食管鳞状细胞癌患者Bin1基因启动子甲基化状态及其在食管癌发生发展中的作用

发布时间：2018-05-09 06:08

  本文选题：食管鳞癌 + 桥接整合因子-1　； 参考：《河北医科大学》2017年硕士论文

【摘要】：第一部分食管鳞状细胞癌患者Bin1基因启动子甲基化状态及其意义目的:通过检测桥接整合因子-1(bridging integrator-1,Bin1)基因在食管鳞癌(esophageal squamous cell cancer,ESCC)患者癌组织和其癌旁组织中表达情况及其启动子甲基化状态,分析在ESCC患者中Bin1甲基化状态与其表达及临床病理特征的关系,探讨Bin1基因甲基化在ESCC临床中的意义,为Bin1基因甲基化作为新靶点应用于ESCC综合治疗提供理论依据。方法:1收集2014年6月至2015年6月期间河北医科大学第四医院胸外科手术切除的58例ESCC患者的癌组织和癌旁组织,并将每例标本分为3份,其中2份标本取下后迅速置于液氮罐中,后放置于-80℃超低温冰箱中贮存以备提取RNA、DNA时使用;另1份以4%甲醛溶液固定以备制作蜡块时使用,用于免疫组织化学。2采用免疫组织化学法(immunohistochemistry,IHC)、甲基化特异性PCR(Methylation-Specific polymerase Chain Reaction,MSP)、实时荧光定量聚合酶链式反应(Quantitative Real-time PCR,qRT-PCR)分别检测58例经病理证实的ESCC患者的癌组织、癌旁组织中Bin1蛋白的表达情况、Bin1基因的表达情况和Bin1基因启动子甲基化状态,分析ESCC中Bin1甲基化状态与Bin1蛋白、基因表达情况及与患者临床病理特征之间的关系。结果:1在58例食管鳞癌患者中,ESCC组织中Bin1蛋白低表达发生率明显高于相应癌旁组织[37/58(63.79%)vs.13/58(22.41%),P0.01];在58例ESCC患者中,ESCC组织中Bin1 mRNA的表达水平明显低于癌旁组织,分别为(0.78±0.05)vs.(1.03±0.03)(t=9.643,P0.01);Bin1发生甲基化的组织中Bin1 mRNA表达水平显著低于未发生甲基化的组织,分别为(0.68±0.04)vs.(0.85±0.07)(t=2.476,p0.05),bin1基因呈低表达的患者有35例,是bin1蛋白低表达的患者的94.59%。这一结果提示,在escc组织中bin1在基因水平和蛋白水平的表达一致。2在35例bin1mrna呈低表达的escc组织中有32例(91.43%)bin1基因启动子发生甲基化,在37例bin1蛋白呈低表达的escc组织中有33例(89.19%)bin1基因启动子发生甲基化;说明在escc组织中bin1启动子区的异常甲基化与bin1的低表达相关,且bin1甲基化是bin1在escc组织中低表达的原因之一。3bin1甲基化状态与患者tnm分期、肿瘤侵犯深度、分化程度、淋巴结转移相关(p0.05),与患者年龄、性别比较无统计学意义(p0.05)。结论:食管癌患者肿瘤组织中bin1呈高甲基化状态,bin1高甲基化状态与食管癌患者肿瘤分化程度、浸润深度、淋巴结转移、tnm分期等临床病理特征密切相关。第二部分bin1基因启动子甲基化状态在食管鳞癌发生发展中的作用目的:通过检测escc细胞yes-2、te13、te1、kyse30和ec109中bin1表达情况和甲基化状态,选择yes-2和te13细胞进行后续实验,应用去甲基化药物5-氮杂-2’脱氧胞苷(5-aza-2’deoxycytidine,5-aza-dc)作用于yes-2和te13细胞,分析去甲基化前后bin1表达水平、escc细胞增殖、凋亡等恶性生物学行为和emt相关蛋白的变化,探讨bin1在escc发生发展的可能机制,为bin1基因甲基化作为新靶点应用于escc综合治疗提供实验依据。方法:1分别采用qrt-pcr、msp方法检测bin1在5种人食管鳞癌细胞yes-2、te13、te1、kyse30和ec109中的表达情况及甲基化状态,选出yes-2和te13进行后续实验。使用去甲基化药物5-aza-dc对两种escc细胞进行干预,并分别用msp、western-blot对两种escc细胞进行检测,分析bin1甲基化状态与其表达的变化情况。2采用mtt、克隆形成实验、流式细胞术检测yes-2和te13细胞去甲基化前后增殖能力的改变情况,分析bin1甲基化对escc细胞增殖能力的影响。3采用流式细胞术(flowcytometryassay,fcm)检测yes-2和te13细胞去甲基化前后增殖、凋亡能力的改变情况,分析bin1甲基化对escc细胞增殖、凋亡能力的影响。4采用细胞划痕实验、transwell实验检测yes-2和te13细胞去甲基化前后迁移、侵袭能力的改变情况,分析bin1甲基化对escc细胞迁移、侵袭能力的影响。5采用westernblot实验检测yes-2和te13细胞去甲基化前后上皮间质转化(epithelial-mesenchymaltransition,emt)相关蛋白e钙粘蛋白(e-cadherin,e-cad)、n钙粘蛋白(n-cadherin,n-cad)、基质金属蛋白酶2(matrixmetalloproteinase-2,mmp-2)、基质金属蛋白酶9(mmp-9)等表达的改变情况,探讨bin1甲基化影响escc细胞的上皮间质转化的发生情况。结果:1bin1mrna在te13、te1、kyse30、ec109和yes-2五种escc细胞中均呈低表达状态(p0.01);五种escc细胞均为甲基化状态,其中yes-2和te1两种细胞为完全甲基化状态;经5-aza-dc去甲基化处理后,yes-2、te13、te1为非甲基化状态;筛选出yes-2和te13细胞进行后续实验;经5-aza-dc去甲基化处理后yes-2和te13两种人escc细胞bin1蛋白表达提高(p0.01)。2mtt结果显示,经不同浓度(30μm、60μm、90μm)5-aza-dc干预yes-2细胞后d2天,细胞的增殖抑制率分别为6.7±0.9%、12.3±1.7%和15.4±2.1%,培养d7天时,细胞的增长抑制率分别为7.3±1.0%、49.3±5.7%和59.6±6.3%,与浓度为0μm的对照组相比,细胞增殖率显著降低;经不同浓度(30μm、60μm、90μm)的5-aza-dc干预te13细胞后d2天,细胞的增殖抑制率分别为6.4±0.8%、11.9±1.7%和15.1±2.0%,培养d7天时,细胞的增长抑制率分别为7.0±1.1%、47.2±5.6%和57.2±5.9%,与浓度为0μm的对照组相比,细胞增殖率显著降低(p0.01)。克隆形成实验结果显示:经不同浓度(0μm、30μm、60μm、90μm)5-aza-dc干预yes-2细胞后,细胞的克隆数分别为(233±29)、(189±23)、(105±16)和(71±11),与浓度为0μm的对照组相比,克隆数明显降低;经不同浓度(0μm、30μm、60μm、90μm)5-aza-dc干预te13细胞后,细胞的克隆数分别为(281±33)、(235±28)、(139±19)和(83±12),与浓度为0μm的对照组相比,克隆数明显降低。说明yes-2和te13细胞经去甲基化处理后增殖活性均显著下降(p0.01)。3流式细胞术结果显示,经不同浓度(0μm、30μm、60μm、90μm)5-aza-dc干预yes-2细胞后,细胞处于g0/g1期的百分比分别为:(37.2±3.1)%、(46.5±3.9)%、(58.2±4.3)%和(70.7±4.9)%,处于s期的百分比分别为:(50.6±4.2)%、(42.7±3.6)%、(26.6±3.1)%和(20.5±2.4)%,与浓度为0μm的对照组相比,细胞处于g0/g1期的百分比明显升高,处于s期的百分比明显降低;经不同浓度(0μm、30μm、60μm、90μm)5-aza-dc干预te13细胞后,细胞处于g0/g1期的百分比分别为:(33.7±2.8)%、(39.4±3.6)%、(47.3±3.9)%和(56.3±4.5)%,处于s期的百分比分别为:(70.2±4.5)%、(58.3±4.2)%、(45.2±3.1)%和(32.6±2.8)%,与浓度为0μm的对照组相比,细胞处于g0/g1期的百分比明显升高,处于s期的百分比明显降低。说明经去甲基化处理后,escc细胞周期发生改变,处于s期的细胞显著下降,阻滞于g0/g1期的细胞显著上升(p0.01)。流式细胞术结果显示,经不同浓度(0μm、30μm、60μm、90μm)5-aza-dc干预yes-2细胞后,处于凋亡的细胞百分比为:(4.12±0.89)%、(7.49±1.34)%、(10.03±2.13)%和(13.07±2.47),与浓度为0μm的对照组相比,处于凋亡的细胞百分比明显提高(p0.01);经不同浓度(0μm、30μm、60μm、90μm)的5-aza-dc干预te13细胞后,处于凋亡的细胞百分比为:(3.57±0.78)%、(8.09±1.43)%和(10.15±2.08)%、(15.64±2.55),与浓度为0μm的对照组相比,处于凋亡的细胞百分比明显提高(p0.01)。上述实验结果说明bin1基因去甲基化后可以抑制yes-2和te13细胞的增殖能力和促进其凋亡,对于escc细胞的恶性生物学行为具有抑制作用。4划痕实验结果显示:发现经5-aza-dc去甲基化处理后,yes-2细胞的48h迁移距离为(57.7±4.5)μm,未经去甲基化处理的48h迁移距离为(122.4±9.4)μm,去甲基化处理后的yes-2细胞迁移距离明显短于处理前,其结果具有统计学意义(p0.01);te13细胞经5-aza-dc去甲基化处理后的48h迁移距离为(64.2±4.8)μm,未经去甲基化处理的48h迁移距离为(133.4±8.1)μm,去甲基化处理后的te13细胞迁移距离明显短于处理前,其结果具有统计学意义(P0.01)。YES-2和TE13细胞迁移距离显著缩短,说明Bin1经去甲基化处理后能够抑制YES-2和TE13细胞的迁移能力。Transwell实验结果显示,经不同浓度(0μM、30μM、60μM、90μM)的5-Aza-d C去甲基化处理后的YES-2细胞,穿过Matrigel滤膜的细胞数分别为(206±35)、(137±29)、(78±18)和(39±9),与浓度为0μM的对照组相比,穿过Matrigel滤膜细胞术明显减少(P0.01)。经不同浓度(0μM、30μM、60μM、90μM)的5-Aza-dC去甲基化处理后的TE13细胞,穿过Matrigel滤膜的细胞数分别为(289±42)、(145±33)、(57±16)和(48±12),与浓度为0μM的对照组相比,穿过Matrigel滤膜细胞术明显减少(P0.01)。YES-2和TE13细胞穿膜细胞数显著减少,说明Bin1经去甲基化处理后能够抑制两种细胞的侵袭能力。上述实验说明说明Bin1经去甲基化后能够抑制YES-2和TE13细胞的迁移、侵袭能力。5 Western blot检测结果显示:ESCC细胞YES-2和TE13经5-Aza-dC处理后,EMT相关蛋白E-cad表达上调(P0.01),N-cad、Snail、MMP-2和MMP-9蛋白的表达下调(P0.01),说明Bin1去甲基化处理后能够改变YES-2和TE13细胞EMT相关蛋白的表达从而抑制上皮间质转化的发生。结论:体外实验证实Bin1甲基化影响食管癌细胞增殖、凋亡、迁移和侵袭能力,通过抑制食管癌细胞上皮间质转化过程影响食管癌发生发展。
[Abstract]:Methylation status of Bin1 gene promoter in the first part of esophageal squamous cell carcinoma (ESCC) and its significance Objective: to detect the expression of bridging integrator-1 (Bin1) gene in the cancerous tissue and the para cancerous tissue of the esophageal squamous cell carcinoma (esophageal squamous cell cancer, ESCC) and its promoter methylation status, and analyze the status of the methylation status of the promoter of the esophageal squamous cell carcinoma (esophageal squamous cell cancer, ESCC). The relationship between Bin1 methylation status and its expression and clinicopathological features in ESCC patients, to explore the significance of Bin1 gene methylation in the clinical ESCC, and to provide a theoretical basis for Bin1 gene methylation as a new target for ESCC comprehensive treatment. Methods: 1 to collect the Department of thoracic surgery operation in the fourth hospital of Hebei Medical University from June 2014 to June 2015. 58 cases of ESCC patients were removed from the cancer tissue and para cancerous tissue, and each sample was divided into 3 samples, of which 2 specimens were quickly placed in a liquid nitrogen tank and then stored in -80 C ultra low temperature refrigerators for RNA, DNA use; the other 1 were used with 4% Formaldehyde Solution to prepare wax blocks and used immuno histochemical.2 for immunization. Immunohistochemistry (IHC), methylation specific PCR (Methylation-Specific polymerase Chain Reaction, MSP), real-time fluorescent quantitative polymerase chain reaction (Quantitative Real-time PCR, qRT-PCR) were used to detect the expression of the protein in the cancerous tissues of 58 pathologically confirmed patients. The relationship between Bin1 methylation status and Bin1 protein, gene expression and the clinicopathological features of patients in ESCC were analyzed. Results: 1 in 58 cases of esophageal squamous cell carcinoma, the incidence of Bin1 protein low expression in ESCC tissues was significantly higher than that of the corresponding para cancerous tissue [37/58 (63.79%) vs.13/58 (2). 2.41%), P0.01]; in the 58 patients with ESCC, the expression level of Bin1 mRNA in ESCC tissues was significantly lower than that of the paracancerous tissue, respectively (0.78 + 0.05) vs. (1.03 + 0.03) (t=9.643, P0.01), and Bin1 mRNA expression in the Bin1 methylation tissues was significantly lower than that of the non methylation tissues, respectively (0.68 + 0.04) vs. (0.85 + 0.07), respectively. There are 35 patients with low expression of gene, 35 cases of low expression of Bin1 protein, the result of 94.59%. in patients with low expression of protein suggests that the expression of Bin1 at the gene level and protein level in ESCC tissues is consistent with.2 in 35 cases of bin1mrna with low expression of ESCC tissue, 32 cases (91.43%) Bin1 gene promoter methylation, and 37 cases of low expression of Bin1 protein in the ESCC group. 33 (89.19%) Bin1 gene promoter methylation occurred in the fabric, indicating that the abnormal methylation of the Bin1 promoter region in the ESCC tissue is associated with the low expression of Bin1, and Bin1 methylation is one of the reasons for the low expression of Bin1 in the ESCC tissue, which is associated with the patient's TNM staging, the depth of the tumor invasion, the degree of differentiation, and lymph node metastasis (p0.). 05), there was no statistical significance between the age of the patients and the sex (P0.05). Conclusion: the methylation of Bin1 in the tumor tissues of the patients with esophageal cancer is highly methylation, and the hypermethylation of Bin1 is closely related to the clinicopathological features of the tumor differentiation, infiltration depth, lymph node metastasis, and TNM staging. The methylation status of the Bin1 gene promoter in the second part of the esophageal cancer patients. The purpose of the development of esophageal squamous cell carcinoma is to detect the Bin1 expression and methylation status in ESCC cells, yes-2, te13, te1, kyse30 and Ec109, select yes-2 and te13 cells for subsequent experiments, and apply the demethylation drug 5- nitrogen -2 'deoxycytidine (5-Aza-2'). Bin1 expression level, ESCC cell proliferation, apoptosis and other malignant biological behaviors and changes of EMT related proteins before and after methylation, explore the possible mechanism of Bin1 in ESCC development, and provide experimental basis for Bin1 gene methylation as a new target for ESCC comprehensive treatment. Methods: qRT-PCR and MSP methods were used to detect Bin1 in 5 kinds of human esophageal scales at 1. The expression and methylation status of cancer cells yes-2, te13, te1, kyse30 and Ec109 were selected, and yes-2 and te13 were selected for follow-up experiments. Two ESCC cells were intervened with the demethylation drug 5-aza-dC, and the two kinds of ESCC cells were detected by MSP and Western-blot, and the changes of the methylation status and expression were analyzed. Clone formation experiment, flow cytometry to detect the changes of proliferation ability of yes-2 and te13 cells before and after demethylation, and analyze the effect of Bin1 methylation on the proliferation of ESCC cells..3 uses flowcytometryassay (FCM) to detect the proliferation of yes-2 and te13 cells before and after demethylation, the change of apoptosis ability and analysis of Bin1 methyl. Effect of ESCC cell proliferation and apoptosis ability.4 use cell scratch test, Transwell test to detect the migration of yes-2 and te13 cells before and after demethylation, change of invasion ability, analyze the effect of Bin1 methylation on ESCC cell migration and invasion ability,.5 adopts Westernblot test to detect the epithelium of yes-2 and te13 cells before and after demethylation. Epithelial-mesenchymaltransition (EMT) related protein E cadherin (E-cadherin, E-cad), n cadherin (N-cadherin, n-cad), matrix metalloproteinase 2 (matrixmetalloproteinase-2, MMP-2), matrix metalloproteinase 9 (MMP-9), etc. Results: 1bin1mrna showed low expression state (P0.01) in five ESCC cells, te13, te1, kyse30, Ec109 and yes-2, and the five ESCC cells were methylation state, and yes-2 and te1 two cells were completely methylation state. After 5-aza-dC demethylation, the expression of Bin1 protein expression in yes-2 and te13 two kinds of human ESCC cells increased (P0.01).2mtt results showed that the proliferation inhibition rates of cells were 6.7 + 0.9%, 12.3 + 1.7% and 15.4 + 2.1%, respectively, by different concentrations (30 u m, 60 u m, 90 micron M). 7.3 + 1%, 49.3 + 5.7% and 59.6 + 6.3% respectively, compared with the control group with a concentration of 0 m, the cell proliferation rate was significantly reduced, and the proliferation inhibition rate of cell proliferation was 6.4 + 0.8%, 11.9 + and + 6.3% after D2 days after te13 cells with different concentrations (30 mu, 60 u m, 90 mu m), and the growth inhibition rate of cell growth was respectively %, 47.2 + 5.6% and 57.2 + 5.9%, compared with the control group with a concentration of 0 micron m, the cell proliferation rate decreased significantly (P0.01). The clone formation experimental results showed that the number of cell clones was (233 + 29), (189 + 23) and (189 + 0), compared with the control group with concentration of M, after 5-aza-dC intervention in yes-2 cells with different concentrations (0 m, 30, m, 60, m, 90 u m). The number of clones was significantly reduced, and the number of clones was (281 + 33), (235 + 28), (139 + 19) and (83 + 12) after the intervention of te13 cells with different concentrations (0 m, 30 m, 60 m, 90 m). The number of clones decreased significantly compared with the control group with concentration of 0 mu m. The proliferation activity of yes-2 and te13 cells after demethylation was significantly decreased (P0.01).3. The results of flow cytometry showed that the percentage of 5-aza-dC cells in g0/g1 phase was (37.2 + 3.1)%, (46.5 + 3.9)%, (58.2 + 4.3)% and (70.7 + 4.9)%, respectively (37.2 + 3.1)%, (58.2 + 4.3)% and (70.7 + 4.9)%) after yes-2 cells were interfered with yes-2 cells at different concentrations (0 m, 30, m, 90 m). Compared with the control group, the percentage of cells in the g0/g1 stage increased significantly, and the percentage in the S phase decreased obviously, and the percentage of the cells at g0/g1 (33.7 + 2.8)%, (39.4 + 3.6)%, (47.3 + 3.9)% and (56.3 + 4.5)%, respectively (70.2 + 60), respectively (33.7 + 2.8)%, (47.3 + 3.6)%, (47.3 + 3.9)% and 56.3 + 4.5) by different concentrations (0 u m, 30 m, 60 mu m, 90 mu m) were respectively .5)%, (58.3 + 4.2)%, (45.2 + 3.1)% and (32.6 + 2.8)%, compared with the control group with the concentration of 0 m, the percentage of cells in the g0/g1 phase increased obviously, and the percentage in the S phase decreased obviously. It indicated that the ESCC cell cycle changed after demethylation treatment, the cells in the S phase decreased significantly, and the cells blocked in g0/g1 phase increased significantly (P0.01). Flow cytometry showed that the percentages of apoptotic cells were (4.12 + 0.89)%, (7.49 + 1.34)%, (10.03 + 2.13)%, (10.03 + 2.13)% and (13.07 + 2.47) after interfering with yes-2 cells with different concentrations (0 m, 30 m, 60 u m, 90 m), and the percentage of apoptotic cells increased significantly (P0.01), compared with the control group with the concentration of 0 mu m (P0.01). The percentage of apoptotic cells in the apoptotic cells was (3.57 + 0.78)%, (8.09 + 1.43)% and (10.15 + 2.08)%, (15.64 + 2.55), and the percentage of apoptotic cells increased significantly (P0.01). The results indicated that Bin1 gene demethylation could inhibit yes-2 and te13 finer after Bin1 gene demethylation, after 5-aza-dC intervention in te13 cells was (3.57 + 0.78)%, (8.09 + 1.43)% and (10.15 + 2.08)%, (15.64 + 2.55), and (15.64 + 2.55), compared with the control group with a concentration of 0 m. The proliferation ability and apoptosis of ESCC cells and the inhibitory effect of.4 on the malignant biological behavior of ESCC cells showed that after 5-aza-dC demethylation treatment, the migration distance of yes-2 cells was (57.7 + 4.5) mu m, 48h migration distance was (122.4 + 9.4) mu m without demethylation treatment, and yes-2 after demethylation treatment. The migration distance of the cells was significantly shorter than before, and the results were statistically significant (P0.01); the migration distance of 48h after 5-aza-dC demethylation was (64.2 + 4.8) mu m, and the migration distance of 48h without demethylation was (133.4 + 8.1) mu m. The migration distance of te13 cells after demethylation treatment was significantly shorter than that before treatment, and the result was a result. The migration distance of.YES-2 and TE13 cells was significantly shortened, indicating that Bin1 could inhibit the migration ability of YES-2 and TE13 cells after demethylation treatment.Transwell experiment results showed that the cell number of the cells passed through the filter membrane was divided into 0 micron (0 u M, 30 u M, 60 mu M, 90 Mu M). Not (206 + 35), (137 + 29), (78 + 18) and (39 + 9), compared with the control group with a concentration of 0 M, Matrigel filtration membrane cells were significantly reduced (P0.01). The number of TE13 cells after 5-Aza-dC demethylation treated by different concentrations (0 mu M, 30 mu M, 60 mu M, 90 M), and the number of cells passing through Matrigel filter membrane were respectively Compared with the control group with a concentration of 0 M, the number of transmembrane cells in.YES-2 and TE13 cells decreased significantly in.YES-2 and TE13 cells through Matrigel filtration cells, indicating that Bin1 could inhibit the invasive ability of the cells after demethylation. The experiments indicated that Bin1 can inhibit the migration and invasion of YES-2 and TE13 cells after the demethylation of Bin1. The results of.5 Western blot detection showed that the E-cad expression of EMT related protein was up regulation (P0.01), N-cad, Snail, Snail, and protein expression after 5-Aza-dC treatment, indicating that the expression of the proteins could be changed after demethylation to inhibit the occurrence of epithelial mesenchymal transition. In vitro experiments: in vitro experiments have proved that Bin1 methylation affects the proliferation, apoptosis, migration and invasion of esophageal cancer cells, and affects the development of esophageal cancer by inhibiting the process of epithelial mesenchymal transition in esophageal cancer cells.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

【参考文献】

相关期刊论文 前2条

1 Laura Lattanzio;Cristiana Lo Nigro;;Epigenetics and DNA methylation in cancer[J];World Journal of Translational Medicine;2015年01期

2 Ru Liu;Xiao-Huan Zhang;Kun Zhang;Wei Li;Wen-Jun Wang;Di-Xian Luo;Ling Gao;;5-Aza-2'-deoxycytidine inhibits retinoblastoma cell by reactivating epigenetically silenced RASSF1A gene[J];International Journal of Ophthalmology(English Edition);2014年01期

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本文编号：1864899