Bit1在食管鳞癌上皮间质转化中的作用及机制初探
发布时间：2018-08-08 17:36
【摘要】：背景与目的食管鳞癌(esophageal squamous cell carcinoma,ESCC)是具有中国特色的高发恶性肿瘤,其发生、发展涉及到多个信号途径的改变,包括一些抗凋亡因子功能的激活或者凋亡因子功能的抑制。Bit1(Bcl-2 inhibitor of transcription 1)于2004年发现,有研究认为Bit1定位在线粒体发挥促凋亡作用,细胞在受到失黏附刺激时,Bit1蛋白会转移释放到胞浆内并与其中的AES(amino-terminalenhancer of split)蛋白作用形成复合物,抑制Bcl-2的转录从而参与细胞的失巢凋亡。也有报道认为Bit1蛋白富集在高尔基体,活化MAPK信号途径发挥抗凋亡作用。Bit1和肿瘤关系的相关研究甚少,并且观点也不一致,而其在ESCC中的作用,除了本课题组的研究外鲜有报道。研究表明上皮间质转化与肿瘤的侵袭转移有着密切的联系。上皮间质转化(Epithelial-Mesenchymal Transition,EMT)即上皮细胞转化为具有活性的间充质细胞,该过程主要特征是上皮标志蛋白表达降低而间质标志蛋白表达升高、上皮细胞极性缺失、细胞与基底膜间的连接溶解及细胞运动增强等,从而使得细胞的侵袭转移、抗凋亡能力增强。因此,EMT是恶性肿瘤细胞增强侵袭与迁移能力从而发生转移的关键生物学过程。本课题组前期研究表明,Bit1在ESCC中表达升高且和淋巴结转移密切相关,下调ESCC细胞中Bit1表达后可抑制ESCC细胞的增殖、侵袭和迁移能力,但具体机制不清,为了进一步探讨Bit1和ESCC侵袭转移的关系及其分子机制,本实验通过(1)ESCC细胞中Bit1表达水平的变化与EMT相关蛋白水平表达的关系研究;(2)具有高侵袭能力ESCC细胞亚系的筛选及其形态、生物学行为变化的检测;(3)Bit1在转化生长因子β1(transforming growth factor-β1,TGF-β1)诱导ESCC细胞发生EMT中的作用研究;(4)初步验证前期基因芯片所筛选的Bit1下游效应分子等四个部分来研究Bit1在食管鳞癌细胞发生EMT中的作用及其分子机制,以期可以为Bit1过表达可促进食管鳞癌侵袭转移提供佐证,同时也可为发现ECSS临床转移及其预后评价的分子标志物提供新思路。方法1 si RNA转染EC9706细胞与EC1细胞下调Bit1的表达,western blot检测Bit1、Bcl-2、Bax、CDK4、Snail、E-cadherin、N-cadherin蛋白表达水平的变化。2通过三次Transwell实验筛选具有侵袭力强的细胞亚系EC9706-I3并与亲本细胞EC9706-I0进行形态学、生物学行为的比较。2.1形态观察EC9706-I3细胞并与其亲本细胞EC9706-I0进行比较。2.2 CCK-8检测两种细胞的增殖能力。2.3划痕实验比较二者的迁移能力。2.4流式细胞术检测二者细胞周期的不同。2.5 Western blot检测二者Bit1、Snail、N-cadherin蛋白水平的变化。3 TGF-β1诱导EC9706细胞建立上皮间质转化模型并检测Bit1在其中的作用。3.1形态观察TGF-β1不同浓度梯度(0、5、10、15、20、25ng/ml)作用下EC9706细胞形态变化。3.2 Western blot检测TGF-β1不同浓度梯度诱导下Bit1、Snail、N-cadherin蛋白表达的变化从而确定TGF-β1的最佳诱导浓度。3.3以最佳浓度诱导EC9706发生EMT后si RNA下调Bit1的表达,观察细胞形态的变化。3.4 TGF-β1诱导EC9706发生EMT后下调Bit1的表达,western blot检测Bit1、Snail、N-cadherin蛋白水平的变化。4 Western blot初步验证前期基因芯片所筛选的Bit1下游效应分子(Paxillin、FAK)。4.1 Western blot检测EC9706-I0与EC9706-I3细胞中Paxillin、p-Paxillin、FAK、p-FAK蛋白表达的变化。4.2 Western blot检测不同浓度TGF-β1诱导下EC9706细胞中Paxillin、FAK、p-FAK蛋白表达的变化。4.3以最佳浓度诱导EC9706细胞后si RNA下调Bit1表达,western blot检测Paxillin、FAK、p-FAK蛋白表达水平的变化。结果1 Bit1-si RNA可下调EC9706与EC1细胞中Bit1的表达,干扰效率可达66%;其中E-cadherin、Bax、CDK4的表达随着Bit1的降低而升高,而Bcl-2、Snail、N-cadherin的表达随着Bit1的下调而下调(均P0.05)。2三次Transwell实验筛选之后EC9706-I3细胞亚系与其亲本细胞EC9706-I0相比:(1)EC9706-I3形态变长、有伪足、细胞之间疏散;(2)EC9706-I3在24h、48h、72h的增殖能力均强于其亲本细胞EC9706-I0(P0.001);(3)EC9706-I3的迁移能力强于其亲本细胞EC9706-I0(P0.05);(4)EC9706-I3细胞的S期(DNA合成期)相对于亲本细胞有所延长并且EC9706-I3的增殖指数较大(P0.05);(5)EC9706-I3细胞中Bit1的表达量明显升高且和N-cadherin、Snail的表达呈正相关(均P0.05)。3 TGF-β1诱导EC9706细胞建立上皮间质转化模型并检测Bit1在其中的作用:(1)TGF-β1浓度为5-15ng/ml之间细胞形态变长,以10ng/ml时最为显著,20-25ng/ml时细胞形态逐渐接近正常形态;(2)Western blot结果显示TGF-β1为10ng/ml时Bit1、Snail、E-cadherin蛋白的表达量最高(均P0.05);(3)10ng/ml TGF-β1诱导EC9706发生EMT之后再用Bit1-si RNA下调Bit1的表达后,细胞形态逐渐变圆,脱落较多;(4)EC9706发生EMT之后下调Bit1的表达,N-cadherin、Snail蛋白的表达与Bit1呈正相关(均P0.05)。4 Western blot初步验证前期基因芯片所筛选的Bit1下游效应分子(Paxillin、FAK):(1)Western blot结果显示EC9706-I0与EC9706-I3细胞中Paxillin、p-Paxillin、FAK、p-FAK蛋白的表达均与Bit1的表达正相关(均P0.05);(2)TGF-β1诱导浓度为10ng/ml时,EC9706细胞中Paxillin、FAK、p-FAK蛋白的表达量最高且与Bit1表达呈正相关关系(均P0.05);(4)TGF-β1诱导之后下调Bit1的表达,Paxillin、FAK、p-FAK的表达也随之下降且与Bit1表达呈正相关关系(均P0.05)。结论1 Bit1可能参与了ESCC细胞EMT的发生过程,其高表达有助于ESCC细胞维持间质表型特征,而下调其表达可抑制该过程。2 Paxillin、FAK可能作为Bit1的下游效应分子促进食管鳞癌EMT的发生。
[Abstract]:Background and objective esophageal squamous cell carcinoma (ESCC) is a high incidence of malignant tumor with Chinese characteristics. Its development involves changes in multiple signal pathways, including the activation of some anti apoptotic factor functions or the inhibition of.Bit1 (Bcl-2 inhibitor of transcription 1) (Bcl-2 inhibitor of transcription 1) in 2004, It is considered that Bit1 is located in the mitochondria to promote apoptosis, and when the cells are stimulated by the loss of adhesion, the Bit1 protein will transfer to the cytoplasm and form a complex with the AES (amino-terminalenhancer of split) protein, inhibit the transcription of Bcl-2 and participate in the cell loss of nesting apoptosis. It is also reported that the accumulation of Bit1 protein is also enriched. In Golgi body, there are few studies on the relationship between anti apoptotic.Bit1 and tumor activation by activating MAPK signal pathway, and the view is not consistent, but its role in ESCC is rarely reported in addition to the study in this group. The study shows that epithelial mesenchymal transformation has a close relationship with the invasion and migration of tumor. Epithelial mesenchymal transformation (Epithelia L-Mesenchymal Transition, EMT) is the transformation of epithelial cells into active mesenchyme cells. This process is characterized by a decrease in the expression of epithelial marker proteins, an increase in the expression of interstitial marker proteins, the deletion of the epithelial cells, the dissolution of the cells and the basement membrane, and the enhancement of cell transport, which makes the cell invasion and metastasis and resistance to withering. Therefore, EMT is the key biological process for the metastasis of malignant tumor cells to enhance invasion and migration. Previous studies in our group have shown that Bit1 is highly expressed in ESCC and is closely related to lymph node metastasis. The expression of Bit1 in ESCC cells can inhibit the proliferation, invasion and migration of ESCC cells, but it has the ability to inhibit the proliferation, invasion and migration of cells. In order to further investigate the relationship and molecular mechanism of Bit1 and ESCC invasion and metastasis, the relationship between the changes of Bit1 expression level and the expression of EMT related protein in ESCC cells was studied in this experiment. (2) screening and morphology of high invasive ESCC cell sublines, detection of biological behavior changes, and (3) Bit1 in the transformation of ESCC cells. The role of growth factor beta 1 (transforming growth factor- beta 1, TGF- beta 1) induced EMT in ESCC cells; (4) preliminarily validates the role and molecular mechanism of Bit1 in the occurrence of EMT in esophageal squamous cell carcinoma cells and its molecular mechanism by the preliminary verification of the four parts of the Bit1 downstream effector selected by the earlier gene chip, so that the esophagus can be overexpressed in the esophagus to promote the esophagus. The invasion and metastasis of squamous cell carcinoma can provide evidence, and also provide new ideas for finding molecular markers of ECSS clinical metastasis and evaluation of prognosis. Methods 1 Si RNA transfected EC9706 cells and EC1 cells down the expression of Bit1. Western blot detected Bit1, Bcl-2, Bax, CDK4, and protein expression levels through three times. A strong invasive cell subline EC9706-I3 was screened and the morphology of the parent cell EC9706-I0 was observed. The comparison of the biological behavior between the EC9706-I3 cells and the parent cell EC9706-I0 was compared with the parent cell EC9706-I0..2.2 CCK-8 was used to detect the proliferation ability of the two cells. The migration ability of the two groups was compared to.2.4 flow cytometry. Detection of the cell cycle of two people with different.2.5 Western blot detection of Bit1, Snail, N-cadherin protein level changes.3 TGF- beta 1 induced EC9706 cells to establish an epithelial mesenchymal transition model and detect the role of Bit1 in the.3.1 morphology observation of TGF- beta 1 under the action of different concentration gradient RN blot detected the expression of Bit1, Snail, N-cadherin protein under the induction of TGF- beta 1 in different concentration gradients to determine the optimal concentration of TGF- beta 1 at the optimal concentration.3.3. The changes in the level of Bit1, Snail and N-cadherin protein.4 Western blot preliminary verification of the Bit1 downstream effectors selected by the early gene chip (Paxillin, FAK).4.1 Western blot. The expression of Paxillin, FAK, and p-FAK protein in the cell was changed by.4.3 at the best concentration of EC9706 cells, and Si RNA decreased the expression of Bit1. Western blot detected the Paxillin, FAK, and the expression level of.4.3. The expression of T1 increased, while the expression of Bcl-2, Snail, N-cadherin decreased with the downregulation of Bit1 (P0.05).2 three Transwell experiments. The EC9706-I3 cell sublines were compared with their parent cells EC9706-I0: (1) EC9706-I3 morphology became longer, there were pseudo feet and scattered between cells. (2) EC9706-I3 was stronger than its parent. Cell EC9706-I0 (P0.001); (3) the migration ability of EC9706-I3 was stronger than that of its parent cell EC9706-I0 (P0.05); (4) the S phase of EC9706-I3 cells (DNA synthesis period) was prolonged compared with the parent cells and the EC9706-I3 proliferation index was larger (P0.05); (5) the expression of Bit1 was significantly higher in EC9706-I3 cells and was positively correlated with the expression (both of them) 0.05).3 TGF- beta 1 induced EC9706 cells to establish an epithelial mesenchymal transformation model and detected the role of Bit1 in it: (1) the cell morphology between TGF- beta 1 was longer between 5-15ng/ml and was the most significant in 10ng/ml, and the cell morphology gradually approached the normal form when 20-25ng/ml; (2) Western blot results showed that TGF- beta 1 was 10ng/ml. The expression was the highest (all P0.05); (3) after 10ng/ml TGF- beta 1 induced EMT to reduce the expression of Bit1 with Bit1-si RNA, the cell morphology gradually became round and dropped more; (4) EC9706 occurred after EMT and downregulated the expression of Bit1. Bit1 downstream effector (Paxillin, FAK): (1) Western blot results showed that the expression of Paxillin, p-Paxillin, FAK, p-FAK protein in EC9706-I0 and EC9706-I3 cells were all positively related to the expression of Bit1. There was a positive correlation (all P0.05); (4) the expression of Bit1 was down regulated by TGF- beta 1, and the expression of Paxillin, FAK and p-FAK decreased and was positively correlated with the expression of Bit1 (all P0.05). Conclusion 1 Bit1 may be involved in the occurrence of EMT in ESCC cells, and its high expression helps the ESCC cells to maintain the interstitial phenotypic characteristics, and the downregulation of its expression can be inhibited. .2 Paxillin and FAK may be used as downstream effectors of Bit1 to promote the occurrence of EMT in esophageal squamous cell carcinoma.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
[Abstract]:Background and objective esophageal squamous cell carcinoma (ESCC) is a high incidence of malignant tumor with Chinese characteristics. Its development involves changes in multiple signal pathways, including the activation of some anti apoptotic factor functions or the inhibition of.Bit1 (Bcl-2 inhibitor of transcription 1) (Bcl-2 inhibitor of transcription 1) in 2004, It is considered that Bit1 is located in the mitochondria to promote apoptosis, and when the cells are stimulated by the loss of adhesion, the Bit1 protein will transfer to the cytoplasm and form a complex with the AES (amino-terminalenhancer of split) protein, inhibit the transcription of Bcl-2 and participate in the cell loss of nesting apoptosis. It is also reported that the accumulation of Bit1 protein is also enriched. In Golgi body, there are few studies on the relationship between anti apoptotic.Bit1 and tumor activation by activating MAPK signal pathway, and the view is not consistent, but its role in ESCC is rarely reported in addition to the study in this group. The study shows that epithelial mesenchymal transformation has a close relationship with the invasion and migration of tumor. Epithelial mesenchymal transformation (Epithelia L-Mesenchymal Transition, EMT) is the transformation of epithelial cells into active mesenchyme cells. This process is characterized by a decrease in the expression of epithelial marker proteins, an increase in the expression of interstitial marker proteins, the deletion of the epithelial cells, the dissolution of the cells and the basement membrane, and the enhancement of cell transport, which makes the cell invasion and metastasis and resistance to withering. Therefore, EMT is the key biological process for the metastasis of malignant tumor cells to enhance invasion and migration. Previous studies in our group have shown that Bit1 is highly expressed in ESCC and is closely related to lymph node metastasis. The expression of Bit1 in ESCC cells can inhibit the proliferation, invasion and migration of ESCC cells, but it has the ability to inhibit the proliferation, invasion and migration of cells. In order to further investigate the relationship and molecular mechanism of Bit1 and ESCC invasion and metastasis, the relationship between the changes of Bit1 expression level and the expression of EMT related protein in ESCC cells was studied in this experiment. (2) screening and morphology of high invasive ESCC cell sublines, detection of biological behavior changes, and (3) Bit1 in the transformation of ESCC cells. The role of growth factor beta 1 (transforming growth factor- beta 1, TGF- beta 1) induced EMT in ESCC cells; (4) preliminarily validates the role and molecular mechanism of Bit1 in the occurrence of EMT in esophageal squamous cell carcinoma cells and its molecular mechanism by the preliminary verification of the four parts of the Bit1 downstream effector selected by the earlier gene chip, so that the esophagus can be overexpressed in the esophagus to promote the esophagus. The invasion and metastasis of squamous cell carcinoma can provide evidence, and also provide new ideas for finding molecular markers of ECSS clinical metastasis and evaluation of prognosis. Methods 1 Si RNA transfected EC9706 cells and EC1 cells down the expression of Bit1. Western blot detected Bit1, Bcl-2, Bax, CDK4, and protein expression levels through three times. A strong invasive cell subline EC9706-I3 was screened and the morphology of the parent cell EC9706-I0 was observed. The comparison of the biological behavior between the EC9706-I3 cells and the parent cell EC9706-I0 was compared with the parent cell EC9706-I0..2.2 CCK-8 was used to detect the proliferation ability of the two cells. The migration ability of the two groups was compared to.2.4 flow cytometry. Detection of the cell cycle of two people with different.2.5 Western blot detection of Bit1, Snail, N-cadherin protein level changes.3 TGF- beta 1 induced EC9706 cells to establish an epithelial mesenchymal transition model and detect the role of Bit1 in the.3.1 morphology observation of TGF- beta 1 under the action of different concentration gradient RN blot detected the expression of Bit1, Snail, N-cadherin protein under the induction of TGF- beta 1 in different concentration gradients to determine the optimal concentration of TGF- beta 1 at the optimal concentration.3.3. The changes in the level of Bit1, Snail and N-cadherin protein.4 Western blot preliminary verification of the Bit1 downstream effectors selected by the early gene chip (Paxillin, FAK).4.1 Western blot. The expression of Paxillin, FAK, and p-FAK protein in the cell was changed by.4.3 at the best concentration of EC9706 cells, and Si RNA decreased the expression of Bit1. Western blot detected the Paxillin, FAK, and the expression level of.4.3. The expression of T1 increased, while the expression of Bcl-2, Snail, N-cadherin decreased with the downregulation of Bit1 (P0.05).2 three Transwell experiments. The EC9706-I3 cell sublines were compared with their parent cells EC9706-I0: (1) EC9706-I3 morphology became longer, there were pseudo feet and scattered between cells. (2) EC9706-I3 was stronger than its parent. Cell EC9706-I0 (P0.001); (3) the migration ability of EC9706-I3 was stronger than that of its parent cell EC9706-I0 (P0.05); (4) the S phase of EC9706-I3 cells (DNA synthesis period) was prolonged compared with the parent cells and the EC9706-I3 proliferation index was larger (P0.05); (5) the expression of Bit1 was significantly higher in EC9706-I3 cells and was positively correlated with the expression (both of them) 0.05).3 TGF- beta 1 induced EC9706 cells to establish an epithelial mesenchymal transformation model and detected the role of Bit1 in it: (1) the cell morphology between TGF- beta 1 was longer between 5-15ng/ml and was the most significant in 10ng/ml, and the cell morphology gradually approached the normal form when 20-25ng/ml; (2) Western blot results showed that TGF- beta 1 was 10ng/ml. The expression was the highest (all P0.05); (3) after 10ng/ml TGF- beta 1 induced EMT to reduce the expression of Bit1 with Bit1-si RNA, the cell morphology gradually became round and dropped more; (4) EC9706 occurred after EMT and downregulated the expression of Bit1. Bit1 downstream effector (Paxillin, FAK): (1) Western blot results showed that the expression of Paxillin, p-Paxillin, FAK, p-FAK protein in EC9706-I0 and EC9706-I3 cells were all positively related to the expression of Bit1. There was a positive correlation (all P0.05); (4) the expression of Bit1 was down regulated by TGF- beta 1, and the expression of Paxillin, FAK and p-FAK decreased and was positively correlated with the expression of Bit1 (all P0.05). Conclusion 1 Bit1 may be involved in the occurrence of EMT in ESCC cells, and its high expression helps the ESCC cells to maintain the interstitial phenotypic characteristics, and the downregulation of its expression can be inhibited. .2 Paxillin and FAK may be used as downstream effectors of Bit1 to promote the occurrence of EMT in esophageal squamous cell carcinoma.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1
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