The Effects Of Regulation Of Wnt5a/JNK Pathway On The Proliferation,Invasion And Metastasis Of Melanoma Cells

[Background]The Wnt signaling pathway is a highly conserved signal transduction pathway in vivo involved in the regulation of embryonic growth and development as well as cell proliferation, migration and apoptosis. Initiating protein of this pathway is encoded by the Wnt gene, which plays a vital role in normal development of embryos and even the development and progression of tumors. Wnt proteins can be roughly divided into two categories, one category is Wnt1, including Wnt1, Wnt3 a, Wnt8 a and Wnt8 b, which can activate the canonical Wnt signaling pathway, namely the Wnt/?-catenin signaling pathway; the other is Wnt5 a, including Wnt5 a, Wnt4 and Wnt11, which can activate non-canonical Wnt signaling pathways, including the Wnt/Ca2+ pathway and the Wnt/JNK signaling pathway.The Wnt-mediated Wnt/JNK signaling pathway directs the collaborative polarization of cells, regulates the cell polarity and guides the asymmetric cytoskeleton formation,cytoskeletal rearrangement and cell migration. This pathway involves the binding of the Wnt5 a and frizzelds receptors, phosphorylation of the two phosphorylation sites on the threonine(Thr) and tyrosine(Tyr) of the c-Jun kinase(JNK). The activated JNK may be translocated into the nucleus, leading to the phosphorylation of two N-terminal amino acid residues of Ser63 and Ser73 on the c-Jun protein, thereby causing the activation of some of the nuclear transcription factors, such as Elk-1, ATF2, DPC4, NFAT4 and ets-2, etc., or the direct activation of proteins regulating cytoskeleton in the cytoplasm, such as paxillin.The Wnt5a/JNK signaling pathway is closely associated with cancers, and many studies have shown that the Wnt5a/JNK signaling pathway plays a key role in the proliferation, invasion and metastasis of many malignancies, such as gastric cancer,esophageal cancer, colon cancer and breast cancer, etc. In malignant tumors, Wnt5 a can activate JNK, and the activated JNK signaling pathway is associated with the drug resistance of tumor cells, the regulation of cell apoptosis and promotion of cell survival or growth. According to the existing literature and our previous study, Wnt5 a is in the active state in malignant melanoma, and it may play an important role in the development and progression of melanoma. While whether Wnt5 a plays its role through the JNK pathway is unclear. Therefore, lentivirus transfection was employed in this study to upregulate or downregulate the Wnt5 a in the two melanoma cell lines. Real-time PCR was used to detect the expression level of Wnt5 a, Dvl, JNK, Paxillin, CD44, ICAM, MMP1, MMP2,cyclin A and cyclin D mRNA in cells, Western blot to detect the expression of proteins of Wnt5 a, JNK, p-JNK, Paxillin and p-Paxillin in cells, CCK-8 assay to detect the cell proliferation, the scratch assay to detect the metastasis cells and the Transwell chamber assay to detect the invasion capability of cells, which further revealed the role and significance of the Wnt5a/JNK signaling pathway in the development and progression of melanoma.[Objective]Investigation of the effects of regulation of the Wnt5a/JNK pathway on the proliferation and invasion abilities of melanoma cells.[Methods]1) Culture of lentivirus transfected melanoma cell lines stably in vitro.2) Real-time PCR was used to detect the expression level of Wnt5 a, Dvl, JNK,Paxillin, CD44, ICAM, MMP1, MMP2, cyclin A and cyclin D mRNA in cells, Westernblot to detect the expression of proteins of Wnt5 a, JNK, p-JNK, Paxillin and p-Paxillinin cells3) CCK-8 assay was used to detect the cell proliferation, the scratch test to detectthe metastasis cells and the Transwell chamber assay to detect the invasion capabilityof cells.[Result]1)The changes in the expression of relevant molecules of Wnt5a-mediated JNK signaling pathway in lentivirus transfected stably cell linesWe previously examined six strains of melanoma cells and selected the WM793 B cells with high expression of Wnt5 a and A2058 cells with low expression of Wnt5 a. And the lentiviral expression vector was adopted to establish four control lentivirus stably transfected cell lines, including the WM793B-pLKO.1-shWnt5 a lentivirus stably transfected cell lines(shwnt5a lentivirus transfected vector the particle transfected were utilized and the results showed the low expression of Wnt5a), the WM793B-pLKO.1-Scrambal control lentivirus stably transfected cell lines(Scrambal lentivirus transfected empty vector versus the particle transfected empty vector were utilized and the results showed that the expression of wnt5 a did not change,with high expression of Wnt5a), the A2058-pLenti-Wnt5 a lentivirus stably transfected cell lines(pLenti6.3-Wnt5 a lentivirus transfected vector the particle transfected were utilized and the results showed the high expression of Wnt5a), the A2058-pLenti-cherry control lentivirus stably transfected cell lines(pLenti6.3-Cherry lentivirus transfected empty vector versus the particle transfected empty vector were utilized and the results showed that the expression of wnt5 a did not change,with low expression of Wnt5a). We used thereal-time PCR to detect the expression levels of mRNAs of genes of the four lentivirus transfected stably cell lines in the downstream Wnt5 a and JNK pathways, and it was found that the expression of Wnt5 a is positively correlated with the expression of the downstream Dvl, JNK, Paxillin, CD44, ICAM, MMP1, MMP2, cyclin A and cyclin D genes in the JNK pathway. Western blot was performed to detect the expressions of Wnt5 a,JNK, p-JNK, Paxillin and p-Paxillin proteins, and it was found that the changes of the JNK and Paxillin proteins were not obvious, while the expression of Wnt5 a was positively correlated with the expressions of the p-JNK and p-Paxillin proteins, and that Wnt5 a can regulate the invasion and metastasis of melanoma cells through the phosphorylation of JNK and Paxillin. Therefore, we believe that the Wnt5a/JNK pathway exists in melanoma WM793 B and A2058 cell lines.2) Wnt5a/JNK regulates the proliferation, invasion and metastasis ofmelanoma cells.To detect the effect of the Wnt5a/JNK signaling pathway on the proliferation,invasion and metastasis of melanoma, we employed the CCK-8 assay to detect the proliferation of stably transfected cell lines, and we found that Wnt5 a upregulation can enhance the proliferation of melanoma cells and that its downregulation can lower the proliferation of melanoma cells. We employed the scratch test to detect the metastasis of stably transfected cell lines, and we found that Wnt5 a upregulation can enhance the metastasis of melanoma cells and that its downregulation can lower the metastasis of melanoma cells. Furtherly, we employed the Transwell chamber assay to detect the invasion of stably transfected cell lines, and we found that Wnt5 a upregulation can enhance the invasion of melanoma cells and that its invasion can lower the proliferation of melanoma cells.[Conclusion]1) The high expression of Wnt5 a can enhance the expression of relevant molecules of the JNK signaling pathway, and the lower expression of Wnt5 a can reduce the expression of relevant molecules of the JNK signaling pathway. Therefore, theWnt5a/JNK pathway exists in melanoma WM793 B and A2058 cell lines.2) The regulation of the Wnt5a/JNK signaling pathway can cause changes in the JNK signaling molecules, and the capacities of melanoma cells' proliferation, invasion and metastasis will change, suggesting that Wnt5 a can affect the JNK signaling pathway and that the Wnt5a/JNK signaling pathway may be involved in the proliferation, invasion and metastasis of melanoma cells.In this project, the relationship between the Wnt5a/JNK signaling pathway and the growth, invasion and metastasis of melanoma cells was preliminarily investigated,providing a theoretical basis for the precise diagnosis and the targeted therapy of melanoma.