Expression of survivin, a novel apoptosis inhibitor and cell cycle regulatory protein, in human gliomas

Recently, a novel anti-apoptosis gene, named survivin,was identified as a structurally unique member of the inhibitor of apoptosis protein (lAP) family. The gene is located on chromosome 17q25. Survivin is a 16.5 kDa protein that is expressed in vivo in common human cancers, but not in normal adjacent tissue,1 during the G2/M phase of the cell cycle. Survivin expression is turned off during fetal development and not found in nonneoplastic adult human tissue, and it is turned on in most common human cancers. We investigated the expression of survivin in 50 patients with human gliomas, and determined its association with cell apoptosis and cell proliferation, and its impact on tumor progression and prognosis.     

Cloning the full-length cDNA of a gene responsible for vascular smooth muscle cell proliferation in atherogenesis and study of function

Objective To clone the full-length cDNA of a gene responsible for vascular smooth muscle cell(v-SMC)proliferation in atherogenesis,and study its function.Methods Oxidized low density lipoprotein(ox-LDL)at optimal concentration was used as the stimulant to induce v-SMC proliferation in culture medium.A cDNA subtractive library of v-SMC proliferation specific to ox-LDL stimulation was established using subtractive hybridization technique.Methods,including blotting,Northern hybridization and gene sequencing,were used to clone new gene fragments.By using full-length cDNA screening and protein expression techniques,one full-length cDNA was cloned and its function was studied.Results One full-length cDNA was cloned.The new gene(Genbank AF 174647) expressed a 44 kDa protein,which mijht be associated with the activity of ox-LDL.Conclusion The new gene cloned may be associated with SMC proliferation in atherogenesis.     

PPM1D silencing by lentiviral-mediated RNA interference inhibits proliferation and invasion of human glioma cells

To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesium-dependent(PPM1D) gene and detect its effectiveness of gene silencing in human gliomas,specific siRNA targets with short hairpin frame were designed and synthesized.DNA oligo was cloned into the pFU-GW-iRNA lentiviral expression vector,and then PCR and sequencing analyses were conducted to verify the constructs.After the verified plasmids were transfected into 293T cells,the lentivirus was produced and the titer of virus was determined.Real-time quantitative PCR and Western blot were performed to detect the PPM1D expression level in the infected glioma cells.PCR and Western blot analyses revealed the optimal interfering target,and the virus with a titer of 6×108 TU/mL was successfully packaged.The PPM1D expression in human glioma cells was knocked down at both mRNA and protein levels by virus infection.The expression of PPM1D mRNA and protein was decreased by 76.3% and 87.0% respectively as compared with control group.The multiple functions of human glioma cells after PPM1D RNA interference were detected by flow cytometry and cell counting kit-8(CCK-8).Efficient down-regulation of PPM1D resulted in significantly increased cell apoptosis and reduced cell proliferation and invasion potential in U87-MG cells.We have successfully constructed the lentiviral shRNA expression vector capable of stable PPM1D gene silencing at both mRNA and protein levels in glioma cells.And our data gave evidence that the reduced cell growth observed after PPM1D silencing in glioma cells was at least partly due to increased apoptotic cell death.     

Expression and biological function of N-myc down-regulated gene 1 in human cervical cancer

The expression of N-myc down-regulated gene 1 (NDRG1) has previously been reported to be involved in the proliferation,differentiation,invasion and metastasis of cancer cells,but its role in cervical cancer is still unclear.This study aimed to investigate the expression of NDRG1gene in human cervical cancer and its effect on aggressive tumor behaviors.The NDRG1 expression in cervical tissues and cells was detected by RT-PCR.Specific expression plasmid pEGFP-N1-NDRG1-GFP was used to enhance the expression of NDRG1 in human cervical cancer cell lines.The mRNA and protein level of NDRG1 was assessed by RT-PCR and Western blotting,respectively.Its effects on cell proliferation,migration,invasion,cell cycle and apoptosis were detected by MTT,transwell migration assay and flow cytometry (FCM),respectively.The results showed that the expression of NDRG1 in cervical cancer tissues and cells was significantly lower than in normal cervical tissues (P<0.001).After transfection with pEGFP-N1-NDRG1-GFP,the mRNA and protein expression of NDRG1 was up-regulated in Siha cells,which suppressed cell proliferation (P<0.001),induced cell cycle arrest (P<0.05),reduced invasion and migration of Siha cells (P<0.05),but caused no cell apoptosis.Moreover,vascular endothelial growth factor (VEGF),a tumor-induced angiogenesis factor,was markedly reduced and E-cadherin,a cell adhesion molecule,was increased in the cells transfected with pEGFP-N1-NDRG1-GFP.It was concluded that up-regulated NDRG1 may play a role in the suppression of malignant cell growth,invasion and metastasis of human cervical cancer.     

Biological function of a novel gene overexpressed in human hepatocellular carcinoma

Objective To clone the full-length of a differentially expressed cDNA fragment, LC27, and study its biological function tentatively. Methods Northern blot was used to analyze the expression pattern of LC27 in hepatocellular carcinoma, matched nontumor liver tissues, fetal liver and normal adult liver tissues, as well as BEL-7402 hepatocellular carcinoma cell line ESTs splicing and 5’ rapid amplification of cDNA ends (5’ RACE) were used to clone the full-length of LC27 cDNA.An antisense oligodeoxynucleotide approach was used to investigate the biological role of the gene in the proliferation of BEL-7402 cells. Results A 2186 bp novel cDNA with an open reading frame encoding a 283 amino acid protein was cloned.Analysis of the deduced amino acid sequence indicated that it is 38% (88/229) identical to human Golgi 4-transmembrane spanning transporter MTP.The gene and the encoded protein was termed hepatocellular carcinoma overexpressed transmembrane protein (hotp) and HOTP, respectively.Hotp mRNA was almost undetectable in normal adult liver and fetal liver tissues.However, it was significantly up-regulated in hepatocellular carcinoma and some matched nontumor liver tissues, as well as BEL-7402 cells.The proliferation of BEL-7402 cells was suppressed by an antisense oligodeoxynucleotide against hotp mRNA at a concentration of 50 μg/ml. Conclusion HOTP may be an integral membrane transporter protein.The overexpression of the gene in hepatocellular carcinoma may play an important role in hepatocarcinogenesis and disease progression.     

卵巢癌紫杉醇耐药的蛋白质组学研究

目的：研究与卵巢癌紫杉醇耐药相关的蛋白质。方法：应用双向凝胶电泳（2-DE）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）寻找紫杉醇耐药细胞和敏感细胞的差异表达蛋白质。使用Western印迹技术对其中2个蛋白质进行验证。结果：通过对2组细胞总蛋白质双向凝胶电脉图谱进行分析,找到差异蛋白质点40个;通过质谱分析,24个蛋白质得到鉴定。这些蛋白质包括增殖细胞核抗原（PCNA）、nm23蛋白、prohibitin（PHB）分子伴侣蛋白、脂皮质素（annexin）、α-烯醇化酶（α-enolase）以及热休克蛋白（HSP）等。结论：通过蛋白质组学技术,发现了卵巢癌紫杉醇耐药细胞系和敏感细胞系之间差异表达蛋白质24个,这些差异蛋白质可能参与卵巢癌细胞紫杉醇耐药过程。     

抗增殖蛋白抑制转化生长因子-β1诱导的肾脏成纤维细胞增殖和表型改变

目的 研究抗增殖蛋白（prohibitin,PHB）在肾间质纤维化发生中的作用。方法（1）检测48例原发性肾小球肾炎患儿肾组织中PHB蛋白表达,并比较其与肾小管间质损伤程度的相关性。（2）观察PHB在大鼠肾脏成纤维细胞（NRK-49F）中亚细胞定位,以Western印迹和RT-PCR测定NRK-49F受到转化生长因子B1（TGF-β1）刺激后PHB表达的变化。（3）构建PHB表达质粒并转染,观察PHB对NRK-49F细胞周期以及表达α-平滑肌肌动蛋白（α-SMA）蛋白质和mRNA的影响。结果 （1）PHB蛋白主要表达于肾间质细胞和肾小管上皮细胞的胞质,随肾小管间质损伤程度加重而逐渐减弱（组间比较,均P〈0．01）,PHB表达量与肾小管间质损伤程度显著负相关（r=-0．802,P〈0．01）。（2）激光共焦显微镜下见PHB主要分布于NRK49F的细胞质,细胞核亦有较弱表达。TGF-β1刺激后PHB蛋白和mRNA表达均下调,呈现时间和剂量依赖关系（组间比较,P〈0．01）。（3）成功构建PHB真核表达质粒,转染48h细胞中PHB蛋白量升高约2．54倍（与未转染组比较,P〈0．01）。（4）转染PHB基因明显抑制TGF—β1所诱导的细胞增殖,使更多的细胞处于G0／G1期（与TGF-β1组比较,P〈0．01）,而对未受刺激的细胞无影响（P〉0．05）。（5）转染PHB基因明显抑制TGF—β1所诱导的α-SMA蛋白质和mRNA表达（与TGF-β1组比较,P〈0．01）,而对α-SMA基础表达无影响（与TGF-β1组比较,P〉0．05）。结论 PHB在肾组织中的表达水平可以反映肾小管间质损伤程度．外源性PHB显著抑制TGF-β1诱导的成纤维细胞增殖和表型改变。     

野生型及Thr187突变型p27Kip1 蛋白对HepG2细胞周期的影响

目的p27kip1氨基端第187号位置的苏氨酸(Thr187)磷酸化位点是该蛋白分子中重要的磷酸化位点,探讨人工诱变该位点苏氨酸为丙氨酸(T187A)后对肝癌细胞株HepG2细胞周期以及细胞增殖的影响。方法应用脂质体转染法将含人野生型和突变型p27kip1基因质粒DNA转染HepG2细胞,免疫细胞荧光化学检测p27kip1蛋白的表达,并用流式细胞仪分析细胞周期的变化。结果野生型和突变p27kip1基因转染HepG2细胞后均能在细胞核表达,并且可以明显抑制细胞增殖。p27kip1基因转染的HepG2细胞发生G0期阻滞,且突变型G0期阻滞作用明显强于野生型(P<0.01)。结论转染的p27kip1能够在HepG2细胞过度表达并能抑制HepG2细胞的增殖,Thr187磷酸化位点的人工诱变可能有利于p27kip1蛋白促使细胞G0期阻滞。     

H3N2流感病毒非结构蛋白-1真核表达载体的构建与表达及其对293T细胞增殖的影响

目的:构建甲型H3N2流感病毒非结构蛋白-1( nonstructal-1,NS1)真核表达载体并表达其编码蛋白;探讨NS1对细胞增殖的影响?方法:从江苏省甲型H3N2流感病毒毒株提取病毒RNA,采用RT-PCR技术扩增NS1全长基因,将其克隆至pMD18-T Simple Vector中构建pMD18-T/NS1质粒,双酶切pMD18-T/NS1与pXJ40-HA后,构建真核表达载体pXJ40-HA-NS1,经酶切及测序鉴定后将质粒转染到293T细胞中,通过免疫印迹法鉴定NS1蛋白的表达?3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide,MTT]法检测NS1转染细胞后对细胞增殖的影响? 结果:经双酶切?测序鉴定证实NS1基因的真核表达载体构建成功;免疫印迹法证实NS1蛋白的表达?MTT法证实转染NS1质粒后对细胞增殖有抑制作用?结论:成功克隆了NS1全长基因,构建了其真核表达载体,并初步验证了NS1蛋白过表达后能抑制细胞的增殖,该表达载体的构建为后期建立稳定表达NS1的细胞模型和NS1蛋白对细胞增殖和凋亡作用机制的进一步研究提供了基础?     

siRNA介导的新趋化因子VCC-1沉默对人SMMC7721肝癌细胞生长的影响

 目的 探讨siRNA靶向抑制VCC-1表达对人肝癌细胞株SMMC27721生长的影响。方法 采用脂质体法将siRNA质粒导入SMMC7721细胞,建立VEGF相关的趋化因子1（(VEGF correlated chemokine 1,VCC-1）)VCC-1基因表达沉默的肝癌细胞系,用Western blot检测细胞中VCC-1的表达水平,用MTT法、软琼脂克隆形成实验等研究siRNA靶向抑制VCC-1表达对SMMC7721细胞生长的影响。结果 Western blot 结果显示RNA干扰组（shVCC1-1组）细胞VCC-1蛋白表达水平明显低于对照组（P<0.01）; ,MTT法结果显示shVCC1-1组细胞增殖速度、克隆形成率均低于对照组（P<0.05）。结论 siRNA靶向抑制 VCC-1表达可以抑制人SMMC7721肝癌细胞增殖能力及克隆形成能力。     

靶向抑制 DNMT1对人脑胶质瘤细胞增殖的影响

目的：应用靶向 DNA 甲基转移酶1（DNMT1）基因的小干扰 RNA（siRNA）重组质粒来转染体外培养的人脑胶质瘤细胞,观察其对胶质瘤细胞增殖活性的影响。方法实验组予以 siRNA-DNMT1序列进行转染,对照组仅予以 siRNA-阴性对照序列。应用 qRT-PCR 分析 DNMT1基因表达变化, Western blot 法分析 DNMT1、PCNA 和 Cyclin D1蛋白表达变化,MTT 法检测细胞增殖生长能力,细胞克隆法分析细胞增殖克隆形成能力。结果与对照组比较,qRT-PCR 结果表明实验组 DNMT1 mRNA 表达明显减少（ P <0.01）;Western blot 法表明实验组 DNMT1、PCNA 和 Cyclin D1蛋白表达水平明显降低（P <0.01）;MTT 法检测表明实验组中活细胞数明显低于对照组（P <0.05）;细胞克隆法表明实验组细胞的增殖克隆形成能力明显低于对照组（ P <0.01）。结论靶向 DNMT1基因的 siRNA 重组质粒可减少人脑胶质瘤细胞内 DNMT1基因的表达,从而抑制胶质瘤细胞的增殖。     

神经干细胞增殖与分化过程中Bmi1与NSPc1的作用

多梳蛋白(PcG)家族是非常保守的表观遗传调节因子。近年来对其成员的功能研究发现,PcG家族对干细胞的自我更新及增殖与分化都有很重要的作用。哺乳动物PRC1成员Bmi1和NSPc1在神经系统发育早期高表达。最近的研究提示它们不仅在细胞增殖分化相关基因转录调控中发挥重要作用,而且在神经干细胞自我更新及分化过程中同样有重要作用。     

siRNA抑制DJ-1基因表达对肺鳞癌SK-MES-1细胞生物学行为的影响

目的:采用RNA 干扰技术下调DJ-1 基因在肺鳞癌细胞SK-MES-1 中的表达, 探讨DJ-1 表达下调对SKMES-1 细胞生物学行为的影响, 以期探讨DJ-1 基因的功能。方法:构建靶向DJ-1 基因siRNA 慢病毒载体( 重组慢病毒中有绿色荧光蛋白真核表达框), 感染SK-MES-1 细胞(DJ-1 siRNA 组), 并设立慢病毒载体对照组(Control-siRNA 组)及空白对照组。荧光显微镜下观察并计算感染效率, Western 印迹检测各组细胞中DJ-1 蛋白表达水平, MTT法检测细胞增殖能力, 流式细胞术测定细胞周期, Transwell 小室实验检测细胞体外迁移侵袭能力。结果:与Control-siRNA组及空白对照组比较, DJ-1 siRNA 组的DJ-1 蛋白表达受到抑制、细胞增殖能力明显减弱(P<0.01)、G1/G2 期细胞数增多和S 期细胞数减少表明细胞周期受阻、细胞体外迁移和侵袭能力显著减弱(P<0.01)。结论:DJ-1 基因具有促进肺鳞癌细胞SK-MES-1 细胞的增殖和体外迁移侵袭的作用。     

Prohibitin 2 调节肌细胞增强因子2转录因子活性的分子机制

目的：探讨Prohibitin 2 (PHB2)抑制肌细胞增强因子2(myocyte enhancer fact or, MEF2)转录因子活性的分子机制,为进一步研究PHB2在其他MEF2阳性表达细胞中的调控作用提供依据。方法：利用脂质体转染方法将表达PHB2和MEF2的真核表达载体与荧光素酶报告基因载体共转染入体外培养的Hela细胞中,转染36 h后,裂解细胞获取细胞裂解上清,Western blotting检测上清中重组蛋白的表达及利用发光检测仪检测上清中荧光素酶的生物发光活性。结果：转染PHB2细胞组与未转染PHB2细胞组中,MEF2调控的荧光素酶生物发光活性比分别为0.28±0.02和1.00±0.02,两组比较差异具有显著性（P<0.01）,且降低程度与PHB2的表达量呈正比;在未转染PHB2细胞组, MEF2转录激活区调控的荧光素酶生物发光活性比为9.53±1.06,而在转染PHB2细胞组其为2.24±0.21,两组比较差异具有显著性（P<0.01）;组蛋白去乙酰化酶（ HDAC）抑制剂TSA处理组与未处理组,共转染PHB2细胞中MEF2调控的荧光素酶生物发光活性比分别为0.98±0.12和0.38±0.04,两组比较差异具有显著性（P<0.01）,提示TSA解除了PHB2对MEF2的抑制作用。结论：PHB2抑制MEF2转录活性的分子机制是通过作用于MEF2的转录激活区由HDAC介导完成的,该抑制作用非依赖于成肌调节因子MyoD,且与MEF2的DNA结合功能无关。     

CDK2-AP1基因过表达对乳腺癌MCF-7细胞增殖及周期的影响

目的:通过过表达手段上调细胞周期调节蛋白依赖性激酶2- 关联蛋白1(CDK2-AP1) 基因在乳腺癌细胞MCF-7 中的表达, 并观察其对MCF-7 细胞生长和细胞周期调控的作用。方法:将CDK2-AP1 基因的编码框构建于慢病毒表达载体, 导入MCF-7 细胞, 应用实时定量PCR 和Western 印迹验证CDK2-AP1 基因mRNA 和蛋白的表达效率。利用M 法绘制生长曲线、克隆形成实验观察CDK2-AP1 基因过表达后MCF-7 细胞生长的变化, PI 染色流式细胞仪检测MCF-7 细胞周期的改变。通过Western 印迹检测CDK2-AP1 过表达后, 细胞周期相关蛋白(CDK2, CDK4, P16^Ink4A, P21^Cip1/Waf1) 的表达。结果:过表达CDK2-AP1 基因的慢病毒感染MCF-7 细胞可上调其mRNA 表达6.94 倍, 蛋白表达也十分显著地增高, 两者相一致。生长曲线显示MCF-7 细胞过表达CDK2-AP1 基因后, 增殖能力显著降低(P<0.05);克隆形成实验表明, 其形成的克隆数目同样显著减少(P<0.05);流式细胞仪检测证实MCF-7 细胞过表达CDK2-AP1 能够使细胞周期出现G₁期阻滞, 并且出现凋亡峰;CDK2-AP1 基因表达上调导致P21^Cip1/Waf1和P16^Ink4A蛋白表达上调, CDK2 和CDK4 蛋白表达下调。结论:CDK2-AP1 基因具有抑癌基因的功能, 在乳腺癌MCF-7 细胞过表达该基因能够抑制细胞的生长和克隆形成能力, 并且使细胞阻滞于G₁期。