Wnt5a/Ca²⁺/Calcineurin/NFAT Signaling Pathway Affect The Proliferation And Invasion Of Melanoma Cell

The main biological characteristics of malignant melanoma is its proliferation andinvasion, which also contribute in large to the nature of melanoma: difficult to cure, easyto recur and poor prognosis. Current investigation on the molecular mechanism ofproliferation and invasion, especially on the shutting down certain molecular pathway andgenetic therapy, constitute the basis in the hope of curing melanoma. Research findingsindicate that Wnt5a/Ca2+/Calcineurin/NFAT pathway is closely involved in the onset andprogression of melanoma. As a Ca2+dependent transcription factor, NFAT is associatedwith the onset and progression of multiple malignant tumors. To clarify the impact ofWnt5a/Ca2+/Calcineurin/NFAT pathway on the melanoma’s biological characteristics andits regulatory mechanism will lend further reference to the targeted cancer therapy in bothbasic science and clinical applications.. This investigation group initially examined theexpression of key signaling molecule in Wnt5a/Ca2+/Calcineurin/NFAT signaling pathwayby RT-PCR, western blot and laser confocal microscopy, validated the activation of thispathway. In this project, the focus proceeds to investigate the impact ofWnt5a/Ca2+/Calcineurin/NFAT signaling pathway on the biology of melanoma. Byconstructing lentiviral vectors overexpressing Wnt5a and shWnt5a targeting Wnt5a, melanoma cells were transduced by lentivirus to assess the proliferation, metastasis andkey signaling molecules expression in the transduced melanoma cells. The results showedthat overexpression of Wnt5a is capable of stimulating the proliferative and metastasispotential of melanoma cells by activating downstream signaling molecules. This findinghas broadened the therapeutic approaches to metastatic melanomas and laid foundationsfor further elucidating the pathogenesis of melanoma and advancing molecular targetedcancer therapy.一．Construction of melanoma with expression and expression suppression wnt5ageneWe designed human wnt5a primers and wnt5a siRNA based on its mRNA sequence,chose WM793B melanoma cell lines expressing high levels of wnt5a to extract RNA.After RT-PCR, the wnt5a cDNA was obtained and ligated into shuttle vector pENTR3C toconstruct pENTR3C-Wnt5a. By LR, Wnt5a was inserted into pLenti6.3/V5-DEST toobtain recombinant plasmid pLenti6.3-Wnt5a. By gateway lentivirus packaging systemand HEK293T cell lines, we prepared lentivirus particles named plenti6.3-wnt5a andinfected wnt5a melanoma cell lines A2058which originally expresses low level of wnt5a.Hence, A2058-pLenti6.3-Wnt5a cell line was constructed. Based on the sequence ofhuman Wnt5a gene in GeneBank, Wnt5asiRNA Wnt5a siRNA was designed andsynthesized. The single-stranded primers were annealed to double-strandedoligonucleotide sequences and subcloned into linear pLKO.1lentiviral plasmid digestedby enzyme to produce pLKO.1-shwnt5a lentiviral vector. After being identified by PCRand sequencing, plasmid pLKO.1-shwnt5a was transfected into HEK293T cells to packagelentiviral particles. Human melanoma WM793B cells were infected by lentiviral particles.Western blot analysis of wnt5a expression in the two cell lines indicated A2058-pLenti6.3-Wnt5a cell line express high level of wnt5a while WM793B-pLKO.1-shWnt5acell line expresses low level of wnt5a compared with controls. This results confirm thesuccessful construction of wnt5a cell lines with either low or high expression of wnt5a,and provide experimental models to study the impact of Wnt5a/Ca2+/Calcineurin/NFATpathway on melanoma cell biology 二．the effect of activating and repressing Wnt5a signaling pathway on proliferationand invasion of melanomaBy western blot, we examined the key signaling molecule VEGF, CAN, NFATexpression in the Wnt5a/Ca2+Pathway in the stabling tranduced cells overexpressing orlow expressing Wnt5a. The results indicated that signaling molecule VEGF, CAN, NFAT’sexpression in the Wnt5a/Ca2+pathway is increased by different levels in the Wnt5aoverexpression cell lines. Using the two cell line model: A2058-pLenti6.3-Wnt5a andWM793B-pLKO.1-shWnt5a, we employed MTT method to detect cell reproduction,transwell to assay metastasis, flow cytometry to detect cell cycle. The results indicated thatoverexpression of wnt5a leads to increased reproduction, metastasis ability and cell cycleparameters. However, down-regulation of wnt5a gene expression exhibits a significantinhibitory effect on the metastasis and reproduction ability of melanoma cells. Usingcalcineurin inhibitor cycloporin A treatment on corresponding transduced melanoma cells,the impact of CsA on biological characteristics of melanoma was examined. By evaluatingthe biological characteristics of CsA treated malignant melanoma cell lines, we ascertainedthat down-regulation of Calcineurin expression could inhibit the invasiveness andmetastasis malignant melanoma cells.Results：1. Overexpression of Wnt5a in melanoma cells is positively correlated with enhancedability of reproduction, metastasis and reproduction parameters, vice versa.2. The expression of key signaling molecules in Wnt5a/Ca2+/Calcineurin/NFAT pathwayis positively correlated with the expression of Wnt5a by different levels, and viceversa.3. Successfully construction of melanoma cells stably overexpressing or downexpressingWnt5a, provide model and platform to study the role of wnt5a in melanoma.4. the reproduction parameters of melanoma conditioning by CsA is decreased, At thesame time, the invasive power and proliferative ability presented a decrease.