Expression Of NFATc3 In Human Malignant Melanoma And Its Mechanism Of Anti-apoptosis

Malignant melanoma is the most malignant skin tumor,the incidence of which is increasing at a rate of 3%per year.At present,only early surgical resection can have a relatively better prognosis,advanced malignant melanoma has a very poor prognosis due to its generally high anti-apoptosis level,insensitivity to routine clinical radiotherapy and chemotherapy,and once distant metastasis occurs,the 5-year survival rate is less than 15%.In recent years,the application of molecular targeted therapy has brought about breakthroughs.However,for the Asian population with a high incidence of acral malignant melanoma,its response rate is low and it is prone to acquired drug resistance,which greatly limits the clinical benefit of patients.Therefore,exploring new molecular therapeutic targets for malignant melanoma and inhibiting the expression of its anti-apoptotic phenotype is the focus of current research.Nuclear factor of activated T cells（NFAT）is a member of the[Ca²⁺]related transcription factor family.In the resting state,NFAT is phosphorylated and confined to the cytoplasm.When[Ca²⁺]channels open and intracellular[Ca²⁺]concentration is maintained at a high level,NFAT undergoes conformational changes,dephosphorylation,and then exposes the nuclear localization sequence,and subsequently binds to the target promoter element and regulates the transcription of specific responsive genes.Thus,cell functions are widely regulated.Studies have shown that the regulation of NFAT is not limited to the neural development and immune system,but also plays an important role in the cell cycle progression,apoptosis and malignant transformation of tumor cells.Recent studies have demonstrated that the expression level of NFAT and the development of malignant melanoma have close relations,among them,NFATc3 plays an important role in highly aggressive tumors such as esophageal cancer,triple-negative breast cancer and glioblastoma,but the expression and function of NFATc3 in malignant melanoma need to be further studied.This experiment adopts the clinical primary and metastatic malignant melanoma tissue samples and 4 different human melanoma cell lines with diverse malignancy to explore the expression of NFATc3 in melanoma and its correlation with the malignancy.Further through the technique of Crispr/Cas9,knocking down the expression of NFATc3,and then CCK-8,Western Blot,immunofluorescence,and flow cytometry was adopted to explore the impact of NFATc3 on the occurrence and development of malignant melanoma and its possible mechanism.This study provides a theoretical basis and data support for the exploration of potential molecular therapeutic targets for melanoma.This experiment is composed of the following four parts:Part Ⅰ,difference of protein expression and NFATc3 expression in malignant melanoma tissues and cells1.Proteomics analysis was used to detect protein expression differences between malignant melanoma and adjacent tissue samples,and it was found that the differential proteins were mainly distributed in organelles,membranes,cytoplasm and protein-containing complexes,with the main functions of binding,catalytic activity and structural molecular activity,and were involved in biological processes such as metabolism,development,response to stimulation and localization.The GO annotation significance enrichment analysis showed that the differential protein enrichment was most significant at the calcium ion junction.2.NFATc3 is expressed in all malignant melanoma tissues and is highly expressed in metastatic melanoma:NFATc3 expression was detected by immunohistochemical staining in all malignant melanoma Pathological paraffin sections.The result showed that NFATc3was widely expressed in malignant melanoma tissues,and had a higher expression in the metastatic malignant melanoma（P<0.05）,meanwhile had no expression in paracancer tissues（P<0.01）.3.The expression level of NFATc3 in malignant melanoma cells is positively correlated with the malignancy:（1）qPCR detection of different malignant melanoma cell lines showed NFATc3 mRNA expression in malignant melanoma cell lines were higher than normal pigment cell line,in which ganglion metastasis of malignant melanoma cell line Mel-RM was the highest,and the expression of NFATc3 in skin situ malignant melanoma cell line A375 was relatively low（P<0.01）;（2）Immunofluorescence staining of melanoma cells with different malignan cy showed that the fluorescence intensity of NFATc3 was significantly higher in ganglion metastasis cell line Mel-RM than in situ melanoma cell line A375.4.TCGA database analysis confirmed that the expression of NFATc3 was significantly higher in malignant melanoma than normal tissues（P<0.001）.These results suggested that NFATc3 is highly expressed in malignant melanoma,and the expression level of NFATc3 was positively correlated with the malignancy.Part Ⅱ,NFATc3 knockdown plasmid constructed by Crispr/Cas9 technology was transfected and identified in vitroThe synthesized plasmids were transfected into 293T cells,and the results showed that the expression level of NFATc3 was significantly reduced after transfecting the recombinant plasmid sg RNA3-NFATc3 compared with the other two groups（P<0.01）,indicating that the sg RNA3-NFATc3 knockdown effect is the best.The knockdown plasmid sg RNA3-NFATc3 and the control plasmid sg RNA-NC were transfected into Mel-RM cells.After transfection for 48 h,the results of Western blot showed that the expression of NFATc3 in knockdown group was significantly lower than that in control group（P<0.01）,indicating that Mel-RM cells knocked-down model by sg RNA3-NFATc3 were successfully constructed and could be used for subsequent experimentsPart Ⅲ,NFATc3 promotes survival and invasion of malignant melanoma cells by inhibiting apoptosis levels1.Knocking down the expression of NFATc3 in Mel-RM cells can reduce cell viability,but does not affect cell proliferation.（1）CCK-8 assay showed that Mel-RM cell activity was significantly reduced after NFATc3 knockdown（P<0.01）;（2）Ed U staining results showed that there was no significant difference in Ed U positive cells in Mel-RM cells after NFATc3 knockdown compared with the control group（P<0.05）;Immunofluorescence detection showed that there was no significant difference in the ratio of PCNA positive fluorescent cells in Mel-RM cells between the knockdown group and the control group（P<0.05）2.NFATc3 knockdown promoted Mel-RM cell apoptosis（1）Flow cytometry showed that the apoptosis rate of Mel-RM cells increased after NFATc3 knockdown（P<0.01）;（2）TUNEL apoptosis staining showed that decreased NFATc3 expression significantly increased the TUNEL positive rate in Mel-RM cells（P<0.01）;（3）Immunofluorescence detection showed that the Pro-Caspase-3 positive fluorescence signal was distributed in the cytoplasm of Mel-RM cells in the knockdown group,and the positive cell rate was significantly higher than that in the control group（P<0.01）;（4）Western blot results showed that NFATc3 knockdown could significantly increase the expression of Cleaved-Caspase-8 and Cleaved-Caspase-3 proteins at 18 k Da and 17k Da respectively;3.NFATc3 knockdown inhibited the invasion and migration of Mel-RM cells（1）The results of wound healing assay showed that the healing percentage of Mel-RM cells was significantly reduced after NFATc3 knockdown（P<0.01）;（2）Transwell migration assay results showed that the number of cells penetrating the compartment was significantly reduced after NFATc3 knockdown compared with the control group（P<0.01）;（3）Transwell invasion assay showed that 24h after transfection of Mel-RM cells in the knockdown group,the number of cells penetrating the compartment was significantly reduced compared with the control group（P<0.01）;4.pCMV-NFATc3 overexpression plasmid was transfected into A375 cells for reverse verification（1）Western Blot identification showed that the expression of NFATc3 was increased in A375 cells 48h after pCMV-NFATc3 transfection,indicating that A375 cells with overexpression of NFATc3 had been successfully constructed.（2）Flow cytometry showed after treatment of apoptosis inducers,the apoptosis rate of A375 cells overexpressed with NFATc3 was significantly lower than that of the control group.（P<0.05）;（3）TUNEL staining indicated that the amount of TUNEL positive cells in the pCMV-NFATc3 overexpression group was significantly lower than that in the control group after using the apoptosis inducers.（P<0.01）;（4）Western blot results confirmed that overexpressing NFATc3 could significantly reduce the expression of Cleaved-Caspase-8 and Cleaved-Caspase-3 proteins at 18k Da and17k Da respectively;These results indicated that NFATc3 could significantly improve the viability of Mel-RM cells,but had no significant effect on their proliferation.Inhibition of NFATc3expression can enhance the activation of caspase-8 and caspase-3,promote the occurrence of apoptosis,and reduce the migration and invasion ability of Mel-RM cells.Part Ⅳ,NFATc3 affects the apoptosis of malignant melanoma cells by regulating the expression of cLIP1.NFATc3 was positively correlated with cLIP expression（1）cLIP qPCR detection in different strains of malignant melanoma cell lines and normal pigment cell line,it was found that cLIP expression in malignant melanoma cell lines were higher than in normal pigment cell line.Besides,in ganglion metastatic melanoma cell line Mel-RM,the expression of cLIP was the highest,while in the primary skin melanoma cell line A375 was relatively low（P<0.05）.（2）Bioinformatics confirmed that NFATc3 and cLIP expression were positively correlated in malignant melanoma（R=0.51,P<0.01）.2.cLIP can promote cell survival and inhibit cell apoptosis in malignant melanoma cells（1）After cLIP reduction in Mel-RM cells,Western blot results showed that the expressions of Cleaved-Caspase-8 and Cleaved-Caspase-3 in Mel-RM cells were increased;TUNEL apoptosis staining confirmed that the proportion of TUNEL positive cells increased significantly.Flow cytometry showed that the apoptosis level of Mel-RM cells increased after cLIP knockdown.（2）After overexpression of cLIP in A375 cells,the expression of Cleaved-Caspase-8and Cleaveed-Caspase-3 in A375 cells was decreased by Western blot.TUNEL apoptosis staining confirmed that the proportion of TUNEL positive cells decreased signif icantly.Flow cytometry showed that apoptosis level of A375 cells was inhibited after cLIP overexpressed.These results suggested that the expression of cLIP could inhibit the activation of Caspase-8 and Caspase-3,and reduce the occurrence of apoptosis.3.NFATc3 could inhibit apoptosis by upregulating the expression of cLIPIt was confirmed by co-transfection that cLIP could reverse the up-regulation of apoptosis in Mel-RM cells after NFATc3 knockdown,indicating that NFATc3 could enhance the ability of anti-apoptosis by up-regulating the expression of cLIP.In conclusion,this study found that1.The expression of NFATc3 is increased in malignant melanoma tissues,and is higher in metastatic tumor tissues than in situ tumor tissues,suggesting a p ositive correlation between its expression level and malignancy.2.Crispr-Cas9 technology was used to knockdown the expression of NFATc3 in metastatic malignant melanoma cells,and then the activation of Caspase-8 and Caspase-3was enhanced,the level of apoptosis was increased,the cell activity was decreased,and the ability of cell migration and invasion was weakened.Overexpression of NFATc3 in in-situ cell lines showed decreased activation of Caspase-8 and Caspase-3,inhibited apoptosis,and increased cell activity.These results indicated that NFATc3 could negatively regulate the apoptosis of malignant melanoma by inhibiting the activation levels of Caspase-8 and Caspase-3,promote cell survival and create conditions for invasion and metastasis.3.The knockdown of cLIP can induce the apoptosis of highly malignant Mel-RM cells,and the antagonistic ability of in-situ A375 cells to apoptosis inducer is enhanced by the overexpression of cLIP.Finally,the co-transfection complement experiment further confirmed that NFATc3 in malignant melanoma cells mainly suppressed the activation levels of Caspase-8 and Caspase-3 by upregulating the expression of cLIP,thus promoting the expression of anti-apoptotic phenotype in malignant melanoma cells.