SHARPIN Promotes Melanoma Progression Via Rap1 Signaling Pathway

Melanoma is an invasive and easily metastasized tumor.In recent years,it has been reported in the literature that SHARPIN is a new tumor-associated gene involved in the development of various tumors.This topic will study the function of SHARPIN and molecular mechanisms of how SHARPIN regulate the development of melanoma.Objective1.To identify the expression level of SHARPIN in GEO database,clinical samples and melanoma cell lines,also to clarify the mutations of SHARPIN in melanoma;To analyze the relation between SHARPIN and BRAF or N-RAS expression,the bioinformatics tool GEPIA(http://gepia.cancer-pku.cn/detail.php?clicktag=correlation###)was used.2.To explore the role of SHARPIN in regulating proliferation,migration,invasion and cells resistance to cisplatin and dacarbazine;3.To explore the role of SHARPIN in tumorigenicity assay and lung metastasis assay in vivo;4.To study the pathways and moleculars of how SHARPIN affected melanoma cells’ function.Methods·The study of SHARPIN gene and protein expression in melanoma,and the relation with prognosis in melanoma patients.1.SHARPIN expression and related prognosis in melanoma.We analyzed SHARPIN expression and related prognosis in melanoma by data mining in GEO.2.SHARPIN expression and mutations of SHARPIN in the tissue of melanoma.Detection the expression level of SHARPIN in paired samples and cell lines by qRT-PCR and Western blot(WB).Using immunohistochemical and immunofluorescence technique tested SHARPIN expression in melanoma.The exons of SHARPIN were amplified by PCR,and then analyzed the rate of mutation afterSanger sequencing.·To explore the effect of SHARPIN in cell and functions in tumorigenicity assay and lung metastasis assay in vivo.1.Cells will be infected with lentivirus carrying SHARPIN gene or shRNA of SHARPIN.After seletion of single cell cloning by serial dilution,the efficiency of infection was evaluated by Western Blot and qRT-PCR;2.Evaluate the effect of SHARPIN on proliferation,migration,invasion,cisplatin resistance and dacarbazine resistance by using CCK-8,wound healing assay and transwell assay;3.The function induced by SHARPIN was confirmed using tumorigenicity assay and lung metastasis assay in vivo.·To study the molecular mechanism of how SHARPIN affected cell functions1.The spectrum of gene expression changes in overexpression and inhibition groups of melanoma cell lines were characterized by transcriptome sequencing,which may provide an insight into the potential molecular mechanisms.Using qRT-PCR and WB to detect the hub genes expression of the signaling pathways.2.The expression alteration of the hub genes in signaling pathways were used by WB after treated with the activator or inhibitors.The alteration of cell numbers was quantified by transwell assay after treated withe activator or inhibitors.3.The protein expression levels of SHARPIN and Rap1 in xenografts using Western blotsResults1.The expression of SHARPIN was increased in melanoma and elevated SHARPIN expression predicts a poor prognosis.2.Using GEPIA web tool,there was no association between SHARPIN and BRAF or N-RAS mRNA expression3.Mutation of the SHARPIN gene in our cases did not cause related amino acid changes.4.SHARPIN was mainly expressed in the cytoplasm of melanoma cell lines.5.Overexpression of SHARPIN can promote melanoma cell proliferation,migration and invasion.Inhibition of SHARPIN can reduce melanoma cell proliferation,migration and invasion.6.Overexpression of SHARPIN in melanoma cells can increase the resistant to cisplatin and dacarbazine.Inhibition of SHARPIN in melanoma cells can decrease the resistant to cisplatin and dacarbazine.However,this result has no statistically significant difference.7.Overexpression of SHARPIN enhanced tumorigenesis and lung metastasis by using subcutaneous tumor assay and implantation lung cancer model.Inhibition of SHARPIN decreased tumorigenesis and lung metastasis by using subcutaneous tumor assay and implantation lung cancer model.8.Compared with control group,the mRNA expression of Rap 1B,p38MAPK,JNK1 and c-Jun were down-regulated in Sh group.Compared with control group,the protein expression of Rap 1-GTP,p38MAPK,JNK and c-Jun were down-regulated obviously in Sh group.9.The protein expression of Rap1,Rap1-GTP,p38MAPK,JNK and c-Jun were elevated after adding the Rap1 activator.The migration ablity and invasion abliby were rescued by adding the Rap1 activator in Sh and Sh-NC groups.10.The protein expression of Rap1,Rap1-GTP,p38MAPK,JNK and c-Jun were decreased after adding the Rap1 inhibitors.The migration ablity and invasion abliby were rescued by adding the Rap1 inhibitors in EXP and EXP-NC groups.11.Compared to the control groups,the protein expression of SHARPIN and Rap1 were decreased in Sh group whearas increased in EXP group.Conclusion1.SHARPIN expression is increased in melanoma and it is related with a poor prognosis of melanoma.2.It has been shown that SHARPIN is inolved in melanoma proliferation,migration and invasion in vitro and in vivo.3.SHARPIN promotes Rap1 and its downstream pathways JNK/c-Jun、p38 pathways to regulates melanoma migration and invasion.