TNFα通过NF-κB调控TWIST表达诱导下咽癌Fadu细胞发生EMT和转移的机制研究

发布时间：2018-06-08 21:12

本文选题：下咽肿瘤 + 鳞状细胞癌　；参考：《山东大学》2015年博士论文

【摘要】：第一部分TWIST诱导下咽癌FaDu细胞发生EMT并促进其转移的机制研究实验目的：一方面探讨TWIST的表达变化对下咽癌FaDu细胞发生上皮细胞-间充质转化(EMT)的影响及其促进下咽癌FaDu细胞发生迁移、侵袭、转移的内在机制；另一方面探讨TWIST在下咽癌组织中的表达及其与临床病理资料的关系。实验方法：体外实验部分,实验分组为TWIST过表达载体组(pcDNA 3.1-TWIST)及其对照组,TWIST沉默载体组(MicroRNA-TWIST)及其对照组,利用脂质体2000转染下咽癌FaDu细胞,并利用Western blot的方法,验证TWIST蛋白在上述两种载体及对照组中的表达情况；利用倒置相差显微镜,观察TWIST过表达对下咽癌FaDu细胞形态的影响：利用Transwell小室实验,观察TWIST过表达对下咽癌FaDu细胞的迁移和侵袭能力的影响；Western blot检测TWIST过表达和沉默时分别对E-cadherin和N-cadherin表达的影响；体内实验部分,采用免疫组织化学的方法,检测TWIST在下咽癌肿瘤组织和癌旁组织中的表达情况,并分析其表达与临床病理资料(年龄,性别,肿瘤的大小、分化、淋巴结转移)的关系。实验结果：Western blot结果显示：TWIST在pcDNA3.1-TWIST组中较对照组表达升高,在miR-TWIST组中较对照组表达降低；倒置显微镜观察Fadu细胞的形态改变,结果显示：在pcDNA3.1-TWIST组中,细胞表现为长条梭形,细胞处于松散状,细胞间的极性丢失,细胞间的粘附性降低；TranswellTM小室实验发现,迁移实验中,pcDNA3.1-TWIST组的细胞迁移数是对照组的1.8倍(P0.05),表明TWIST过表达能增强Fadu细胞迁移能力；侵袭实验中,pcDNA3.1-TWIST组中,细胞浸润数是对照组的1.6倍(P0.05),表明TWIST过表达同样能增强Fadu细胞侵袭能力。检测TWIST变化对E-cadherin和N-cadherin的影响,Western blot结果显示：在pcDNA3.1-TWIST组中,TWIST表达升高时,E-cadherin表达降低,N-cadherin升高；在miR-TWIST组表达,TWIST表达降低时,E-cadherin升高,N-cadherin表达降低,表明TWIST促进Fadu细胞发生了EMT。利用免疫组织化学的方法,检测下咽癌组织和癌旁组织中TWIST的表达,结果显示：TWIST在下咽癌组织中有表达,癌旁组织中未见表达。TWIST在下咽癌组织的细胞核和细胞质均有表达,但细胞质中表达更明显；研究TWIST表达与临床病理资料的关系,结果显示：TWIST表达与下咽癌的分化(P=0.038),肿瘤大小(P=0.048),淋巴结转移(P=0.044)有关；而TWIST表达与病人性别、年龄无关(P0.050)。实验结论：本实验,体外实验首先证实了TWIST在pcDNA3.1-TWIST组中较对照组表达升高,在miR-TWIST组中较对照组表达降低,表明TWIST过表达载体和沉默载体构建成功并在细胞内发挥作用,其作为后续的基础。TWIST的过表达能影响Fadu细胞的形态改变,并促进Fadu细胞的迁移和侵袭能力；TWIST变化导致E-cadherin和N-cadherin的表达变化,表明TWIST诱导Fadu细胞发生EMT；体内实验部分,研究发现TWIST在下咽癌组织中表达,且其表达与下咽癌组织的分化、肿瘤大小、淋巴结转移有关,与年龄、性别无关。通过体内外实验发现,TWIST的表达与下咽癌组织的分化、肿瘤大小、淋巴结转移有关,且能促进下咽癌Fadu细胞发生迁移、侵袭、转移的内在机制,是TWIST通过促进下咽癌发生EMT和调控E-cadherin和N-cadherin表达实现的。第二部分TNFα通过NF-κB通路调控TWIST表达诱导下咽癌Fadu细胞发生EMT的机制研究实验目的：上皮细胞-间充质转化(EMT)的发生在肿瘤转移中发挥重要作用,TNFa能促进肿瘤的侵袭和转移,其机制可能与EMT的发生有关。但是具体的机制并不清楚,所以,本实验研究意在探讨TNFa对下咽癌细胞Fadu发生EMT及侵袭转移能力的影响,并深入阐释其内在可能的机制。实验方法：以下咽癌Fadu细胞为实验材料,TNFa(10ng/ml)处理组为实验组,TNFa(10ng/ml)未处理组为对照组。TNFa(10ng/ml)作用Fadu细胞24 h,利用细胞划痕-愈合实验,检测不同组细胞的运动能力,观察TNFa(10 ng/ml)对FaDu细胞的运动能力的影响；采用Transwell实验,检测不同组细胞的迁移和侵袭能力,观察TNFa(10 ng/ml)对下咽癌FaDu细胞迁移和侵袭能力的影响；利用倒置相差显微镜观察细胞形态变化,探讨TNFa(10 ng/ml)对下咽癌FaDu细胞形态的影响；利用细胞免疫荧光染色及共聚焦荧光显微镜检测不同组中上皮间充质标志物E-cadherin, N-cadherin表达,观察TNFa(10 ng/ml)对E-cadherin, N-cadherin表达的影响；为了进一步明确TNFa(10 ng/ml)对E-cadherin, Vimentin的影响,采用Western blot检测NFa (10g/ml)处理FaDu细胞不同时间(0 h,1 h,3 h,5h)时E-cadherin, Vimentin的表达；为了进一步探讨TNFa (10 ng/ml)对E-cadherin, Vimentin影响的机制,采用了Western blot和免疫荧光染色及共聚焦荧光显微镜,检测TNF (10 ng/ml)处理FaDu细胞不同时间(0h,1 h,3 h,5h)时TWIST,p65的表达变化；为了研究TNFa对p65的影响,同时检测’TNFa(10 ng/ml)处理FaDu细胞不同时间(Oh,1 h,3 h,5h)时p-Ikk及p-IκBa表达变化；为了研究p65对TWIST的影响,利用NF-κB特异性抑制剂(BAY11-7082)和siRNA-p65质粒,降低p65表达,同时利用Western blot检测各组中TWIST的表达变化。实验结果：TNFa作用细胞24 h后,TNFa处理组中,细胞的运动速度比对照组明显加快；迁移实验中,处理组和对照组细胞数分别是276±38,124±15,差异具有统计学意义(P0.05)；浸润实验中,TNFa处理组和对照组细胞数分别是95±3,63±6,差异具有统计学意义(P0.05)；TNFa(10 ng/ml)作用后,Fadu细胞形态发生明显变化,TNFa处理组的细胞极性丢失,细胞呈长梭形,分散状,细胞间的粘附性降低等间充质细胞表型,而对照组细胞呈椭圆形,细胞间排列紧密,细胞间的粘附性高。此外,免疫荧光及Western blot检测发现TNFa处理组中,上皮标志物E-cadherin降低,间充质标志物Vimentin和N-cadherin表达升高；TNFa (10 ng/ml)作用Fadu细胞不同时间(0 h,1 h,3 h,5h)后,利用Western blot检测作用不同时间的TWIST表达,结果显示随着作用时间的延长,TWIST的表达升高；TNFa (10 ng/ml)作用Fadu细胞不同时间(0 h,1 h,3h,5h)后,利用Western blot检测作用不同时间的p65, p-Ikk和p-IκBα的表达,结果显示：随着作用时间的延长,其表达均升高；免疫荧光和Western blot检测p65和TWIST表达,结果显示：与对照组比较,实验组核内p65和TWIST表达升高；利用siRNA-p65和BAY11-7082降低p65表达,Western blot检测p65和TWIST表达,结果显示：siRNA-p65和BAY11-7082均能降低p65表达,p65降低的同时TWIST表达降低。实验结论：fNFa能够促进FaDu田胞运动,迁移及浸润能力；TNFa能诱导FaDu细胞形态由细胞间排列紧密、椭圆形,极性存在的上皮样表型,向细胞松散、呈梭形、极性丢失的间充质表型变化,同时伴有上皮标志物E-cadherin降低,间充质标志物Vimentin和N-cadherin表达升高,表明TNFa诱导FaDu细胞发生具有较强迁移及侵袭能力的EMT。此外,TNFa诱导TWIST表达升高。这正是TNFa通过升高TWIST表达,诱导FaDu细胞发生EMT,从而增强细胞的迁移及侵袭能力的机制。TNFa诱导TWIST表达升高的机制是TNFa促进p-Ikk, p-IκBa的表达,激活NF-κB通路中p65表达,进一步促进TWIST表达升高。总之,TNFa通过NF-κB通路调控TWIST表达,进而促进下咽癌发生EMT和增强细胞的转移能力。
[Abstract]:The first part TWIST induces the mechanism of EMT in hypopharyngeal cancer FaDu cells and the mechanism to promote its metastasis. The objective of this study is to investigate the effect of TWIST expression on the epithelial cell mesenchymal transition (EMT) of hypopharyngeal carcinoma FaDu cells and the internal mechanism of promoting the migration, invasion and metastasis of the FaDu cells of hypopharynx cancer; on the other hand, TW is discussed. The expression of IST in hypopharyngeal carcinoma and its relationship with the clinicopathological data. Experimental methods: experimental part in vitro, the experimental group was divided into TWIST overexpression vector group (pcDNA 3.1-TWIST) and its control group, TWIST silencing carrier group (MicroRNA-TWIST) and its control group, using liposome 2000 to transfect the hypopharyngeal carcinoma FaDu cells, and use Western blot recipe. The expression of TWIST protein in the two carriers and control groups was tested, and the effect of TWIST overexpression on the morphology of FaDu cells in hypopharyngeal carcinoma was observed by inverted phase contrast microscope: the effect of TWIST overexpression on the migration and invasion ability of FaDu cells in hypopharyngeal carcinoma was observed by Transwell chamber test; Western blot detected TWIST over table. The effects of E-cadherin and N-cadherin on the expression of E-cadherin and N-cadherin respectively; in the experimental part of the body, immunohistochemistry was used to detect the expression of TWIST in the tumor tissues and para cancerous tissues of hypopharyngeal carcinoma, and to analyze the relationship between the expression and the clinicopathological data (age, sex, tumor size, differentiation, lymph node metastasis). Results: the results of Western blot showed that the expression of TWIST in the group pcDNA3.1-TWIST was higher than that in the control group, and the expression in the miR-TWIST group was lower than that in the control group. The morphological changes of Fadu cells were observed by the inverted microscope. The results showed that in the pcDNA3.1-TWIST group, the cells displayed a long shuttle shape, the cells were in the loose shape, the cell polarity was lost, and the cells were lost. In the TranswellTM lab experiment, the number of cell migration in group pcDNA3.1-TWIST was 1.8 times that of the control group (P0.05), indicating that TWIST overexpression could enhance the migration ability of Fadu cells. In the invasive experiment, the number of cell infiltration in the pcDNA3.1-TWIST group was 1.6 times as much as that of the control group (P0.05), indicating that the over expression of TWIST was equally possible. The effects of TWIST changes on E-cadherin and N-cadherin were enhanced. The results of Western blot showed that the expression of E-cadherin decreased and N-cadherin increased when the expression of TWIST increased in the pcDNA3.1-TWIST group, and in miR-TWIST group, when the TWIST expression decreased, the expression decreased. The expression of TWIST in hypopharyngeal and paracancerous tissues was detected by EMT. in U cells. The results showed that TWIST was expressed in the hypopharyngeal carcinoma tissue. The expression of.TWIST in the nucleus and cytoplasm of the hypopharyngeal carcinoma was not expressed in the para cancer tissues, but the expression of the cytoplasm in the cytoplasm was more obvious; the expression of TWIST was studied. The relationship with the clinicopathological data showed that the expression of TWIST was related to the differentiation of hypopharyngeal carcinoma (P=0.038), tumor size (P=0.048) and lymph node metastasis (P=0.044), and the expression of TWIST was not related to the sex of the patients and age (P0.050). Experimental conclusion: in this experiment, the expression of TWIST in the group of pcDNA3.1-TWIST was first confirmed in the pcDNA3.1-TWIST group than in the control group. The expression of TWIST over expression vector and silencing carrier was successfully constructed and played a role in the cells in the miR-TWIST group. The overexpression of.TWIST, as a follow-up basis, could affect the morphological changes of Fadu cells and promote the migration and invasion of Fadu cells, and the TWIST changes led to the expression of E-cadherin and N-cadherin. The results showed that TWIST induced the occurrence of EMT in Fadu cells. In the experimental part of the body, the expression of TWIST in hypopharyngeal carcinoma was found, and its expression was related to the differentiation of hypopharyngeal carcinoma, the size of the tumor, the metastasis of lymph nodes, and the age and sex. The expression of TWIST and the differentiation of the hypopharyngeal carcinoma, the size of the tumor, the lymph nodes, and the lymph nodes were found in the body and the body. Metastasis is related, and it can promote the migration, invasion and metastasis of hypopharyngeal carcinoma Fadu cells, which is realized by TWIST by promoting EMT and regulating E-cadherin and N-cadherin expression in hypopharyngeal carcinoma. The second part TNF alpha regulates TWIST expression by NF- kappa B pathway to regulate the EMT mechanism of Fadu cell occurrence in hypopharyngeal carcinoma: epithelial fine Cellular mesenchyme transformation (EMT) plays an important role in tumor metastasis. TNFa can promote tumor invasion and metastasis, and its mechanism may be related to the occurrence of EMT. However, the specific mechanism is not clear. Therefore, this study is intended to explore the effect of TNFa on the EMT and invasion and metastasis of hypopharynx cancer cells, and to explain it in depth. The possible mechanism. Experimental methods: the Fadu cells of the following pharynx cancer are the experimental material, the TNFa (10ng/ml) treatment group is the experimental group, the TNFa (10ng/ml) untreated group is 24 h of the.TNFa (10ng/ml) action of the control group, and the cell scratch healing experiment is used to detect the movement ability of the different groups of cells, and the movement ability of TNFa (10 ng/ml) on the FaDu cells is observed. The effect of Transwell test was used to detect the migration and invasion ability of different groups of cells and to observe the effect of TNFa (10 ng/ml) on the migration and invasion ability of FaDu cells in hypopharyngeal carcinoma. The morphological changes of the cells were observed by inverted phase contrast microscope and the effects of TNFa (10 ng/ml) on the morphology of the FaDu cells in hypopharyngeal carcinoma; and cell immunofluorescence staining was used. The effects of TNFa (10 ng/ml) on the expression of E-cadherin and N-cadherin were detected by TNFa (10 ng/ml), and the effects of TNFa (10 ng/ml) on E-cadherin and Vimentin were detected by confocal fluorescence microscopy, and the effects of TNFa (10 ng/ml) on E-cadherin and Vimentin were determined by Western blot (0, 1, 1). H, 3 h, 5H) the expression of E-cadherin, Vimentin; in order to further explore the mechanism of TNFa (10 ng/ml) on E-cadherin and Vimentin, Western blot and immunofluorescence staining and confocal fluorescence microscopy were used to detect the changes in the expression of TNF (1, 3, 3). In order to study the effect of TNFa (10 ng/ml) on the expression of p-Ikk and p-I kappa Ba at different times of FaDu cells (Oh, 1 h, 3 h, 5H), the expression of p65 and p-I kappa Ba were detected. Results: after 24 h of TNFa cells, the speed of cell movement in the TNFa treatment group was significantly faster than that in the control group, and the number of cells in the treatment group and the control group were 276 + 38124 + 15, respectively, and the difference was statistically significant (P0.05). In the infiltration experiment, the number of TNFa treatment group and the irradiated group was 95 + 3,63 + 6, and the difference was statistically significant. Meaning (P0.05); after the action of TNFa (10 ng/ml), the morphology of Fadu cells changed obviously. The cell polarity of the TNFa treatment group was lost, the cells showed long spindle shape, scattered, and the adhesion between cells decreased, while the cells in the control group were elliptical, the cells were arranged closely, and the adhesion between cells was high. In addition, immunofluorescence and Western Blot detection found that in the TNFa treatment group, the epithelial markers E-cadherin decreased and the expression of mesenchymal markers Vimentin and N-cadherin increased, and TNFa (10 ng/ml) was used to detect the expression of Fadu cells at different time (0 h, 1 h, 3 h, 5H). The results showed that the expression increased with the prolongation of action time. After TNFa (10 ng/ml) action of Fadu cells at different time (0 h, 1 h, 3h, 5H), Western blot was used to detect the expression of p65, p-Ikk and p-I kappa alpha. The results showed that the expression increased with the prolongation of action time, and the results showed that the experimental group was compared with the control group, and the experimental group was compared with the control group. The expression of p65 and TWIST in the nucleus was increased, p65 expression was reduced by siRNA-p65 and BAY11-7082, and Western blot was used to detect p65 and TWIST expression. The results showed that siRNA-p65 and BAY11-7082 could reduce the p65 expression and decrease the expression at the same time. The cell morphology is characterized by a compact, elliptical and polar epithelioid phenotype that is loose, spindle shaped, and an interstitial phenotypic change, accompanied by a decrease in the epithelial marker E-cadherin, and the increase in the expression of Vimentin and N-cadherin in the mesenchymal markers. It is indicated that TNFa induced FaDu cells have strong migration and invasion ability. In addition, TNFa induced the increase of TWIST expression. This is the mechanism that TNFa induces EMT in FaDu cells by increasing TWIST expression, thus enhancing the migration and invasion of cells. The mechanism.TNFa induces the increase of TWIST expression is TNFa promoting p-Ikk, p-I kappa expresses, and further promotes the increase of expression. TNFa regulates TWIST expression through NF- kappa B pathway, thereby promoting EMT and enhancing cell migration in hypopharyngeal carcinoma.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.6

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