【作者】 陈峰；

【导师】 高下；

【作者基本信息】 南京医科大学 ， 耳鼻咽喉科学（专业学位）， 2019， 博士

【摘要】 目的1.探究SIRT6在鼻咽癌细胞系中的表达;2.观察SIRT6对鼻咽癌细胞系迁移能力的影响;3.探索SIRT6对鼻咽癌细胞系迁移能力影响的作用机制。方法1.通过qPCR和Western Blot实验方法,对高转移性5-8F和低转移性6-10B两种鼻咽癌细胞系,分别从m RNA和蛋白水平来研究SIRT6在不同的鼻咽癌细胞系中的表达;2.通过质粒转染,在5-8F细胞系中过表达SIRT6,在6-10B细胞系中敲低SIRT6,构建调控SIRT6表达的鼻咽癌细胞模型;3.在建立的细胞模型中,通过细胞划痕实验、Trans well实验(Matrigel细胞侵袭试验和细胞迁移试验)检测SIRT6对鼻咽癌细胞迁移能力的影响;4.通过免疫共沉淀(co-immunoprecipitation,co-IP)、染色质免疫共沉淀(chromatin immunoprecipitation,Ch IP)实验,检测并探讨SIRT6对鼻咽癌迁移能力的影响机制。结果1.在6-10B鼻咽癌细胞系中,SIRT6基因/蛋白表达水平均明显高于5-8F细胞系。通过质粒转染使5-8F细胞系中过表达SIRT6后,细胞划痕实验检测划痕48h后细胞的迁移能力,发现在过表达SIRT6的5-8F细胞系中,细胞的迁移能力明显降低,Trans well实验则发现过表达SIRT6后5-8F细胞系的侵袭能力和迁移能力也都明显降低。随后我们又在6-10B细胞系中下调SIRT6的表达,同样采用了细胞划痕实验和Trans well实验来检测下调SIRT6的影响,细胞划痕实验显示划痕48h后下调SIRT6的6-10B细胞的愈合能力明显提高,Matrigel侵袭实验表明下调SIRT6后6-10B细胞系的侵袭能力和迁移能力明显增强。从而表明SIRT6的表达和鼻咽癌细胞迁移能力呈负相关。2.我们通过细胞转染实验,在5-8F细胞系过表达SIRT6,结果细胞内SNAIL的表达降低,而在6-10B细胞系中下调SIRT6的表达,SNAIL的表达却明显升高,表明SNAIL可能受SIRT6负调控。当在6-10B细胞系中敲低SNAIL的表达后,Trans well实验结果显示在敲低SNAIL表达的细胞系中,细胞的侵袭和迁移能力都明显降低,这说明SIRT6可能是通过SNAIL影响鼻咽癌细胞迁移能力的。继而我们进行了co-IP检测,该实验结果显示在鼻咽癌细胞系中SIRT6与p65的确存在相互作用。我们再进一步通过Ch IP实验发现,SIRT6与p65相互作用,它们在SNAIL启动子区域去乙酰化H3K9和H3K56组蛋白来抑制SNAIL的表达,进而降低鼻咽癌细胞的迁移能力。结论1.SIRT6在高转移性的5-8F和低转移性的6-10B鼻咽癌细胞系中都有表达,在6-10B细胞系中的表达明显高于在5-8F细胞系中的表达;2.SIRT6的表达和鼻咽癌细胞迁移能力呈负相关,高表达SIRT6可以抑制鼻咽癌细胞系的迁移,而减少SIRT6会促进鼻咽癌细胞系的迁移;3.SIRT6可以与p65相互作用,在SNAIL的启动子区域使H3K9和H3K56去乙酰化,进而抑制SNAIL的表达。更多还原

【Abstract】 Objective1.To explore the expression of SIRT6 in nasopharyngeal carcinoma cells.2.To observe the effect of SIRT6 on the migration ability of nasopharyngeal carcinoma cells.3.To explore the mechanism of SIRT6 on the migration ability of nasopharyngeal carcinoma cells.Methods1.We used q PCR and Western Blot to detect the expression of SIRT6 in 5-8F and 6-10 B nasopharyngeal carcinoma cell lines.2.Overexpression of SIRT6 in 5-8F cell lines and downregulated the expression of SIRT6 in 6-10 B cell lines by plasmid transfection to construct a nasopharyngeal carcinoma cell model that regulates SIRT6 expression.3.The effect of SIRT6 on the migration ability of nasopharyngeal carcinoma cells was detected by wound healing assay and trans-well test.4.The mechanism of the effect of SIRT6 on the migration ability of nasopharyngeal carcinoma was detected by co-IP and Ch IP experiments.Results1.We first tested the effects of increased SIRT6 expression in 5-8F cells.Indeed,the migratory and invasive abilities of 5-8F-SIRT6 cells were significantly impaired,as demonstrated by delayed wound closure in a scratch wound healing assay,and decreased invasion rate in a Matrigel invasion assay as well as reduced migration rate in a trans-well migration assay.Next,we investigated whether downregulating SIRT6 expression in 6-10 B cells could promote metastasis or not.The scratch wound healing assay showed that reduced SIRT6 expression markedly enhanced wound closure in6-10B-sh SIRT6 cells compared with 6-10B-sh GFP cells.In addition,Matrigel invasion assay and trans-well migration assay revealed that inhibition of SIRT6 expression significantly upregulated the metastatic capacity of 6-10 B cells.2.The highly expression of SNAIL is related to the high metastasis potential of colorectal cancer,liver cancer,lung cancer and nasopharyngeal cancer.Through cell transfection,we knock down the expression of SNAIL in 6-10B-sh GFP and6-10B-sh SIRT6 cell lines.The result of trans-well assay showed that the invasion and migration ability of 6-10 B cells were significantly reduced in 6-10B-sh GFP and6-10B-sh SIRT6 cell lines after knocked down SNAIL,suggesting that SIRT6 may play a role through SNAIL in nasopharyngeal carcinoma cell lines.Meanwhile,we also performed co-IP to detect the interaction between SIRT6 and SNAIL in nasopharyngeal carcinoma cell lines.In addition,we found that SIRT6 functions as a histone deacetylase to suppress SNAIL expression through deacetylating histone H3K9 and H3K56 at the promoter of SNAIL gene.Conclusions1.SIRT6 is expressed in both 5-8F and 6-10 B nasopharyngeal carcinoma cell lines,and the expression in the 6-10 B cell line is significantly higher than in the 5-8F cell line.2.The expression of SIRT6 is negatively correlated with the migration ability of nasopharyngeal carcinoma cells.High expression of SIRT6 can inhibit the migration of nasopharyngeal carcinoma cell lines,while reducing SIRT6 can promote the migration of nasopharyngeal carcinoma cell lines.3.SIRT6 can interact with P65 to deacetylate H3K9 and H3K56 in the promoter region of SNAIL,thereby inhibiting the expression of SNAIL.更多还原