HOXD13调控SOX9基因可能参与先天足部软组织畸形的发生

目的　研究HOXD13与SOX9基因在单纯性马蹄内翻足（idiopathic congenital talipes equinovarus,ICTEV）足部软组织畸形发生中的分子机制。 方法　软件预测SOX9基因5′上游3个HOXD13结合位点,构建SOX9基因上游不同长度片段以及结合位点野生和突变序列载体,并测定其荧光素酶活性。在体内用ChIP验证HOXD13与SOX9基因启动子区3位点结合作用。干扰HOXD13基因表达,qRT-PCR检测HOXD13和SOX9表达水平的改变。 结果　荧光素酶检测发现在SOX9基因5′上游-1158至-770 bp（位点1）和-512至-368 bp之间（位点3）为负调控区;-770至-512 bp之间（位点2）是正调控区。ChIP检测显示HOXD13蛋白可与SOX9基因位点3结合。干扰HOXD13表达,SOX9基因mRNA表达上调。 结论　HOXD13结合SOX9基因5′上游负调控区可上调SOX9基因表达。干扰HOXD13会上调SOX9基因表达,高表达SOX9基因可能与足踝部软组织的挛缩发生相关。     

GLI3基因在单纯性马蹄内翻足发生中的调控机制

目的 探讨在单纯性马蹄内翻足发生过程中GLI3基因的凋控机制.方法 构建荧光素酶报告基因表达载体,分析大鼠Gli3基因5′侧翼启动子区域的活性.用P-Match软件预测Gli3基因上游序列中转录因子的结合位点,并通过染色质免疫沉淀实验、凝胶迁移实验验证.用RNA干扰实验以及构建Hoxd13表达载体,观察其在L6细胞中对Gli3基因表达的影响.结果 在大鼠Gli3基因序列的启动子区域发现2个Hoxd13的结合位点,染色质免疫沉淀和凝胶迁移实验证实Hoxd13结合于结合位点2上.Hoxd13表达下调时,Gli3基因表达明显上调.Hoxd13基因表达上调时,Gli3基因则表达下调.结论 在大鼠胚胎肢体发育中,Hoxd13蛋白可能与Gli3基因启动子区的Hoxd13结合位点2结合,调控Gli3的表达.     

γ-谷氨酰半胱氨酸合成酶基因调控的初步研究

目的研究抗氧化基因——大鼠γ-谷氨酰半胱氨酸合成酶（γ-GCS）基因的功能区及其调控表达。方法克隆1．76kb大鼠γ-GCS基因的重链亚单位——GCLC基因的上游调控序列，并构建含荧光素酶基因的报道载体GCLC—Luc（GCLC／pGL-3）。利用嵌顿缺失方法构建GCLC基因的缺失体，并将其表达载体转染大鼠肺泡上皮细胞，在细胞中分析该基因的调控区域。通过该方法初步发现GCLC基因的上游调控序列的-745～-705bp属负性调控区域。利用EMSA和supershift证实大鼠肺泡上皮细胞GCLC基因负调控区域的E—box元件能与转录因子USF1／USF2特异性的结合。将USF的真核表达载体和逆转录病毒表达载体分别导入肺泡上皮细胞，观察GCLC基因的转录活性和GCLC蛋白的表达情况。结果GCLC基因的-403～-111bp和-705～-613bp区段属正调控区域；-745～-705bp属负调控区域。EMS和supershifl证实上游刺激因子（USF）能与该负调控区域的E—box元件结合抑制GCLC基因的转录和表达。结论发现GCLC基因其中2个正调控区域（-403～-111bp与-705～-613bp）和1个负调控区域（-745～-705bp），其中负调控区域-745～-705bp上的E—box元件通过与USF结合介导GCLC基因的负性表达调控。     

hper1基因3′端UTR区双荧光素酶报告系统构建

目的：构建hper1基因3′端UTR区双荧光素酶报告系统pmiR-RB-REPORT^TM-hper1-3′UTR（PMIR-3′UTR），检测其表达，为研究hperl基因的表达调控及microRNA调控靶基因的结合位点提供基础平台。方法：用PCR扩增的方法提取hper1基因3′UTR序列，并连接至pmiR—IBREPORT^TM（PMIR）双荧光素酶报告载体的多克隆位点，测序验证插入序列并转染入A549细胞检测其荧光素酶活性。结果：测序结果表明PMIR-3′UTR的插入序列正确，在转染入A549细胞后其荧光素酶能正常表达。结论：PMIR-3′UTR双荧光素酶报告系统构建成功。     

NOX1基因荧光素酶报告基因系统的建立

目的：对炎症细胞因子TNF-α及IFN-γ刺激下,人NOX1基因的表达调控进行初步分析。方法：将NOX1基因5′-端上游序列连接到无启动子的PGL3-BASIC质粒,构建了PGL3-BASIC／NOX1报告质粒。PGL3-BASIC／NOX1质粒转染A549细胞,用TNF-α、IFN-γ刺激12h,双荧光素酶报告基因系统检测基因表达情况。结果：克隆的NOX1片段具有较强的启动子活性,在TNF-α和IFN-γ共同刺激下,转染报告基因的A549细胞萤光素酶活性与对照相比有明显的增高（约4．3倍）。分析显示NOX1基因5′端上游序列片段含有NF-KB结合位点,提示细胞因子刺激的荧光素酶表达增强可能与NF-KB位点激活相关。结论：NOX1基因表达水平明显受到炎症细胞因子的调控,提示该基因可能参与机体免疫防御（特别是上皮细胞免疫防御）,值得进行深入研究。     

γ-谷氨酰半胱氨酸合成酶基因调控的初步研究

目的 研究抗氧化基因--大鼠γ-谷氨酰半胱氨酸合成酶(γ-GCS)基因的功能区及其调控表达.方法 克隆1.76 kb大鼠γ-GCS基因的重链亚单位--GCLC基因的上游调控序列,并构建含荧光素酶基因的报道载体GCLC-Luc (GCLC/pGL-3).利用嵌顿缺失方法构建GCLC基因的缺失体,并将其表达载体转染大鼠肺泡上皮细胞,在细胞中分析该基因的调控区域.通过该方法初步发现GCLC基因的上游调控序列的-745～-705 bp属负性调控区域.利用EMSA和supershift证实大鼠肺泡上皮细胞GCLC基因负调控区域的E-box元件能与转录因子USF1/USF2特异性的结合.将USF的真核表达载体和逆转录病毒表达载体分别导入肺泡上皮细胞,观察GCLC基因的转录活性和GCLC蛋白的表达情况.结果 GCLC基因的-403～-111 bp和-705～-613 bp区段属正调控区域;-745～-705 bp属负调控区域.EMS和supershift证实上游刺激因子(USF)能与该负调控区域的E-box元件结合抑制GCLC基因的转录和表达.结论 发现GCLC基因其中2个正调控区域(-403～-111 bp与-705～-613 bp)和1个负调控区域(-745～-705 bp), 其中负调控区域-745～-705 bp上的 E-box元件通过与USF结合介导GCLC基因的负性表达调控.     

人LAIR-1/CD305基因启动子的生物信息学分析

目的:研究人LAIR-1/CD305启动子及其5′上游调控序列。方法:通过检索NCBI中的人类基因组数据库,获得LAIR-1的转录本序列及翻译起始位点上游2500bp的序列。利用Promoter2.0等软件预测LAIR-1的启动子序列,然后利用MatInspector等软件对启动子序列的转录因子结合位点和启动子功能模块进行预测。结果:LAIR-1基因的核心启动子区位于翻译起始密码子上游的600～200bp区域内。在转录调控区发现了一些重要的转录因子结合位点,并预测到13种启动子功能模块。结论:LAIR-1基因的表达可能受到多种转录因子和5′端上游调控序列的调控。     

人血红素加氧酶基因近端启动子区研究进展

分析血红素加氧酶(heme oxygenase,HO)基因mRNA加帽位点上游1416bp及mRNA5′端未翻译区24bp全长1.44kb片段。通过大片段缺失,发现与诱导剂相关的mRNA加帽位点近端上游121bp,可使基因的瞬时表达提高3～5倍,同时诱导作用发生在SV_(40)增强子元件位于启动子序列上游。另外在1.44kb片段间mRNA加帽位点上游有沉默子或负调控元件及NF-κB-和AP-2结合位点,而在1.44kb之外还有附加的诱导增强子元件。     

人支气管上皮细胞γ-谷氨酰半胱氨酸合酶催化亚单位基因转录的调控

目的： 研究人支气管上皮细胞谷胱甘肽（GSH）合成的限速酶γ-谷氨酰半胱氨酸合酶（γ-GCS）催化亚单位（GCLC）基因转录调控。方法: 通过PCR的方法克隆GCLC基因的部分转录调控区基因，构建表达虫荧光素酶报告基因的质粒；采用外切核酸酶Ⅲ结合S1核酸酶的方法构建了GCLC基因部分转录调控区基因的嵌套缺失体；基因转染上述质粒，通过虫荧光素酶报告基因的表达分析揭示嵌套缺失体突变基因的转录调控功能。结果: 人GCLC基因转录起始位点上游-2 515~-2 236 bp、-864~-838 bp、-769~-538 bp、-421~-341 bp区间的转录调控区为正性转录调控区，为新发现的转录调控区域；而-810~-769 bp区间特别是-782~-769 bp区间为负性转录调控区，它的定位范围较原来更精确。结论: 本研究有助于进一步深入探讨人支气管上皮细胞GCLC基因的转录调控，为γ-GCS 及GSH的合成调控研究提供了良好的前期实验基础。     

HCV5′NCR片段调控荧光素酶表达质粒的构建及其在H …

目的 构建ＨＣＶ５′ＮＣＲ片段调控荧光素酶表达质粒,并在ＨｅｐＧ２细胞中表达。方法 ＰＣＲ扩增,获得中国人ＨＣＶ基因组５′非编码区（ｎｏｎｃｏｄｉｎｇ ｒｅｇｉｏｎ,ＮＣＲ）完整序列与Ｃ区部分序列的目的基因片段（５′ＮＣＲ－Ｃ片段）。将此片段插入ｐＧＬ３荧光素酶报告载体蝗荧光素酶基因起始密码上游,构建受５′ＮＣＲ片段调控的荧光素酶表达质粒。应用脂质体介导基因转染技术将８个质粒转染ＨｅｐＧ２肝癌细胞     

Genomic structure of HOXD13 gene: a nine polyalanine duplication causes synpolydactyly in two unrelated families

Synpolydactyly (SPD) is a limb malformation that shows a characteristic manifestation in both hands and feet. This condition is inherited as an autosomal dominant trait with reduced penetrance. We have recently mapped this locus centromeric to the HOXD8 intragenic marker and suggested the HOXD13 gene as a potential candidate for this condition. The genomic structure of HOXD13 established in this study consists of two exons that encodes a polypeptide of 335 amino acids. The downstream exon at the 3' end of this gene contains the homeodomain sequences that are highly conserved. Sixty-three bp upstream of this exon lies a stretch of intronic CA-repeats that proved to be polymorphic in two different populations. The upstream exon encodes 75% of the entire protein and contains a stretch of 15 normal alanines at its 5' end. Sequence comparison at this position in the homozygous affected individuals identified a total of 24 alanine residues that resulted from a duplication of nine polyalanines. In two unrelated SPD families, this duplication was directly transmitted from the affected parents to their affected, but not unaffected, offspring; in one family its size has remained constant for at least 150 years spanning over seven generations. The presence of this duplication confirmed the status of four normal gene carriers, one incomplete penetrance and two affected individuals who were recombinants for HOXD8 or HOXD13-CA repeat markers. This duplication was not present in 150 chromosomes of unrelated healthy subjects of two different populations.      

HOXD13 methylation status is a prognostic indicator in breast cancer

Homeobox protein Hox-D13 is encoded by HOXD13 gene which is frequently methylated in cancer and has been recognized as a tumor suppressor in pancreatic cancer. In this study, we examined HOXD13 mRNA expression in 40 pairs of breast cancers and corresponding normal breast tissues. Bisulfite sequencing of HOXD13 promoter was performed in 6 pairs of breast tumors and corresponding normal breast tissues to examine the potential HOXD13 CpG methylated sites. HOXD13 DNA methylation frequency analysis was performed using MethyLight in 196 pairs of breast cancers and corresponding normal breast samples. DNA methylation status and clinico-pathological features were investigated. Kaplan-Meier survival analysis and Cox proportional hazards models were utilized to assess the effect of methylation status on overall survival. We found that 60% (24/40) of breast cancers showed low HOXD13 mRNA expression when compared with corresponding normal breast tissue. The predicted CpG island was located in the -1325 bp to +675 bp region. Next, the -332 bp site in HOXD13 gene promoter was further examined and in 57.7% (113/196) samples methylation was detected at this site. HOXD13 methylation was correlated with larger tumor size (P = 0.004), but not with other clinico-pathological parameters. In addition, patients with methylated -HOXD13 promoter had worse overall survival (OS) (P = 0.005). Based on our results we conclude that HOXD13 methylation is a common event in primary breast cancer and is associated with poor survival of breast cancer patients. HOXD13 methylation could therefore potentially be used as a prognostic factor for breast cancer.     

miR-128-3p过表达靶向MAPK1抑制膀胱癌5637细胞株的侵袭，迁移和上皮间质转化

目的　研究miR-128-3p过表达对膀胱癌5637细胞株的侵袭,迁移和上皮间质转化影响。 方法　基因预测软件TargetScan筛选出miR-128-3p的靶基因,荧光素酶报告实验验证;RT-PCR检测miR-128-3p和MAPK1的表达,Transwell检测细胞侵袭情况,划痕实验检测细胞迁移能力,Western blot检测E-cadherin、N-cadherin、ERK1/2、c-Myc和c-fos的表达,免疫荧光检测Vimentin的表达;裸鼠皮下注射建立移植瘤模型,30 d后检测瘤重量,绘制存活曲线,检测移植瘤中Vimentin、miR-128-3p、MAPK1、ERK1/2、c-Myc和c-fos的量。 结果　miR-128-3p靶向抑制MAPK1表达;miR-128-3p过表达后,侵袭细胞数目、伤口愈合率降低,E-cadherin表达上调,N-cadherin表达下调,Vimentin阳性率减少,p-ERK1/2、c-Myc和c-fos表达下调。经miR-128-3p干预,裸鼠体内移植瘤重量减轻,存活率增加,miR-128-3p表达上调,MAPK1的表达下调,Vimentin阳性率减少,p-ERK1/2、c-Myc和c-fos表达下调。 结论　过表达miR-128-3p通过靶向抑制MAPK1表达来抑制膀胱癌细胞5637的侵袭能力、迁移能力、上皮-间充质转化和ERK1/2、c-Myc和c-fos通路。     

miR-128-3p过表达靶向MAPK1抑制膀胱癌5637细胞株的侵袭，迁移和上皮间质转化

目的　研究miR-128-3p过表达对膀胱癌5637细胞株的侵袭,迁移和上皮间质转化影响。 方法　基因预测软件TargetScan筛选出miR-128-3p的靶基因,荧光素酶报告实验验证;RT-PCR检测miR-128-3p和MAPK1的表达,Transwell检测细胞侵袭情况,划痕实验检测细胞迁移能力,Western blot检测E-cadherin、N-cadherin、ERK1/2、c-Myc和c-fos的表达,免疫荧光检测Vimentin的表达;裸鼠皮下注射建立移植瘤模型,30 d后检测瘤重量,绘制存活曲线,检测移植瘤中Vimentin、miR-128-3p、MAPK1、ERK1/2、c-Myc和c-fos的量。 结果　miR-128-3p靶向抑制MAPK1表达;miR-128-3p过表达后,侵袭细胞数目、伤口愈合率降低,E-cadherin表达上调,N-cadherin表达下调,Vimentin阳性率减少,p-ERK1/2、c-Myc和c-fos表达下调。经miR-128-3p干预,裸鼠体内移植瘤重量减轻,存活率增加,miR-128-3p表达上调,MAPK1的表达下调,Vimentin阳性率减少,p-ERK1/2、c-Myc和c-fos表达下调。 结论　过表达miR-128-3p通过靶向抑制MAPK1表达来抑制膀胱癌细胞5637的侵袭能力、迁移能力、上皮-间充质转化和ERK1/2、c-Myc和c-fos通路。     

Identification of Novel Craniofacial Regulatory Domains Located far Upstream of SOX9 and Disrupted in Pierre Robin Sequence

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ～2 Mb region upstream of SOX9 can recapitulate the CD–DSD phenotype fully or partially, suggesting the existence of an unusually large cis‐regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ～1.2–1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP‐Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS.     

Mouse tumor necrosis factor receptor type I: genomic structure, polymorphism, and identification of regulatory regions

The mouse tumor necrosis factor receptor (TNFR)-I gene was cloned,sequenced, and characterized. The nucleotide sequence analysisshows that the TNFR-I Is composed of 10 exons and nine Introns.The first Intron includes two simple dinucleotide repeat sequences,(GA)8, and (TC)9(TG)19. The (TC)9, (TG)19 tandem repeat wasfound to be polymorphic In its length among various mouse strains.The nucleotide sequence of the 1076 bp 5' flanking region ofthe TNFR-I was also determined. Various possible regulatorysequences were identified in the 5' flanking region of the TNFR-Igene. For functional analysis, the 5' flanking region of theTNFR-I gene was isolated, ligated upstream of the luciferasereporter gene, and translently transfected Into L929, Hela,and a T cell hybridoma cell line. The results show that theIsolated 5' flanking region has functional promoter activityand is responsible for constitutive expression of the TNFR-Igene. A series of truncated promoter constructs were generatedand studied in a translent transfectlon system. Analysis oftranslent expression in L929 cells shows that the regions-1076/-939,-615/-425,and-425/-198 include positive regulatory elements, while theregion-939/-615 may contain negative cis-actlng elements forthe constitutive expression of the TNFR-I. The shortest constructcontaining 198 bp of the 5' flanking region still has significantpromoter activity, suggesting that the two GC-rich elementsin this region may play an important role in the constitutiveexpression of the TNFR-I gene.     

CK13基因5′旁侧的初步功能分析

目的　探讨细胞角蛋白 13(cytokeratin13,CK13)基因表达调控的机理 ,研究 CK13基因 5′旁侧不同基序对其转录活性的影响。　方法　采用分子克隆结合报告基因分析的方法 ,构建 CK 13基因 5′旁侧 5 13bp内不同基序与氯霉素乙酰转移酶 (chloramphenicol acetyltransferase,CAT)报告基因增强子载体p CAT的重组体 ,通过脂质体介导的转染技术导入 He L a细胞 ,检测各报告基因载体 CAT的相对活性。　结果　 CK13基因 5′旁侧起始密码子 ATG上游 - nt.32 5～ - nt.2 0 7间 119bp中具有某种抑制子元件 ,-nt.2 0 6～ - nt.94间 113bp中具有某种增强子元件。　结论　 CK13基因 5′旁侧 5 13bp内存在促进及抑制CK13基因表达的反应元件 ,进一步定位这些顺式反应元件并研究与之相互作用的反式作用因子 ,可望阐明 CK13基因表达调控及组织特异性表达的详细机理。