Expression Of SOX4 And Its Role In Regulation And Development Of Malignant Melanoma

BackgroundMalignant melanoma is a skin neoplasm stemmed from epidermal melanocytes and characterized by highly aggressiveness and poor prognosis in its advanced stage. Nearly 20, 000 patients are diagnosed with melanoma in China and the incidence is rising worldwide. Recently, some studies have suggested that mutant genes and abnormal activated pathways, such as MAPK, MEK, PI3 K, may be the crucial events in the carcinogenesis of melanocytes and finally contribute to tumor invasion and metastasis.The SOX family of transcription factors plays important roles in the controlling of cell fate and differentiation in many processes, such as male differentiation, stemness, neurogenesis and skeletogenesis. SOX4, which belongs to the highly conserved group C SOX transcription factors, has been demonstrated to be important for numerous aspects of embryogenesis and various facets of tumor initiation and progression. Recently, large-scale gene expression studies have demonstrated abnormal SOX4 expression in various major human cancers, indicating a central role in the development of many tumors. In the development of human cancers, SOX4 has paradoxical roles acting as either a tumor suppressor or an oncogene and influences key processes related to tumor biology, including cell proliferation, migration, metastasis, apoptosis, EMT and cancer stemness. Notably, the lung tumor-specific overexpression and validation of a comprehensive Ag-specific immune response specified for SOX4 has been investigated in vaccination strategies against lung cancer. Besides, the recognition and identification of naturally processed T cell and Ab epitopes from SOX4 makes ideal and valuable tools for the further development of peptide-based vaccination strategies as well as to monitor and master the SOX4-specific responses in vaccinated lung cancer patients. Therefore, we could not help to asking the exact role of SOX4 in malignant melanoma and the underlying specific mechanisms. We also want to explore the mechanisms related to therapy strategies against melanoma. SOX4 may be a potential target for treatment or vaccination.Our study is aim to investigate the expression of SOX4 in metastatic melanoma tissues, primary melanoma tissues and pigmented nevus tissues and in step wise to explore the possible role of SOX4 in melanoma. Next, we investigated the effects of si RNA mediated SOX4 gene silencing on apoptosis and cell cycle arrest state of human malignant melanoma cell lines(A2058 and WM35). Effects of SOX4 si RNA on proliferation of melanoma cells are also studied. Further, a lentiviral vector expressing sh RNAs targeting SOX4 gene was successfully established in A2058 cell lines. Finally, we made an initial exploration on the effect of deregulation of SOX4 gene on EMT related biological markers.Methods1. Analyze the expressions of SOX4 in invasion melanoma, melanoma in situ and pigmented nevus with western blot and real-time PCR.2. Analyze the expression of SOX4 protein with western blot in melanoma cell lines.3. We transfected WM35 cell lines and A2058 cell lines with SOX4 si RNA, then analyzed the expression of SOX4 with real-time PCR and western blot.4. Analyzing the cell cycle and apoptosis of A2058 cells and WM35 cells transfected with SOX4 si RNA with flow cytometry.5. A lentiviral vector expressing sh RNAs targeting SOX4 gene was successfully established in A2058 cells, A2058 cells transfected with blank vector and A2058 cells transfected with lentiviral vector expressing sh RNAs targeting SOX4 gene.6. Analyzing the ability of migration and invasion in vitro of A2058 cells transfected with lentiviral vector expressing sh RNAs targeting SOX4 gene by Transwell.Results1. The expressions of SOX4 in metastasis melanoma were higher significantly than in pigment nevus(P < 0.05), and were higher than that in melanoma in situ(P < 0.05) on m RNA level. The expressions of SOX4 in metastasis melanoma and melanoma in situ were higher significantly than in pigment nevus(P < 0.05). These results showed that the expression of SOX4 was elevated in metastasis melanoma, indicating that SOX4 could participate in the invasion and metastasis of melanoma.2. A2058 cells were transfected with SOX4 si RNA, and it was verified with real-time PCR and western blot that the expression of SOX4 was inhibited by si RNA. Our results showed the proliferation slowed down and the rate of apoptosis was increased when compared to A2058 cells transfected with liposomes(P < 0.01). And the same with WM 35 cell lines.3. A lentiviral vector expressing shRNAs targeting SOX4 gene was successfully established. A2058 cells transfected with blank vector and A2058 cells transfected with lentiviral vector expressing sh RNAs targeting SOX4 gene were prepared for further experiments.4.Transwell test show that the number of migrated cells on the lower surface of the membrane was fewer in A2058 cells transfected with SOX4 si RNA than that in A2058 cells transfected with liposomes(P < 0.05). ConclusionWe investigated the expression of SOX4 in melanoma tissues and figured out that the expression of SOX4 on both the m RNA and protein level was elevated in the metastasis melanoma compared to pigmented nevus(p<0.05). Then we transfected melanoma cell lines, A2058 and WM35, with siRNA of silencing on SOX4 gene and we found that the abilities of migration, invasion and promoting the genesis and metastasis of melanoma were decreased significantly. Melanoma cells with si RNA of silencing on SOX4 genes showed higher apoptosis rates and shorter cell cycle. These results showed that SOX4 played an important role in the tumor genesis and metastasis of melanoma at least partly by controlling the apoptosis and cell cycle. We also successfully established a lentiviral vector expressing sh RNAs targeting SOX4 gene A2058 cell lines. We assured the downregulation of SOX4 in A2058 cell lines on m RNA and protein level by real-time PCR and western blot. We want to explore the underlying mechanisms of SOX4 and melanoma by the established A2058 cell lines. Then, we tested the expression of E-cadherin genes in SOX4-deregulated A2058 cells. As we all know, the downregulation of E-cadherin is one marker of EMT processing, which lead to the migration and invasion more easily and frequent in tumor metastasis. We found the lower expression of SOX4 was accompanied with upregulated expression of E-cadherin, which indirectly demonstrate that SOX4 might work as an oncogene gene in melanoma by initiating an epithelial-mesenchymal transition. But more work is warranted to clarify the detailed mechanism of melanoma and transcription factors.