SHARPIN参与皮肤基底细胞癌发病的功能学研究

发布时间：2018-02-01 17:46

本文关键词： 基底细胞癌 SHARPIN 细胞增殖 JUN GLI　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：近年来的研究发现,SHARPIN参与肝癌、肾癌、骨肉瘤、前列腺癌、乳腺癌等肿瘤的发生发展,提示SHARPIN是一个新的肿瘤相关基因。SHARPIN表达于多种组织和器官,参与调控NF-κB、JNK信号通路、角质形成细胞凋亡等,具有调节炎症、细胞增殖与凋亡等多种生物学功能。目前尚无SHARPIN与皮肤肿瘤发病关系的研究。皮肤基底细胞癌(Basal cell carcinoma,BCC)是人类最常见的皮肤肿瘤,其发病与Hedgehog信号通路有很大关联,Hedgehog信号通路下游分子GLI1和GLI2可以直接调控c-JUN,有研究证实c-JUN在BCC组织中高表达,JUN是JNK信号通路下游信号分子。因此我们提出疑问,SHARPIN是否通过调控经典的JNK信号通路参与BCC的发生发展?本课题拟研究SHARPIN在BCC中所起的功能及其相关的信号通路。研究目的1.明确SHARPIN基因在BCC中的突变情况;2.明确SHARPIN蛋白在BCC组织和细胞株中的表达情况;3.研究SHARPIN表达变化对BCC细胞增殖、凋亡、侵袭和迁移的影响;4.研究SHARPIN表达变化通过哪些信号通路和分子影响BCC细胞功能。研究内容和方法1.SHARPIN基因突变及表达变化与BCC的相关性研究1)SHRPIN基因在BCC石蜡组织中的突变情况采用PCR对SHARPIN外显子进行扩增,Sanger测序后分析SHARPIN基因的突变情况。2)SHARPIN蛋白在BCC组织中的表达情况采用免疫组化技术,检测SHARPIN蛋白在BCC和正常皮肤中的表达情况;采用Western Blot检测BCC细胞株TE354.T和人永生化角质形成细胞HaCaT中SHARPIN蛋白表达差异;采用免疫荧光检测SHARPIN在BCC组织中的定位。2.SHARPIN对BCC细胞株TE354.T细胞功能的影响1)构建SHARPIN过表达和沉默慢病毒载体,转染TE354.T细胞,采用WB和qRT-PCR检测转染效率;2)利用EdU、CCK-8检测SHARPIN表达变化对细胞增殖的影响;3)利用TUNEL和Annexin V检测SHARPIN表达变化对细胞凋亡的影响;4)利用Transwell检测SHARPIN表达变化对细胞侵袭和迁移的影响。3.SHARPIN表达变化影响细胞功能的分子机制研究1)根据第2部分结果利用WB检测SHARPIN表达变化对细胞功能相关蛋白表达的影响;2)利用WB检测SHARPIN表达变化对JUN和GLI蛋白表达的影响。实验结果1.BCC中SHARPIN基因存在高频突变,突变率达28.1%;SHARPIN蛋白在BCC癌组织中表达降低或丧失;2.SHARPIN低表达可以促进TE354.T细胞的增殖;3.SHARPIN低表达使Cyclin D1和CDK4表达升高;c-JUN表达降低,磷酸化c-JUN表达升高;GLI1表达无明显变化,GLI2表达升高。结论1.SHARPIN在BCC组织中表达显著降低甚至丧失,且存在高频突变,提示SHARPIN在BCC发病中具有肿瘤抑制基因的特征,可作为基底细胞癌鉴别诊断的分子标记;2.SHARPIN低表达可以促进TE354.T细胞的增殖,提示SHARPIN表达下降加快细胞增殖可能是调控BCC发生发展的主要机制;3.SHARPIN低表达促进JUN磷酸化,激活GLI2,从而高表达CyclinD1和CDK4,导致BCC细胞周期的S期细胞比例增多,促进细胞增殖。
[Abstract]:In recent years, it has been found that SHARPIN is involved in the occurrence and development of liver cancer, renal cancer, osteosarcoma, prostate cancer, breast cancer and so on. It is suggested that SHARPIN is a new tumor-related gene. SHARPIN is expressed in various tissues and organs, and it is involved in the regulation of NF- 魏 B, JNK signal pathway, keratinocyte apoptosis and so on. Has the ability to regulate inflammation. There are many biological functions, such as cell proliferation and apoptosis. There is no research on the relationship between SHARPIN and skin tumor. Basal cell carcinoma of skin basal cell carcinoma. BCCs are the most common skin tumors in humans. The pathogenesis of BCCs is closely related to the Hedgehog signaling pathway. GLI1 and GLI2, downstream molecules of Hedgehog signaling pathway, can directly regulate c-JUN. it has been confirmed that c-JUN is highly expressed in BCC tissues. JUN is a downstream signal molecule of JNK signaling pathway. Therefore, we ask whether SHARPIN participates in the development of BCC by regulating the classical JNK signaling pathway. The purpose of this study is to study the function of SHARPIN in BCC and its related signaling pathways. Objective 1. To determine the mutation of SHARPIN gene in BCC. 2.The expression of SHARPIN protein in BCC tissues and cell lines was determined. 3. To study the effect of SHARPIN expression on proliferation, apoptosis, invasion and migration of BCC cells. 4. To study the signal pathways and molecules by which SHARPIN expression changes affect the function of BCC cells. 1. The mutation of SHARPIN gene and the correlation between the changes of SHARPIN gene expression and BCC. Research 1). The mutation of SHRPIN gene in BCC paraffin tissues was amplified by PCR. The expression of SHARPIN gene in BCC was analyzed by immunohistochemical technique after Sanger sequencing. The expression of SHARPIN protein in BCC and normal skin was detected. Western Blot was used to detect the difference of SHARPIN protein expression between BCC cell line TE354.T and human immortalized keratinocytes HaCaT. Immunofluorescence Detection of SHARPIN Localization in BCC tissue. 2. Effect of SHARPIN on the function of BCC cell line TE354.T 1). Construction of SHARPIN overexpression and silencing lentivirus vector. The transfection efficiency of TE354.T cells was detected by WB and qRT-PCR. 2) the effect of SHARPIN expression on cell proliferation was detected by using Edutrol CCK-8. 3) TUNEL and Annexin V were used to detect the effect of SHARPIN expression on apoptosis. 4) using Transwell to detect the effect of SHARPIN expression on cell invasion and migration. 3. The molecular mechanism of SHARPIN expression affecting cell function 1). According to the results of the second part, the effects of SHARPIN expression on the expression of function-related proteins were detected by WB. 2) the effect of SHARPIN expression on the expression of JUN and GLI protein was detected by WB. 1. There was high frequency mutation of SHARPIN gene in BCCs. The mutation rate was 28.1%; The expression of SHARPIN protein was decreased or lost in BCC carcinoma. 2. The low expression of SHARPIN could promote the proliferation of TE354.T cells. 3. The low expression of SHARPIN increased the expression of Cyclin D1 and CDK4. The expression of c-JUN decreased and phosphorylated c-JUN increased. There was no significant change in the expression of GLI1 and GLI2.Conclusion 1. The expression of SHARPIN in BCC tissues was significantly decreased or even lost, and there were high frequency mutations. 2. The results suggest that SHARPIN has the characteristics of tumor suppressor gene in the pathogenesis of BCC and can be used as a molecular marker for differential diagnosis of basal cell carcinoma. 2. The low expression of SHARPIN can promote the proliferation of TE354.T cells, suggesting that the decrease of SHARPIN expression and the acceleration of cell proliferation may be the main mechanism of regulating the occurrence and development of BCC. 3. The low expression of SHARPIN promoted the phosphorylation of JUN and activated GLI2, which resulted in the high expression of CyclinD1 and CDK4, which resulted in the increase of S phase cells in BCC cell cycle. Promote cell proliferation.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5

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