and nuclear staining of β-catenin, with the highest level of the relative quantity of mRNA and intensive immunoblotting NHERF1protein expression; while the A375cells and the M14cells showed less intensive cytoplasmic staining of NHERF1, with the much lower level of the relative quantity of mRNA and less immunoblotting NHERF1protein expression. The level of NHERF1cytoplasm expression in melanoma cell lines may be associated with the proliferation rate of these cells. NHERF1and β-catenin were differentially expressed in the melanoma cell lines. The influence in the expression of β-catenin as well as the cell proliferation and apoptosis of melanoma cell line A375by the shRNA knockdown of NHERF1To investigate the influence of NHERF1knockdown in melanoma cell line A375in the expression of β-catenin and the cell proliferation and apoptosis. The shRNA interference fragment of NHERF1was designed and synthesized, then loaded in the pLVX-shRNA-puro vector. The vector was transfected into E. coli competent. The recombinant plasmids were identified by using Real-time PCR and gene sequencing. Lentiviral packaging, lentivirus infection of target cells, polyclonal cells verification and monoclonal screening of stable NHERF1knockdown A375cell were performed. By using gene sequencing, restriction enzyme digestion, qPCR and Western blotting, the effect of transfection was identified. Then, the expression of NHERF1and β-Catenin in NHERF1-knockdown A375cells were detected with Western blotting, in comparing with the shRNA-control A375cells and the untreated A375cells. MTT assay and flow cytometry were performed to detect cell proliferation and apoptosis. After the designed interference fragment of shRNA-NHERF1was loaded in the pLVX-shRNA-puro vector and transfected A375cells, the expression of NHERF1in the transfected cells was decreased significantly as detected by qPCR and Western blotting, while the expression of β-catenin was increased in the transfected A375cells. The cell proliferation of transfected A375cells was faster than that of the controls. The designed shRNA-NHERF1interference fragment inhibits the expression of NHERF1in A375cells, leading to increased β-Catenin expression, acceleration of cell proliferation, and decrease of cell apoptosis.ConclusionsIn the melanoma cell lines, NHERF1and β-catenin were differentially expressed, and NHERF1may regulate the expression of β-catenin through their interaction, thereby modulating cell proliferation and apoptosis. The expression of NHERF1in cutaneous melanoma tissues and melanoma cell lines showed aberrant cytoplasm positive expression in comparing with benign melanocyte nevi and normal melanocytes, respectively.