【作者】 周助人；

【导师】 孙开来；

【作者基本信息】 中国医科大学 ， 遗传学， 2008， 博士

【摘要】 前言TBX5是T-box转录因子家族成员之一,编码518个氨基酸,在胚胎心脏和发育中的肢体组织中表达,并且在正常心脏和肢体发育中起重要作用。TBX5基因突变可引起心手综合征,后者是以先天性心脏和上肢畸形为特点的常染色体显性遗传病。大多数发生在TBX5羧基端的突变引起心手综合征的分子机制尚不清楚,而且,TBX5羧基端的转录激活机制在很大程度上未知。因此,TBX5羧基端序列特征的研究对探索突变引起的心手综合征致病分子机制和阐明TBX5的转录激活作用很有必要。目前已知,TBX5与其它心脏发育相关转录因子GATA4和NKx2-5存在相互作用。研究表明,发生于TBX5蛋白的G80R,R237Q和R237W突变可减低TBX5与靶基因的DNA结合能力,导致TBX5对靶基因的转录激活水平降低,并且丧失了与NKx2-5的协同转录激活功能;TBX5蛋白的G80R和R237W错义突变可干扰与人类心脏间隔缺损相关转录因子GATA4的相互作用,而Q49K、I54T、G169R和S252I突变则不具有这种作用。因此,进一步确定是否还存在与TBX5相互作用的其它蛋白,对于揭示人类心脏发育机制和先天性心脏病发病机制具有重要意义。与TBX5同属于转录因子的GLI1蛋白介导Hedgehog通路,在果蝇和脊椎动物胚胎发育中起关键作用。GLI1蛋白含有两个已知的结构域,即锌指蛋白区域和假定的转录抑制子SFP1区域。最近报道并命名了GLI1氨基端中一段富含丝氨酸的N-端调控区,它在GLI/Ci家族中高度保守,功能未明。尽管如此,对GLI1蛋白氨基端的序列-功能研究仍然很少,并且该序列对GLI1转录活性的影响也不清楚。据报道,大约三分之一的致死性肿瘤与Hedgehog通路成员突变或者对Hedgehog配体过度反应引起Hedgehog过度激活有关,而GLI1介导Hedgehog通路,在其中起重要作用。GLI1过表达与许多肿瘤发生有关,例如,胶质瘤,黑色素瘤,前列腺癌,食道鳞状细胞癌和胃癌。目前的许多抗肿瘤药物均以信号传导通路中关键的激酶为靶点。MEK、PI3K抑制剂具有应用于临床治疗肿瘤的前景,但由于其特异地抑制MEK、PI3K通路,可能存在着一定的不可预见的毒副作用,并未全面开展其临床实验研究。因此,掌握MEK、PI3K抑制剂对肿瘤细胞中某些肿瘤相关蛋白的表达和细胞侵袭力的影响,将为MEK、PI3K抑制剂应用于肿瘤临床治疗提供理论基础。方法一、实验材料1、全部物种蛋白数据库http://www.ncbi.nlm.nih.gov/sites/entrez?db=protein&cmd=search&term=2、人类非冗余数据库（Non-redundant Database）http://www.ncbi.nlm.nih.gov/RefSeq/3、PSI-BLAST（位置特异性叠代BLAST）网址PSI-BLAST:http://www.ncbi.nlm.nih.gov/blast/Blast.cgi4、FFAS03（折叠和功能预测系统）网址FFAS03:http://ffas.ljcrf.edu/ffas-cgi/cgi/ffas.pl5、HHpred（隐马尔科夫模型的同源检测和结构预测）网址:HHpred:http://toolkit.tuebingen.mpg.de/hhpred6、比对软件M-Coffee:M-Coffee:http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi7、蛋白磷酸化位点预测软件PredPhospho:PredPhospho:http://pred.ngri.re.kr/PredPhospho.htm8、昆明种小白鼠由中国医科大学实验动物部提供9、胃癌细胞SGC7901人胃癌细胞SGC7901常规在1640培养基（10%胎牛血清,含青霉素及链霉素）中,于37℃、5%CO₂及饱和湿度的条件下培养。10、MEK、PI3K抑制剂MEK抑制剂U0126（Camarillo,San Diego,CA）浓度12.51μM,PI3K抑制剂LY29400（Calbiochem,San Diego,CA）浓度25μM。以DMSO溶解以上两种抑制剂。二、实验方法1、应用PSI-BLAST、FFAS03、HHpred生物信息学同源检测工具对非冗余数据库进行TBX5、GLI1同源蛋白检测。2、采用M-Coffee进行不同物种TBX5、GLI1蛋白的多重比对。3、采用PredPhospho进行TBX5羧基端、GLI1氨基端磷酸化位点预测。4、提取小鼠心肌组织核蛋白以TBX5为抗体进行免疫沉淀,目的条带进行质谱鉴定。应用核蛋白提取试剂盒（Pierce）提取核蛋白。采用Pierce免疫共沉淀试剂盒#23600进行免疫沉淀。实验组:有活性gel+15μl Tbx5多克隆抗体,对照组:无活性gel+15μl Tbx5多克隆抗体,IgG组:有活性gel+5μl IgG,分别加入20μl小鼠心肌组织核蛋白进行免疫沉淀。5、质谱分析确定目的蛋白后,应用Western印迹杂交验证免疫共沉淀蛋白的相互作用。6、胃癌细胞SGC7901分组及处理实验分为对照组（正常培养的胃癌SGC7901细胞）、DMSO组（在4ml培养基中加入2μl DMSO）,以及MEK抑制剂组（浓度12.5μM）、PI3K抑制剂组（浓度25μM）和一组两种抑制剂共同作用组（MEK抑制剂与PI3K抑制剂共同作用,浓度同上）,共5组。7、应用RT-PCR和Western印迹杂交分别检测MEK、PI3K抑制剂作用第0、12、24、48、72小时胃癌SGC7901细胞中GLI1基因和GLI1蛋白表达水平。8、流式细胞仪检测MEK、PI3K抑制剂作用第12、24、48、72小时胃癌SGC7901细胞表面受体CD44、CD14和TLR2的表达。9、Transwell实验检测MEK和PI3K抑制剂作用第12、24、48、72小时,对胃癌SGC7901细胞体外侵袭能力的影响。结果1、TBX5羧基端、GLI1氨基端与酵母DNA指导的RNA聚合酶Ⅱ最大亚基羧基端结构域具有显著同源性。2、TBX5羧基端Tyr291和Tyr342是潜在的磷酸化位点,而GLI1氨基端Ser84和Ser102残基是潜在的磷酸化位点。3、小鼠心肌Tbx5蛋白与α-肌球蛋白重链（Myh6）相互作用。4、Western Blot验证小鼠Tbx5与α-肌球蛋白重链存在相互作用。5、MEK、PI3K抑制剂,在作用第48、72小时使胃癌SGC7901细胞中GLI1基因和GLI1蛋白表达水平降低。6、MEK和PI3K抑制剂在作用第72小时使胃癌SGC7901细胞中CD44表达下调,两者具有协同作用。7、MEK和PI3K抑制剂在作用第72小时使胃癌SGC7901细胞中CD14表达下调,两者具有协同作用。8、MEK和PI3K抑制剂,单独或联合使用,在作用第12至72小时,均未对胃癌SGC7901细胞中TLR2表达水平有明显的影响。9、MEK和PI3K抑制剂,单独或联合使用,在作用第72小时使胃癌SGC7901细胞体外侵袭能力下降。结论1、TBX5羧基端残基267-448以及GLI1氨基端残基2-175均与酵母DNA指导的RNA聚合酶Ⅱ最大亚基羧基端结构域高度同源。2、TBX5羧基端的部分功能可能是通过与其它蛋白相互作用或募集其它蛋白,发挥转录活性。GLI1氨基端高度保守的Ser84和Ser102残基是潜在的PKA和/或PKC磷酸化位点。3、小鼠心肌Tbx5蛋白与α-肌球蛋白重链（Myh6）存在相互作用。4、MEK和PI3K抑制剂作用第48、72小时使胃癌SGC7901细胞中GLI1基因和GLI1蛋白表达下降。5、MEK和PI3K抑制剂在第72小时使胃癌SGC7901细胞中CD44、CD14表达水平明显下降,并且两者存在协同作用。MEK、PI3K抑制剂,单独或联合使用,均对胃癌SGC7901细胞中TLR2的表达无影响。6、MEK和PI3K抑制剂,单独或联合使用,在作用第72小时使胃癌SGC7901细胞体外侵袭能力下降。更多还原

【Abstract】 IntroductionTBX5,as a member of the T-box-containing transcription factor family,encodes a protein of 518 amino acids and is expressed in the embryonic heart and developing limb tissues and plays an important role in normal heart and limb development.TBX5 mutations could cause Holt-Oram syndrome,an autosomal dominant condition characterized by congenital cardiac malformations and upper limb anomalies.The molecular mechanism by which most TBX5 mutations at the C-terminal region cause Holt-Oram syndrome remains unclear.Moreover,the mechanism underlying the transactivation by C-terminus of TBX5 is largely unknown.Thus,the sequence-based study of C-terminus of TBX5 is needed for a better understanding of the molecular mechanism underlying TBX5 mutation-related Holt-Oram syndrome,and of TBX5 function as a transcriptional activator.It has been shown that TBX5 interacts with GATA4 and NKx2-5 which are transcriptional factors related to heart development. G80R,R237Q and R237W mutations reduce the TBX5 DNA-binding ability with target genes,leading to decreased transcriptional activation of TBX5 target genes and a loss of synergistic transcriptional activation with NKx2-5.G80R and R237W missense mutations in TBX5 are also found to disrupt the TBX5 physical interaction with GATA4,a cardiac transcriptional factor implicated in human cardiac septal defects, whereas Q49K,I54T,G169R and S252I mutations are not.Thus,to further explore any other TBX5-interacting proteins would shed light on the mechanism for human heart development and congenital heart diseases.Also playing a role as a transcriptional factor like TBX5,GLI1 mediates hedgehog （Hh）pathway and plays a crucial role during development of embryos in Drosophila and vertebrate.GLI1 protein contains two known domains,that is,Zn-finger region and putative transcriptional repressor SFP1 region.A serine-rich region conserved through the GLI/Ci family in the NH₂-terminal region was recently termed as N-terminal Regulatory region,the function of which remains elusive.Nevertheless,the sequence-function study for the NHE-terminal region of GLI1 is rare.The contribution of NHE-terminal region to the transcriptional activity of GLI1 is unclear.About one third of lethal cancers are associated with mutations of the components in Hh pathway or the super activation of Hh pathway by excessive response to its ligands,with GLI1 mediating this process and playing an important role.GLI1 overexpression is related to a variety of neoplasms,for instance,glioma,melanomas,prostate cancer,esophageal squamous cell carcinoma and gastric cancer.So far,many antitumor drugs target the crucial kinases in the signaling pathway.MEK and PI3K inhibitors are promising for their application in clinical cancer therapy.Due to their specific inhibition of MEK and PI3K pathways,however,these two inhibitors may have certain unforeseeable side-effects and the comprehensive clinical application is restricted.Thus,to study the impact of MEK and PI3K inhibitors on the tumor-related protein expression and tumor cell invasiveness would offer theory foundation for their application in clinical tumor therapy.MethodsExperimental materials:1.The protein database of all species http://www.ncbi.nlm.nih.gov/sites/entrez?db=protein&cmd=search&term=2.The non-redundant database of protein http://www.ncbi.nlm.nih.gov/RefSeq/3.PSI-BLAST（Position-Specific Iterated BLAST）website PSI-BLAST:http://www.ncbi.nlm.nih.gov/blast/Blast.cgi4.FFAS03（Fold & Function Assignment System）websites FFAS03:http://ffas.ljcrf.edu/ffas-cgi/egi/ffas.pl5.HHpred（Homology detection & structure prediction by HMM-HMM）websites HHpred:http://toolkit.tuebingen.mpg.de/hhpred6.M-coffee for alignment: http//www.ch.embnet.org/software/TCoffee.html7.Phosphorylation sites prediction: http://pred.ngri.re.kr/PredPhospho.htm8.The kunming mice were provided by China Medical University.9.Gastric cancer SGC7901 cells were cultured in 1640 medium supplemented with 10%（v/v）calf serum,penicillin and streptomycin in humidified 5%（v/v）CO₂ atmosphere at 37℃.10.MEK,PI3K inhibitor concentration:12.5μM and 25μM in DMSO, respectively.Experimental Methods1.Non-redundant database search for TBX5 and GLI1 homologous proteins by bioinformatic tools（PSI-BLAST,FFAS03 and HHpred）2.Multiple alignments of TBX5 and GLI1 proteins from different species by M-Coffee3.Phosphorylation sites prediction for C-terminal region of TBX5 and NH₂-terminal region of GLI1 by PredPhospho4.Immunoprecipitation study of nuclear protein from mouse heart tissue using Tbx5 antibody and the band of interest is subject to mass spectrometry analysis.Pierce nuclear protein extraction protocol and immunoprecipitation protocol（#23600）were used.Experimental group:active gel with 15μl Tbx5 polyclonal antibody,control group:inactive gel with 15μl Tbx5 polyclonal antibody,IgG group:active gel with 5μl IgG.20μl mouse heart tissue nuclear protein were used for immunoprecipitation.5.After the verification of the protein of interest by mass spectrometry,the interaction of the two interacting proteins was confirmed by western blotting.6.The grouping of gastric cancer SGC7901 cells and treatment:The five groups of gastric cancer SGC7901 cells:control group（normal gastric cancer SGC7901 cells）, DMSO group（2μl DMSO in 4ml medium）,MEK inhibitor group（concentration: 12.5μM）,PI3K inhibitor group（concentration:25μM）and co-acting group（MEK inhibitor and PI3K inhibitor added together in the medium,12.5μM and 25μM in concentration,respectively）.7.The GLI1 gene and GLI1 protein expression level in gastric cancer SGC7901 cells detected by RT-PCR and western blotting at the 0,12^th,24^th,48^thand 72^ndhour of MEK and PI3K inhibitors treatment.8.Detection of CD14,CD44 and TLR2 expression by flow cytometry in gastric cancer SGC7901 cells at the 12^th,24^th,48^thand 72^ndhour of MEK and PI3K inhibitors treatment.9.Transwell test to measure in vitro invasiveness of gastric cancer SGC7901 cells at the 12^th,24^th,48^thand 72^ndhour of MEK and PI3K inhibitors treatment.Results1.C-terminus of TBX5 and N-terminus of GLI1 are highly homologous to the C-terminal domain of yeast DNA-direeted RNA polymeraseⅡlargest subunit.2.Tyr291 and Tyr342 at C-terminus of TBX5,and Ser84 and Ser102 at N-terminus of GLI1 are identified as potential phosphorylation sites.3.Tbx5 protein in mouse heart tissue interacts with Myh6.4.Western blotting confirmed the interaction between mouse Tbx5 and Myh6.5.At the 48^thand 72^ndhour of MEK and PI3K inhibitors treatment,the GLI1 gene and GLI1 protein expression in gastric cancer SGC7901 cells were downregulated.6.At the 72^ndhour of MEK and PI3K inhibitors treatment,CD44 protein expression in gastric cancer SGC7901 cells were downregulated.MEK and PI3K inhibitors had synergistic effect.7.At the 72^ndhour of MEK and PI3K inhibitors treatment,CD14 protein expression in gastric cancer SGC7901 cells were downregulated.MEK and PI3K inhibitors had synergistic effect.8.MEK and PI3K inhibitors,alone or in combination,do not affect the TLR2 expression level significantly in gastric cancer SGC7901 cells from 12^thto 72^ndof treatment.9.MEK and PI3K inhibitors,alone or in combination,make the gastric cancer SGC7901 cells less invasive at the 72nd hour of treatment.Conclusion1.Residues 267-448 at C-terminus of TBX5 and residues 2-175 at N-terminus of GLI1 both are highly homologous to the C-terminal domain of yeast DNA-directed RNA polymeraseⅡlargest subunit.2.The partial function of the C-terminus of TBX5 might be to interact with or recruit other proteins for its transcriptional activity.GLI1 Ser84 and Ser102 residues were identified as potential PKA and/or PKC phosphorylation sites. 3.The Tbx5 protein in the mouse heart tissue interacts with Myh6 protein.4.At the 48^thand 72^ndhour of MEK and PI3K inhibitors treatment,the GLI1 gene and GLI1 protein expression in gastric cancer SGC7901 cells were downregulated.5.At the 72^ndhour of MEK and PI3K inhibitors treatment,CD44 and CD14 protein expression in gastric cancer SGC7901 cells were significantly downregulated. MEK and PI3K inhibitors had synergistic effect.MEK and PI3K inhibitors,alone or in combination,do not affect the TLR2 expression level in gastric cancer SGC7901 cells.6.MEK and PI3K inhibitors,alone or in combination,made the gastric cancer SGC7901 cells less invasive at the 72^ndhour of treatment.更多还原