【作者】 李艳梅；

【导师】 赵佳；

【作者基本信息】 吉林大学 ， 遗传学， 2016， 硕士

【摘要】 目的研究HMGB1基因对下咽癌细胞上皮间质转化和侵袭转移的影响,并尝试探讨HMGB对于这一过程的调控机制,以期为临床上治疗下咽癌提供新靶点。方法1、利用TGF-β1诱导下咽癌细胞(Fa Du)发生EMT,并通过检索文献[1-2]确定最佳诱导浓度。将Fa Du细胞分为Normal组和TGF-β1组,显微镜下观察Fa Du细胞经TGF-β1诱导后的形态变化。通过细胞免疫荧光实验检测各组细胞中Vimentin、Snail、HMGB1的表达情况,以RT-PCR和Western Blot方法检测HMGB1蛋白和基因在经TGF-β1诱导前后的Fa Du细胞中的表达情况;2、利用脂质体法将siRNA-HMGB1和siRNA-Control转染至FaDu细胞(分为Normal组、Control组和si HMGB1组),用细胞免疫荧光、RT-PCR和Western Blot检测si RNA对HMGB1的抑制作用。采用细胞免疫荧光观察Vimentin、Snail、HMGB1的表达情况,采用RT-PCR方法分析Vimentin、Snail、E-cadherin的表达水平;Western Blot方法分析转染后下咽癌Fa Du细胞中RAGE蛋白表达变化;应用创伤愈合实验检测Fa Du细胞迁移能力的改变,Transwell实验观察HMGB1基因沉默后Fa Du细胞侵袭能力变化。结果1、Fa Du细胞经过TGF-β1诱导后,细胞形态由规则的路砖状转变为长梭形;细胞免疫荧光结果显示,TGF-β1组Fa Du细胞中的Vimentin、Snail表达上调;.2、细胞免疫荧光结果显示,TGF-β1组细胞中的HMGB1表达升高,同时HMGB1在m RNA(P<0.01)和蛋白(P<0.01)水平的表达也呈现上调现象;3、si RNA-HMGB1转染Fa Du细胞后可在m RNA(P<0.05)和蛋白(P<0.01)水平抑制HMGB1的表达;4、细胞免疫荧光结果显示,转染siRNA-HMGB1后细胞中的Vimentin、Snail表达水平降低;5、RT-PCR结果显示,转染si RNA-HMGB1后细胞中的Vimentin(P<0.05)、Snail(P<0.01)基因表达下调,而E-cadherin(P<0.05)表达上调;6、Western Blot结果显示,转染si RNA-HMGB1后细胞中的RAGE蛋白表达下调(P<0.01);7、创伤愈合实验结果显示,si RNA-HMGB1转染Fa Du细胞后,Fa Du细胞的迁移能力显著下降;8、Transwell实验结果显示,si RNA-HMGB1转染Fa Du细胞后,Fa Du细胞的侵袭能力明显降低(P<0.01)。结论1、可以通过TGF-β1诱导构建下咽癌细胞的EMT模型,HMGB1的表达与下咽癌细胞EMT形成相关;2、利用RNAi技术沉默HMGB1的表达可逆转下咽癌细胞EMT;3、利用RNAi技术沉默HMGB1的表达后,下咽癌细胞Fa Du的侵袭转移能力明显受抑;4、HMGB1可通过与RAGE相互作用调控下咽癌的侵袭转移;5、HMGB1基因可能为临床治疗下咽癌提供新的分子靶点。更多还原

【Abstract】 Objective To investigate the effect of HMGB1 gene on the epithelial-mesenchymal transition and invasion and metastasis in hypopharyngeal carcinoma cell lines.Furthermore,we also try to discuss the regulatory mechanism of HMGB1 in this process.We hope to find the new target of treatment in hypopharyngeal carcinoma.Method1.TGF-β1 induced Fa Du cells that occurred EMT by exploring literature[1-2] and screening optimal induction concentration of TGF-β1.And Fa Du cells were divided into two groups: Normal group and TGF-β1 group. We used microscope to observe the morphologic change of Fa Du cells after the induction of TGF-β1.To detect the expression status of Vimentin 、 Snail 、 HMGB1 protein in each group by Cell immunofluorescence. RT-PCR and Western blot were used to check the expression status of HMGB1 on m RNA and protein levels before and after TGF-β1 induction in FaDu cells.2.We transfected si RNA-HMGB1 and si RNA-Control to Fa Du cells by liposome transfection method(Fa Du cells were divided into three groups: Normal group 、Control group and si HMGB1 group),and detected the inhibition of si RNA by Cell immunofluorescence、Western Blot and Real Time-PCR. The Vimentin、Snail 、HMGB1 pritein expression in Fa Du cells by using Cell immunofluorescence. RT-PCR technique were used to tset Vimentin、Snail、HMGB1、E-cadherin m RNA expression levels. Western blot were used to check the expression change of HMGB1 and RAGE on protein levels after cell transfection. We observed the ability of Fa Du cells migration after silenced HMGB1 by wound healing assay.We observed the ability of Fa Du cells invasion after silenced HMGB1 by Transwell assay.Results1. Fa Du cells transit to elongated fibroblastic phenotype from epithelial cobblestone phenotype after the induction of TGF-β1.Cell immunofluorescence showed that the expression of Vimentin and Snail were up-regulated after the induction of TGF-β1.2. Cell immunofluorescence showed that the expression of HMGB1 was increased in Fa Du cells after the induction of TGF-β1. And In TGF-β1 group, the expression of HMGB1 was up-regulated obviously both on m RNA(P<0.01) and protein(P<0.01)levels.3. The expression of HMGB1 mRNA was inhibited in FaDu cells by siRNA-HMGB1(P<0.05), and also HMGB1 protein expression was inhibited obviously in Fa Du cells by si RNA-HMGB1(P<0.01).4.Cell immunofluorescence results showed the expression level of Vimentin and Snail were decreased after the transfection with si RNA-HMGB1.5.RT-PCR showed that the expression level of m RNA of Vimentin(P < 0.05) and Snail(P<0.01)were down-regulated,but E-cadherin(P<0.01) was increased after the transfection with si RNA-HMGB1.6.Western Blot showed that the expression level of RAGE was decreased after the transfection with si RNA-HMGB1(P<0.01).7.The wound healing assay proved Fa Du cells migration decrease after the transfection with si RNA-HMGB1.8.The Transwell assay proved Fa Du cells invasion decrease after the transfection with si RNA-HMGB1( P<0.01).Conclusion1.Successfully established epithelial-mesenchymal transition model of hypopharyngeal carcinoma cells after Fa Du cells were inducted 48 h by5ng/ml-TGF-β1.And the expression of HMGB1 was associated with epithelial-mesenchymal transition in hypopharyngeal carcinoma cells.2.Silencing HMGB1 gene can reverse the epithelial-mesenchymal transition of FaDu cells.3.Silencing HMGB1 gene can obviously suppress migration and invasion of hypopharyngeal carcinoma Fa Du cell lines.4.HMGB1-RAGE interaction can regulate invasion and metastasis of hypopharyngeal carcinoma Fa Du cell lines.5. HMGB1 gene can be a molecular target for the clinic treatment of hypopharyngeal carcinoma.更多还原