【作者】 张燕齐；

【导师】 丁杰；

【作者基本信息】 第四军医大学 ， 内科学， 2007， 博士

【摘要】 【背景】最早在研究果蝇发育过程中发现,TWIST参与调节多种发育过程,尤其在早期中胚层的形成方面起重要作用。1996年人类TWIST基因被成功克隆,定位于7p21.2,编码203个氨基酸,分子量约28Kd。TWIST蛋白是具有碱螺旋-环-螺旋(basic helix-loop-helix, bHLH)结构的转录因子家族成员,其可以与另外一个同源或异源的bHLH因子结合,形成二联DNA结合域,特异性地结合E-box consensus sequence (CANNTG)从而激活或抑制特异性的靶基因转录。近年来的研究表明TWIST是个新的癌基因,TWIST可以通过p53依赖与非依赖的途径参与细胞的凋亡抵抗。目前研究表明,TWIST过表达在肿瘤的发生、侵袭转移、血管生成与肿瘤细胞耐药中发挥重要作用,但是其中的具体机制尚不清楚。有关TWIST在胃癌发生、发展中的作用尚无深入的研究,参与调控TWIST的分子及其机制未见报道。本研究拟构建TWIST不同表达水平的胃癌细胞亚系,观察TWIST对胃癌细胞生物学行为的影响,探讨TWIST在胃癌发生、进展与血管生成中的作用,以及参与调控TWIST的分子及其机制。【目的】1.研究TWIST在胃腺癌中的表达及其临床意义;2.观察TWIST对胃癌细胞生物学行为及肿瘤血管生成的影响,并探讨可能的机制;3.研究缺氧与TWIST的关系及其调控的分子机制。【方法】1.采用免疫组织化学方法观察TWIST在胃癌组织中表达与细胞内的定位,并分析TWIST蛋白表达与临床病理资料之间的关系。2.采用半定量RT-PCR与Western blot方法检测TWIST在不同胃癌细胞系和永生化的胃上皮细胞中的mRNA和蛋白表达水平,以及新鲜胃癌及癌旁组织中TWIST的蛋白表达情况。3.将含有TWIST全长cDNA的正义真核表达质粒pcDNA3.0-TW与构建的TWIST特异性的小干扰RNA质粒pSilencer-TW1与pSilencer-TW2分别转染胃癌细胞系SGC7901,经G418筛选,建立TWIST不同表达水平的胃癌细胞亚系。4.采用MTT实验,流式细胞仪技术、细胞损伤刮擦实验、细胞侵袭实验、裸鼠移植瘤实验研究不同TWIST表达水平对胃癌细胞SGC7901的生长、细胞周期、细胞凋亡、移动、侵袭以及体内形成肿瘤能力的影响,并且Western blot方法检测凋亡相关分子的变化。5.应用ELISA法检测不同胃癌细胞亚系培养上清中VEGF蛋白含量。6.免疫组织化学方法检测裸鼠移植瘤组织中Ⅷ因子相关抗原的表达,比较分析不同移植瘤组织中微血管密度(MVD)。7.建立体外缺氧模型,检测缺氧条件下胃癌细胞系SGC7901中缺氧诱导因子-1(HIF-1)的表达变化,以及TWIST在基因转录和蛋白翻译水平的变化,利用HIF-1αsiRNA转染的细胞系SGC7901,研究缺氧条件下TWIST的表达变化,初步确立HIF-1α与TWIST之间的调控关系。8.构建TWIST的报告基因载体,利用双荧光素酶报告基因实验检测在缺氧条件下或共转染HIF-1α真核表达质粒时,TWIST启动子活性的变化。9.利用染色质免疫共沉淀(ChIP)实验初步筛选鉴定TWIST启动子上的HIF-1α结合位点。10.利用凝胶电泳迁移率变动实验(EMSA)与超变动分析(Super shift assay)确定TWIST启动子中与HIF-1α结合的功能位点。【结果】1.免疫组化的结果显示:与正常胃黏膜相比,胃癌组织中TWIST染色强度明显增强;TWIST在不同分化程度的胃癌组织、黏液癌及印戒细胞癌、淋巴结转移癌中均有阳性表达,TWIST蛋白在胃癌组织中主要定位于细胞胞浆,也可见部分细胞核着色;76例胃癌中TWIST的阳性率为46.1% (35/76,与正常胃黏膜比较p<0.05),中、低分化腺癌阳性率高于高分化腺癌(p<0.05),在有淋巴结转移的胃癌组织中TWIST阳性率高于无淋巴结转移的胃癌组织(p<0.05)。2.Western blot检测显示:与对应的癌旁组织相比,胃癌组织中TWIST的表达显著升高。3.半定量RT-PCR与Western blot的结果显示:与永生化的胃黏膜上皮细胞GES-1相比,TWIST mRNA与蛋白质在多种胃癌细胞系中高表达。4.成功构建了TWIST特异性的小干扰RNA质粒pSilencer-TW1与pSilencer-TW2,分别转染TWIST正义质粒pcDNA3.0-TW与pSilencer-TW1、pSilencer-TW2到SGC7901细胞,经Western blot鉴定,获得TWIST表达上调、下调SGC7901细胞亚系: SGC7901/pcDNA3-TW和SGC7901/pSi-TW1。5.MTT结果显示: SGC7901/pcDNA3-TW细胞增殖速率较对照细胞加快;SGC7901/pSi-TW1细胞增殖速率较对照细胞减慢。6.流式细胞仪技术检测细胞周期分布的结果提示: SGC7901/pcDNA3-TW细胞增殖指数较对照增加(45.4 vs 40);SGC7901/pSi-TW1细胞增殖指数降低(32.8 vs 39.7)。7.流式细胞仪检测细胞凋亡结果表明: SGC7901/pcDNA3-TW细胞中凋亡细胞所占百分比低于亲本细胞和空载体转染的细胞;SGC7901/pSi-TW1细胞中凋亡细胞所占比例高于亲本细胞和空载体转染的细胞。8.Western blot结果显示: SGC7901/pcDNA3-TW细胞中bcl-2表达升高,bax表达下降,bcl-2/bax比值升高;SGC7901/pSi-TW1细胞中bcl-2表达下降,bax表达升高,bcl-2/bax比值降低。9.损伤刮擦实验的结果显示:相同时间内SGC7901/pcDNA3-TW组细胞进入划痕区域的细胞数目较空载体SGC7901/pcDNA3组多,而SGC7901/pSi-TW1组细胞进入划痕区域的细胞数目较空载体SGC7901/pSi组少。10.Transwell小室实验结果显示: SGC7901/pcDNA3-TW组穿过膜细胞数较对照组增多,而SGC7901/pSi-TW1组穿过膜细胞数目较对照减少,相对侵袭指数差异有显著性(p<0.05)。11.转染细胞裸鼠体内成瘤实验结果:接种31天处死裸鼠取出瘤体,测量裸鼠移植瘤体积(±SD,mm3)示:SGC7901/pcDNA3-TW组为1558.40±270.36,与对照组比较肿瘤体积大(p<0.05),SGC7901/pSi-TW1组为389.46±80.63,与对照组比较肿瘤体积小(p<0.05);瘤体重量(±SD,mg)示:SGC7901/pcDNA3-TW组为1364.40±271.30,SGC7901/ pSi-TW1组为370.25±80.61,组间统计学分析与肿瘤体积结果一致。12. ELISA法检测细胞培养上清中VEGF蛋白含量表明:SGC7901/pcDNA3-TW细胞分泌VEGF的水平明显高于SGC7901与SGC7901/pcDNA3细胞(p<0.05),而SGC7901/pSi-TW1细胞分泌VEGF的水平低于SGC7901与SGC7901/pSi细胞(p<0.05)。13.Ⅷ因子相关抗原免疫组织化学染色显示:胃癌裸鼠移植瘤组织内微血管内皮细胞染色阳性,其中SGC7901/pcDNA3-TW肿瘤组的MVD(31.20±4.30)高于SGC7901组和SGC7901/pcDNA3组(p<0.05),而SGC7901/pSi-TW1组(7.50±1.83)低于SGC7901组和SGC7901/pSi组(p<0.05),对照组间比较差异无显著性。14. RT-PCR与Western-blot结果显示:随着缺氧时间的延长HIF-1α的mRNA表达水平无明显变化而蛋白表达水平升高;缺氧诱导胃癌细胞系SGC7901中TWIST mRNA及蛋白表达上调,并且具有时间依赖性;应用siRNA技术抑制HIF-1α表达后再进行缺氧诱导,与对照细胞系相比TWIST mRNA与蛋白的表达均无明显上调,提示缺氧通过HIF-1α调控TWIST的表达。15.生物信息学分析发现:在TWIST转录启始点附近-1442～+152范围内存在6个HRE的可能结合位点,利用PCR技术克隆了包含6个HRE的长1601bp的启动子片段,通过基因重组技术与PGL3-enhancer载体连接,经酶切、测序证实成功地构建了报告基因载体pGL3-TW。16.双荧光素酶报告基因实验结果显示:缺氧或者转染pcDNA3.1/HIF-1使TWIST的启动子活性升高3.0～4.5倍,说明缺氧通过HIF-1α对TWIST启动子有转录激活作用。17.染色质免疫共沉淀实验结果显示:在TWIST启动子-1014～-799bp区域(包含-1003～-997、-887～-880、-817～-811,3个疑似HRE)扩增出阳性条带,条带大小与预期一致,提示在TWIST启动子-1014～-799bp区域中存在HIF-1的结合位点。18.EMSA和Super shift试验结果显示:在转录起始位点的-1014～-799bp区域内的第一个潜在的HRE结合位点(-1009～-991)与缺氧条件下提取的核蛋白提取物有很强的结合能力,启动子探针电泳滞后,抗HIF-1α抗体可以导致电泳条带的进一步滞后,加入冷探针竞争后HIF-1α/DNA复合物的条带明显减弱,加入突变探针竞争后HIF-1α/DNA复合物的条带无变化,而其余两个潜在的结合位点迁移率变动没有影响。提示:在TWIST启动子距转录起始位点的-1003～-997处存在与HIF-1α结合的功能位点,HIF-1α通过结合该位点发挥作用。【结论】我们的研究结果表明:1.TWIST在胃癌组织、细胞系中表达显著上调,提示其在胃癌的发生及发展中起重要的作用,并且参与了肿瘤的转移,TWIST可能成为判断胃癌预后的指标与治疗的靶点。2. TWIST可通过细胞周期、凋亡、侵袭及血管生成等机制在胃癌的发生发展中发挥作用。3.缺氧可以诱导TWIST的表达,并且是通过HIF-1α与TWIST的启动子直接结合,从而激活TWIST mRNA转录、蛋白翻译。更多还原

【Abstract】 【Background】: TWIST was originally identified in Drosophila as a protein involved in the regulation of diverse developmental processes, particularly in the formation of mesoderm. The human TWIST gene is located at chromosome 7p21.2.The TWIST protein consists of 201 amino acids and its molecular weight is about 28kd. TWIST protein is a transcription factor that belongs to the family of basic helix-loop- helix (bHLH) protein. The mechanism of bHLH molecules require that they either homo- or heterodimerize with other bHLH molecules, form a bipartite DNA-binding domain and bind to a conserved E-box region (CANNTG) on the promoter of genes which activate or inhibit transcription.Recent studies have shown that TWIST is a potential oncogene and be involved in the protection of apoptosis via both p53-dependent and independent pathways. To date several studies revealed that TWIST played crucial roles in tumorigenesis, invasion and metastasis, angiogenesis and drug resistance, but its accurate molecular mechanisms remain unclear. However, there have been little available data on the role of TWIST in the development and progession of gastric cancer; the molecule involved in the regulation of TWIST and its mechanism was still unclear. In this study, we have constructed several gastric cancer cell lines with different expression of TWIST. We observed the effects of TWIST on the biological behavior of gastric cancer cells and explored its roles in the development, progression and angiogenesis of gastric cancer. We researched for the molecule involved in the regulation of TWIST and its mechanisms.【Objectives】: (1) To investigate the expression of TWIST in gastric cancer tissue and cells and analyze the relationship of expression intensity with the clinicopathologic characteristics of gastric cancer. (2) To explore the effects of TWIST protein on malicious biological behaviours and tumor angiogenesis of gastric cancer. (3) To study the relationship of hypoxia with TWIST and the molecular mechanism.【Methods】: (1) The expression and sucellualr location of TWIST protein in gastric cancer tissues was investigated by immunohistochemistry and the relationship between the TWIST expression and the clinicopathologic characteristics was statistically analyzed. (2) The expression of TWIST mRNA and protein in gastric cancer cells and immortalized gastric epithelial cells were measured by semiquantitative RT-PCR and Western blot, respectively. The expression of TWIST protein in fresh gastric cancer tissues and normal gastric tissues was detected by Western blot. (3) The pcDNA3-TW vector which contained the full length of TWIST cDNA and the TWIST siRNA vector were transfected into gastric cancer cells SGC7901 respectively, followed by screening and verifying. (4) MTT assay, Flow cytometry, wound-healing assay, cell invasion assay and nude mice tumor formation assay were used to detect the proliferation, apoptosis, cycle, migration and invasion, and tumorigenesis effects of TWIST on the gastric cancer cells. The apoptosis related molecules were dectede by Western blot. (5) The concentration of VEGF in the supernatant of transfectants was determined by ELISA. (6) The expression of factorⅧrelated antigen in xenograft tumor was detected by immunohistochemistry and the MVD was compared among different xenograft tumors. (7) The hypoxia model in vitro was established and the expression of TWIST mRNA and protein was dected respectively under hypoxia condition. The changes of TWIST in HIF-1αsiRNA transfected cells under hypoxia and the regulation relationship of HIF-1 and TWIST were observed. (8) The TWIST reporter gene vector was constructed; The TWIST promoter activity was evaluated by dual luciferase reporter assay when co-transfected with HIF-1 or under hypoxia condition. (9) The possible binding sites of HIF-1 on the promoter of TWIST were selected by Chromatin Immunoprecipitation Assay (ChIP). (10) The functional binding site of HIF-1αon the promoter of TWIST was determined by Electrophoretic Mobility shift DNA-binding Assay (EMSA).【Results】: (1) Immunohistochemistry carried out on 76 paraffin-embedded gastric cancer sections showed that TWIST was mainly expressed in the cytoplasm of the epithelial cells, and only occasionally in the nuclei. TWIST expression was very low and at the lower limit of detection in most normal epithelial cells. TWIST expression was significantly increased in 35 (46.1%) of the 76 cancers. The positive rate of TWIST was higher in moderately and poorly differentiated gastric cancers compared to that in well-differentiated (p<0.05) and in the tumors with lymph-node metastasis compared to that without metastasis (node positive rate 60.4%; node negative rate 21.4%; P<0.05). (2) The expression of TWIST protein was higher levels in fresh cancer tissues compared to that in adjacent normal tissues. (3) The expression of mRNA and protein of TWIST in gastric cancer cell lines were up-regulated compared to that in GES-1 detected by semiquantitative RT-PCR and Western blot, respectively. (4) The TWIST specific siRNA plasmid pSilencer-TW1 and pSilencer-TW2 were constructed successfully. The plasmid pSilencer-TW1, pSilencer-TW2, and pcDNA3-TW were transfected into SGC7901 cell lines respectively. The cell lines with forced ectopic or knocked out expression of TWIST were established and identified, and named as SGC7901/pcDNA3-TW and SGC7901/pSi-TW1, respectively. (5) The growth of the sense transfectants was accelerated while the RNAi transfectants was slowed down, showed by MTT assay. (6) The FCM indicated that the proliferation index of SGC7901/pcDNA3-TW was higher compared to control group (45.4 vs 40) while the SGC7901/pSi-TW1 was lower than that of control group (32.8 vs 39.7).(7) The FCM also showed that the apoptosis rate was lower in sense transfectants while higher in RNAi transfectants when compared to vector transfectants. (8) Western blot revealed that the Bcl-2 and the Bcl-2/bax ratio increased in sense transfectants, while the Bcl-2 and the Bcl-2/bax ratio decreased in RNAi transfectants. (9) The wound-healing assay showed that the migration ability were elevated in sense transfectants while cut down in RNAi transfectants. (10) The Transwell assay indicated that the number of the invasive cells through the membrane were much more in sense transfectants than in RNAi transfectants (p<0.05). (11) The weights of xenograft tumor from sense transfectants were higer than that from RNAi transfectants (p<0.05); the volumes of xenograft tumor from sense transfectants were bigger than that from RNAi transfectants (p<0.05). (12) The concentration of VEGF in the supernatant of sense transfectants were increased while decreased in RNAi transfectants when compared to the control cells. (13) The stain of factorⅧ-related antigen was positive in the microvessel endothelial of nude mice xenograft tumors. The microvessel density was higher in the sense transfectants while lower in the RNAi transfectants when compared to control groups (p<0.05). (14) RT-PCR and Western blot results indicated that the expression of HIF-1αmRNA was unchanged, yet the HIF-1αprotein was increased under hypoxia condition. Both the expression of TWIST mRNA and protein of SGC7901 cells were upregulated under hypoxia condition while the HIF-1αspecific RNAi transfected SGC7901 cells were not induced. (15) By biological information analysis, six potential binding sites of HRE were located in the upstream region of the initial of transcription (-1442～+152). A length of 1.6 kb promoter of TWIST was cloned into PGL3-enhancer vector and identified right by sequencing and enzyme digestive. (16) The dual luciferase reporter assay demonstrated that the transfection of HIF-1αsense plasmids or under hypoxia could lead to a 3.0～4.5-fold increase of enzyme activety compared to empty vector transfection. (17) ChIP assay indicated that a length of 200bp DNA was amplified which located in -1014～-799bp of the TWIST promoter(containing three potential HRE binding sites). (18) To determine the functional sites of HIF-1αbinding to the TWIST promoter, EMSA and Super shift assay were applied and -1003～-997 site away from the transcriptional start was proved to be the functional binding site of HIF-1α.【Conclusion】: (1) Our results suggest that up-regulation of TWIST plays a role in the neoplastic transformation to gastric cancer and subsequent development of lymph nodes metastasis. TWIST may serve as a useful prognostic marker for predicting the development of metastasis and a target for therapy in gastric cancer. (2) The roles of TWIST in the development and progression of gastric cancer may be associated with cell cycle, apoptosis, invasion, and angiogenesis mechanisms. (3) TWIST expression in the gastric cancer cell can be induced by hypoxia and this is due to the HIF-1αbinding to the HRE site in the TWIST promoter region.更多还原