Functional Study Of SHARPIN In The Development Of Cutaneous Basal Cell Carcinoma

In recent years,numerous studies found that SHARPIN was involved in the development of liver cancer,kidney cancer,osteosarcoma,prostate cancer,breast cancer and so on,these results supported that SHARPIN is a new tumor-associated gene.SHARPIN expressed in multiple tissues and organs,regulated NF-?B,JNK signaling pathways and cell apoptosis of keratinocyte,have a variety of biological functions such as inflammation,cell proliferation and apoptosis,ect.So far,there is no research about SHARPIN and skin tumor.Skin basal cell carcinoma(BCC)is the highest incidence of skin tumor,and its pathogenesis is closely correlated to Hedgehog pathway.GLI1 and GLI2,the downstream molecular of Hedgehog pathway,can directly regulate c-JUN.Some studies have confirmed that the expression of JUN increased in BCC,JUN is a downstream signaling molecule of JNK signaling pathway.So we ask,what is the express level of SHARPIN in BCC and whether SHARPIN participate the development of BCC?This topic will be study the molecular mechanisms of how SHARPIN regulate the development of BCC.Objective1.To clarify the mutations of SHARPIN in BCC;2.To identify the expression level of SHARPIN in BCC and BCC cell line;3.To explore the role of SHARPIN in regulating proliferation,apoptosis,invasion and migration;4.To study the pathways and moleculars of how SHARPIN affected cells function.Methods1.The correlation study of SHARPIN mutation and expression in BCC1)Mutations of SHARPIN in BCC paraffin-embedded tissues The exons of SHARPIN were amplified by PCR,and then analyzed the rate of mutation after Sanger sequencing.2)Expression of SHARPIN in BCC tissuesUsing immunohistochemical technique tested SHARPIN expression in BCC and normal skin;detection the expression level of SHARPIN in BCC cell line TE354.T and keratinocyte cell line HaCaT used Western Blot;using immunofluorescence examined the position of SHARPIN in BCC.2.To explore the effect of SHARPIN in cell functions1)TE354.T cells were transfected with lentivirus vectors carrying SHARPIN gene or shRNA of SHARPIN;then use WB and qRT-PCR detected the transfection efficiency of SHARPIN;2)The effect of SHARPIN on proliferation was used EdU and CCK-8 assays;3)TUNEL and Annexin V assays were performed to detection the influence of SHARPIN on cell apoptosis;4)Transwell was carried to evaluate the effect of SHARPIN on cell invasion and migration;3.To study the molecular mechanism of how SHARPIN affected cell functions1)According to the result of part 2,using WB detected the proteins that regulate cell functions;2)Using WB detected the expression of JUN and GLI.Results1.There existed high frequency mutation of SHARPIN in BCC,and the mutation rate was 28.1%;2.The expression of SHARPIN was decreased and even disappeared in BCC tissues and cell line;3.Knock down of SHARPIN can promote the proliferation of TE354.T cells;4.Knock down of SHARPIN increased expression level of Cyclin D1 and CDK4;the expression of c-JUN was down-regulated,but phosphorylation c-JUN was up-regulated;the expression of GLI1 was no difference,but GLI2 increased.Conclusion1.The expression of SHARPIN was decreased and even disappeared in BCC,and there existed high frequency mutations of SHARPIN,so we suggested that SHARPIN has the characteristic of tumor suppressor gene,and may be a molecular marker of BCC in diagnosis.2.Knock down of SHARPIN increased the proliferation ability of TE354.T,so we infer that this may be the main mechanism in developed of BCC.3.We consider that knock down of SHARPIN may activated GLI2,promoted phosphorylation of c-JUN,and then overexpressed Cyclin D1 and CDK4,increased S phase cells,finally promote proliferation of BCC.