Bit1在食管鱗癌上皮間質(zhì)轉(zhuǎn)化中的作用及機制初探
發(fā)布時間：2018-08-08 17:36
【摘要】：背景與目的食管鱗癌(esophageal squamous cell carcinoma,ESCC)是具有中國特色的高發(fā)惡性腫瘤,其發(fā)生、發(fā)展涉及到多個信號途徑的改變,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年發(fā)現(xiàn),有研究認為Bit1定位在線粒體發(fā)揮促凋亡作用,細胞在受到失黏附刺激時,Bit1蛋白會轉(zhuǎn)移釋放到胞漿內(nèi)并與其中的AES(amino-terminalenhancer of split)蛋白作用形成復(fù)合物,抑制Bcl-2的轉(zhuǎn)錄從而參與細胞的失巢凋亡。也有報道認為Bit1蛋白富集在高爾基體,活化MAPK信號途徑發(fā)揮抗凋亡作用。Bit1和腫瘤關(guān)系的相關(guān)研究甚少,并且觀點也不一致,而其在ESCC中的作用,除了本課題組的研究外鮮有報道。研究表明上皮間質(zhì)轉(zhuǎn)化與腫瘤的侵襲轉(zhuǎn)移有著密切的聯(lián)系。上皮間質(zhì)轉(zhuǎn)化(Epithelial-Mesenchymal Transition,EMT)即上皮細胞轉(zhuǎn)化為具有活性的間充質(zhì)細胞,該過程主要特征是上皮標志蛋白表達降低而間質(zhì)標志蛋白表達升高、上皮細胞極性缺失、細胞與基底膜間的連接溶解及細胞運動增強等,從而使得細胞的侵襲轉(zhuǎn)移、抗凋亡能力增強。因此,EMT是惡性腫瘤細胞增強侵襲與遷移能力從而發(fā)生轉(zhuǎn)移的關(guān)鍵生物學(xué)過程。本課題組前期研究表明,Bit1在ESCC中表達升高且和淋巴結(jié)轉(zhuǎn)移密切相關(guān),下調(diào)ESCC細胞中Bit1表達后可抑制ESCC細胞的增殖、侵襲和遷移能力,但具體機制不清,為了進一步探討B(tài)it1和ESCC侵襲轉(zhuǎn)移的關(guān)系及其分子機制,本實驗通過(1)ESCC細胞中Bit1表達水平的變化與EMT相關(guān)蛋白水平表達的關(guān)系研究;(2)具有高侵襲能力ESCC細胞亞系的篩選及其形態(tài)、生物學(xué)行為變化的檢測;(3)Bit1在轉(zhuǎn)化生長因子β1(transforming growth factor-β1,TGF-β1)誘導(dǎo)ESCC細胞發(fā)生EMT中的作用研究;(4)初步驗證前期基因芯片所篩選的Bit1下游效應(yīng)分子等四個部分來研究Bit1在食管鱗癌細胞發(fā)生EMT中的作用及其分子機制,以期可以為Bit1過表達可促進食管鱗癌侵襲轉(zhuǎn)移提供佐證,同時也可為發(fā)現(xiàn)ECSS臨床轉(zhuǎn)移及其預(yù)后評價的分子標志物提供新思路。方法1 si RNA轉(zhuǎn)染EC9706細胞與EC1細胞下調(diào)Bit1的表達,western blot檢測Bit1、Bcl-2、Bax、CDK4、Snail、E-cadherin、N-cadherin蛋白表達水平的變化。2通過三次Transwell實驗篩選具有侵襲力強的細胞亞系EC9706-I3并與親本細胞EC9706-I0進行形態(tài)學(xué)、生物學(xué)行為的比較。2.1形態(tài)觀察EC9706-I3細胞并與其親本細胞EC9706-I0進行比較。2.2 CCK-8檢測兩種細胞的增殖能力。2.3劃痕實驗比較二者的遷移能力。2.4流式細胞術(shù)檢測二者細胞周期的不同。2.5 Western blot檢測二者Bit1、Snail、N-cadherin蛋白水平的變化。3 TGF-β1誘導(dǎo)EC9706細胞建立上皮間質(zhì)轉(zhuǎn)化模型并檢測Bit1在其中的作用。3.1形態(tài)觀察TGF-β1不同濃度梯度(0、5、10、15、20、25ng/ml)作用下EC9706細胞形態(tài)變化。3.2 Western blot檢測TGF-β1不同濃度梯度誘導(dǎo)下Bit1、Snail、N-cadherin蛋白表達的變化從而確定TGF-β1的最佳誘導(dǎo)濃度。3.3以最佳濃度誘導(dǎo)EC9706發(fā)生EMT后si RNA下調(diào)Bit1的表達,觀察細胞形態(tài)的變化。3.4 TGF-β1誘導(dǎo)EC9706發(fā)生EMT后下調(diào)Bit1的表達,western blot檢測Bit1、Snail、N-cadherin蛋白水平的變化。4 Western blot初步驗證前期基因芯片所篩選的Bit1下游效應(yīng)分子(Paxillin、FAK)。4.1 Western blot檢測EC9706-I0與EC9706-I3細胞中Paxillin、p-Paxillin、FAK、p-FAK蛋白表達的變化。4.2 Western blot檢測不同濃度TGF-β1誘導(dǎo)下EC9706細胞中Paxillin、FAK、p-FAK蛋白表達的變化。4.3以最佳濃度誘導(dǎo)EC9706細胞后si RNA下調(diào)Bit1表達,western blot檢測Paxillin、FAK、p-FAK蛋白表達水平的變化。結(jié)果1 Bit1-si RNA可下調(diào)EC9706與EC1細胞中Bit1的表達,干擾效率可達66%;其中E-cadherin、Bax、CDK4的表達隨著Bit1的降低而升高,而Bcl-2、Snail、N-cadherin的表達隨著Bit1的下調(diào)而下調(diào)(均P0.05)。2三次Transwell實驗篩選之后EC9706-I3細胞亞系與其親本細胞EC9706-I0相比:(1)EC9706-I3形態(tài)變長、有偽足、細胞之間疏散;(2)EC9706-I3在24h、48h、72h的增殖能力均強于其親本細胞EC9706-I0(P0.001);(3)EC9706-I3的遷移能力強于其親本細胞EC9706-I0(P0.05);(4)EC9706-I3細胞的S期(DNA合成期)相對于親本細胞有所延長并且EC9706-I3的增殖指數(shù)較大(P0.05);(5)EC9706-I3細胞中Bit1的表達量明顯升高且和N-cadherin、Snail的表達呈正相關(guān)(均P0.05)。3 TGF-β1誘導(dǎo)EC9706細胞建立上皮間質(zhì)轉(zhuǎn)化模型并檢測Bit1在其中的作用:(1)TGF-β1濃度為5-15ng/ml之間細胞形態(tài)變長,以10ng/ml時最為顯著,20-25ng/ml時細胞形態(tài)逐漸接近正常形態(tài);(2)Western blot結(jié)果顯示TGF-β1為10ng/ml時Bit1、Snail、E-cadherin蛋白的表達量最高(均P0.05);(3)10ng/ml TGF-β1誘導(dǎo)EC9706發(fā)生EMT之后再用Bit1-si RNA下調(diào)Bit1的表達后,細胞形態(tài)逐漸變圓,脫落較多;(4)EC9706發(fā)生EMT之后下調(diào)Bit1的表達,N-cadherin、Snail蛋白的表達與Bit1呈正相關(guān)(均P0.05)。4 Western blot初步驗證前期基因芯片所篩選的Bit1下游效應(yīng)分子(Paxillin、FAK):(1)Western blot結(jié)果顯示EC9706-I0與EC9706-I3細胞中Paxillin、p-Paxillin、FAK、p-FAK蛋白的表達均與Bit1的表達正相關(guān)(均P0.05);(2)TGF-β1誘導(dǎo)濃度為10ng/ml時,EC9706細胞中Paxillin、FAK、p-FAK蛋白的表達量最高且與Bit1表達呈正相關(guān)關(guān)系(均P0.05);(4)TGF-β1誘導(dǎo)之后下調(diào)Bit1的表達,Paxillin、FAK、p-FAK的表達也隨之下降且與Bit1表達呈正相關(guān)關(guān)系(均P0.05)。結(jié)論1 Bit1可能參與了ESCC細胞EMT的發(fā)生過程,其高表達有助于ESCC細胞維持間質(zhì)表型特征,而下調(diào)其表達可抑制該過程。2 Paxillin、FAK可能作為Bit1的下游效應(yīng)分子促進食管鱗癌EMT的發(fā)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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.1
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