本文關(guān)鍵詞： 基底細(xì)胞癌 SHARPIN 細(xì)胞增殖 JUN GLI　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：近年來的研究發(fā)現(xiàn),SHARPIN參與肝癌、腎癌、骨肉瘤、前列腺癌、乳腺癌等腫瘤的發(fā)生發(fā)展,提示SHARPIN是一個(gè)新的腫瘤相關(guān)基因。SHARPIN表達(dá)于多種組織和器官,參與調(diào)控NF-κB、JNK信號(hào)通路、角質(zhì)形成細(xì)胞凋亡等,具有調(diào)節(jié)炎癥、細(xì)胞增殖與凋亡等多種生物學(xué)功能。目前尚無SHARPIN與皮膚腫瘤發(fā)病關(guān)系的研究。皮膚基底細(xì)胞癌(Basal cell carcinoma,BCC)是人類最常見的皮膚腫瘤,其發(fā)病與Hedgehog信號(hào)通路有很大關(guān)聯(lián),Hedgehog信號(hào)通路下游分子GLI1和GLI2可以直接調(diào)控c-JUN,有研究證實(shí)c-JUN在BCC組織中高表達(dá),JUN是JNK信號(hào)通路下游信號(hào)分子。因此我們提出疑問,SHARPIN是否通過調(diào)控經(jīng)典的JNK信號(hào)通路參與BCC的發(fā)生發(fā)展?本課題擬研究SHARPIN在BCC中所起的功能及其相關(guān)的信號(hào)通路。研究目的1.明確SHARPIN基因在BCC中的突變情況;2.明確SHARPIN蛋白在BCC組織和細(xì)胞株中的表達(dá)情況;3.研究SHARPIN表達(dá)變化對(duì)BCC細(xì)胞增殖、凋亡、侵襲和遷移的影響;4.研究SHARPIN表達(dá)變化通過哪些信號(hào)通路和分子影響B(tài)CC細(xì)胞功能。研究?jī)?nèi)容和方法1.SHARPIN基因突變及表達(dá)變化與BCC的相關(guān)性研究1)SHRPIN基因在BCC石蠟組織中的突變情況采用PCR對(duì)SHARPIN外顯子進(jìn)行擴(kuò)增,Sanger測(cè)序后分析SHARPIN基因的突變情況。2)SHARPIN蛋白在BCC組織中的表達(dá)情況采用免疫組化技術(shù),檢測(cè)SHARPIN蛋白在BCC和正常皮膚中的表達(dá)情況;采用Western Blot檢測(cè)BCC細(xì)胞株TE354.T和人永生化角質(zhì)形成細(xì)胞HaCaT中SHARPIN蛋白表達(dá)差異;采用免疫熒光檢測(cè)SHARPIN在BCC組織中的定位。2.SHARPIN對(duì)BCC細(xì)胞株TE354.T細(xì)胞功能的影響1)構(gòu)建SHARPIN過表達(dá)和沉默慢病毒載體,轉(zhuǎn)染TE354.T細(xì)胞,采用WB和qRT-PCR檢測(cè)轉(zhuǎn)染效率;2)利用EdU、CCK-8檢測(cè)SHARPIN表達(dá)變化對(duì)細(xì)胞增殖的影響;3)利用TUNEL和Annexin V檢測(cè)SHARPIN表達(dá)變化對(duì)細(xì)胞凋亡的影響;4)利用Transwell檢測(cè)SHARPIN表達(dá)變化對(duì)細(xì)胞侵襲和遷移的影響。3.SHARPIN表達(dá)變化影響細(xì)胞功能的分子機(jī)制研究1)根據(jù)第2部分結(jié)果利用WB檢測(cè)SHARPIN表達(dá)變化對(duì)細(xì)胞功能相關(guān)蛋白表達(dá)的影響;2)利用WB檢測(cè)SHARPIN表達(dá)變化對(duì)JUN和GLI蛋白表達(dá)的影響。實(shí)驗(yàn)結(jié)果1.BCC中SHARPIN基因存在高頻突變,突變率達(dá)28.1%;SHARPIN蛋白在BCC癌組織中表達(dá)降低或喪失;2.SHARPIN低表達(dá)可以促進(jìn)TE354.T細(xì)胞的增殖;3.SHARPIN低表達(dá)使Cyclin D1和CDK4表達(dá)升高;c-JUN表達(dá)降低,磷酸化c-JUN表達(dá)升高;GLI1表達(dá)無明顯變化,GLI2表達(dá)升高。結(jié)論1.SHARPIN在BCC組織中表達(dá)顯著降低甚至喪失,且存在高頻突變,提示SHARPIN在BCC發(fā)病中具有腫瘤抑制基因的特征,可作為基底細(xì)胞癌鑒別診斷的分子標(biāo)記;2.SHARPIN低表達(dá)可以促進(jìn)TE354.T細(xì)胞的增殖,提示SHARPIN表達(dá)下降加快細(xì)胞增殖可能是調(diào)控BCC發(fā)生發(fā)展的主要機(jī)制;3.SHARPIN低表達(dá)促進(jìn)JUN磷酸化,激活GLI2,從而高表達(dá)CyclinD1和CDK4,導(dǎo)致BCC細(xì)胞周期的S期細(xì)胞比例增多,促進(jìn)細(xì)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.5

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