IQGAP2在肝细胞肝癌中的表达及其临床意义

目的:认识肝细胞肝癌中含IQ模体的GTP酶活化蛋白2(IQ motif containing GTPase activating protein 2,IQGAP2)的亚细胞定位和表达,及其在肝细胞肝癌(hepatocellular carcinoma,HCC)中的作用.方法:分别通过Western blot、免疫荧光和免疫组织化学染色分析IQGAP2在7种人肝癌细胞与正常肝细胞系中的表达、定位,及其在HCC组织样本中的表达与分布.结果:IOGAP2在Bel-7402、Bel-7404、SMMC-7721、SK-HEP-1、HLE和HL-7702等肝癌和正常成人肝细胞系中不表达,仅在HepG2和Hep3B等2种甲胎蛋白(AFP)表达阳性的人肝癌细胞系中表达,IQGAP2主要分布于细胞质,此外,在HepG2细胞中还具有明显的核膜和核仁定位,IQGAP2在HCC组织中表达降低(56.9%,29/51).其中,19例配对HCC组织的IQGAP2表达与肿瘤大小,AJCC分期和血清AFP水平相关(P=0.020,P=0.017,P=0.002);38例配对组织IQGAP2在肿瘤和癌旁正常肝组织中主要定位于胞质,部分细胞伴有胞核和胞膜定位,但是,IQGAP2的表达与肿瘤大小、分化程度、AJCC分期以及血清AFP表达水平无相关性.结论:IQGAP2蛋白可能参与细胞黏附、信号转导等过程,在肝癌的发生发展中发挥重要作用,是一种潜在的抑癌基因.     

5-氮杂-2'-脱氧胞苷对人胃癌SGC-7901细胞株生长及EDNRB基因启动子异常甲基化的影响

目的:探讨去甲基化药物5-氮杂-2'-脱氧胞苷(5-Aza-CdR)对人胃癌细胞系SGC-7901细胞株的生长及EDNRB基因启动子异常甲基化的影响.方法:使用1、2、5、10μmol/L5-Aza-CdR干预胃癌SGC-7901细胞,甲基化特异性PCR(MSP)和逆转录聚合酶链反应(RT-PCR)分别检测药物干预前后EDNRB基因的甲基化状态和EDNRB mRNA的表达,MTT法检测细胞增殖活性,流式细胞术分析细胞周期及细胞凋亡的改变.结果:未经5-Aza-CdR处理的SGC-7901细胞中EDNRB基因启动子区域CpG岛高甲基化,且EDNRB mRNA不表达,经1、2、5、10μmol/L5-Aza-CdR处理4d后,EDNRB基因启动子区域高甲基化状态得到逆转,细胞中EDNRB mRNA表达恢复.4种浓度5-Aza-CdR处理的SGC-7901细胞后,细胞增殖受到抑制,且呈时间和剂量依赖性;并抑制SGC-7901细胞生长周期,其细胞周期阻滞于S期,5、10μmol/L5-Aza-CdR实验组细胞凋亡率显著高于对照组,且差异有统计学意义(7.13%±0.87%,13.34%±1.12% vs 3.69%±...     

母体表达基因3通过miR-125a-5p/TET2途径抑制HepG2细胞载脂蛋白(a)的表达

目的研究发现母体表达基因3(MEG3)对HepG2细胞载脂蛋白(a)[Apo(a)]表达的调控作用及其机制。方法用荧光素酶报告系统分析MEG3与miR-125a-5p的靶向性结合。采用实时定量PCR(qRT-PCR)检测高表达Apo(a)的HepG2细胞和低表达Apo(a)的SMMC7721细胞中MEG3的表达情况;向HepG2细胞转染MEG3,Western blot和qRT-PCR检测Apo(a)、TET2表达情况;采用小干扰RNA技术沉默TET2的表达。结果 (1)MEG3与hsa-miR-125a-5p能互补性结合,荧光素酶报告基因系统分析结果证实了MEG3与hsa-miR-125a-5p结合的存在。(2)miR芯片结果表明,在HepG2细胞中,hsa-miR-125a-5p表达水平升高,是对照组的近1.5倍,MEG3在HepG2细胞和SMMC7721细胞中均有表达,但前者MEG3的表达水平显著低于后者。(3)MEG3抑制Apo(a)表达。(4)MEG3下调miR-125a-5p的表达,上调TET2的表达; miR-125a-5p的mimics可逆转MEG3对Apo(a)的下调作用及TET2的表达,但可被miR-125a-5p的抑制剂逆转; TET2沉默可逆转MEG3对Apo(a)的下调作用。结论 MEG3通过miR-125a-5p/TET2途径下调HepG2细胞Apo(a)的表达。     

亚砷酸钠对HaCaT细胞MGMT基因启动子区甲基化CpG结合蛋白-2、DNA甲基转移酶1、组蛋白去乙酰化酶1结合的影响

目的 观察亚砷酸钠(NaAsO2)对人肤角质形成细胞株(HaCaT细胞)MGMT基因启动子区甲基化CpG结合蛋白-2(MeCP2)、DNA甲基转移酶1(DNMT1)及组蛋白去乙酰化酶1(HDAC1)结合情况的影响,为深化阐释砷毒作用机制提供依据.方法 分别以0.00(空白对照)、3.13、6.25、12.50、25.00 μmol/L NaAsO2重复间隔处理HaCaT细胞72 h(NaAsO2处理24h,隔天再次相同处理,重复3次),以人表皮鳞癌细胞株(A431)作为阳性对照,定量染色质免疫共沉淀技术(Q-ChIP)检测MGMT基因转录调控区ChIP1、ChIP2区域及MGMT基因编码区ChIP3区域MeCP2、DNMT1、HDAC1结合情况.结果 各组HaCaT细胞MGMT基因转录调控区ChIP1、ChIP2区域MeCP2、DNMT1、HDAC1蛋白结合水平比较,差异有统计学意义(F值分别为7.387、84.634、78.442和19.263、69.649、26.546,P均＜0.05);其中各NaAsO2处理组ChIP1、ChIP2区域MeCP2、DNMT1、HDAC1蛋白结合水平[3.13 μmol/L NaAsO2处理组:(136.00±16.97)％、(145.00±2.83)％、(88.50±19.09)％和(106.50±37.48)％、(112.34±8.73)％、(59.71±8.49)％;6.25 μmol/L NaAsO2处理组:(130.00±42.43)％、(154.50±4.95)％、(101.00±1.27)％和(88.50±3.54)％、(134.32±2.82)％、(102.75±19.91)％;12.50 μmol/LNaAsO2处理组:(141.50±23.33)％、(161.50±7.78)％、(125.00±11.31)％和(119.50±24.75)％、(171.59±3.54)％、(167.61±10.61)％;25.00 μmol/NaAsO处理组:(134.50±43.13)％、(472.50±50.20)％、(383.50±30.41)％和(180.09±12.73)％、(348.50±27.58)％、(158.45±12.02)％]均高于空白对照组[(51.50±9.19)％、(82.00±12.73)％、(25.03±2.91)％和(37.02±4.24)％、(91.56±26.16)％、(19.09±2.90)％,P均＜0.05].各组HaCaT细胞MGMT基因编码区ChIP3区域MeCP2蛋白结合水平比较,差异无统计学意义(F=1.670,P＞0.05),而DNMT1、HDAC1蛋白结合水平比较,差异有统计学意义(F值分别为4.404、9.863,P均＜0.05),其中25.00 μmol/L NaAsO2处理组DNMT1、HDAC1蛋白结合水平[(615.85±29.63)％、(306.09±59.40)％]与空白对照组[(99.70±12.02)％、(92.45±48.79)％]比较,差异有统计学意义(P均＜0.05).结论 MeCP2可结合于砷所致高甲基化MGMT基因转录调控区,通过招募DNMT1及HDAC1使组蛋白去乙酰化,同时DNMT1可结合于MGMT基因编码区,以非甲基化DNA结合蛋白(MBD)依赖的方式招募HDAC1,通过染色质重塑方式导致MGMT基因沉默,可能是砷毒性表现的早期分子事件.     

Involvement of methylation-associated silencing of formin 2 in colorectal carcinogenesis

AIM To investigate whether promoter methylation is responsible for the silencing of formin 2(FMN2) in colorectal cancer(CRC) and to analyze the association between FMN2 methylation and CRC. METHODS We first identified the expression levels and methylation levels of FMN2 in large-scale human CRC expression datasets, including GEO and TCGA, and analyzed the relationship between the expression and methylation levels. Then, the methylation levels in four CpG regions adjacent to the FMN2 promoter were assessed by MethylTarget? assays in CRC cells and in paired colorectal tumor samples and adjacent nontumor tissue samples. Furthermore, we inhibited DNA methylation in CRC cells with 5-Aza-2'-deoxycytidine and assessed the expression of FMN2 by q RT-PCR. Last, the association between FMN2 methylation patterns and clinical indicators was analyzed.RESULTS A statistically significant downregulation of FMN2 expression in large-scale human CRC expression datasets was found. Subsequent analysis showed that a high frequency of hypermethylation occurred in the FMN2 gene promoter in CRC tissues; operating characteristic curve analysis revealed that FMN2 gene methylation had a good capability for discriminating between CRC and nontumor tissue samples(AUC = 0.8432, P 0.0001). Methyl Target? assays showed that CRC cells and tissues displayed higher methylation of these CpG regions than nontumor tissue samples. Correlation analysis showed a strong inverse correlation between methylation and FMN2 expression, and the inhibition of DNA methylation with 5-Aza significantly increased endogenous FMN2 expression. Analysis of the association between FMN2 methylation patterns and clinical indicators showed that FMN2 methylation was significantly associated with age, N stage, lymphovascular invasion, and pathologic tumor stage. Notably, the highest methylation of FMN2 occurred in tissues from cases of early-stage CRC, including cases with no regional lymph node metastasis(N0), cases in stages Ⅰ and Ⅱ, and cases with no lymphovascular invasion, but the methylation level began to decrease with tumor progression. Additionally, FMN2 promoter hypermethylation was more common in patients 60 years old and in colon cancer tissue. CONCLUSION FMN2 promoter hypermethylation may be an important early event in CRC, most likely playing a critical role in cancer initiation, and can serve as an ideal diagnostic biomarker in elderly patients with early-stage colon cancer.     

ShRNA-mediated gene silencing of β-catenin inhibits growt of human colon cancer cells

AIM: To observe the gene silencing mediated by the specific shRNA targeted against β-catenin and its effect on cell proliferation and cycle distribution in the human colon cancer cell line Colo205. METHODS: Two shRNA plasmid vectors against β-catenin were constructed and transfected into Colo205 cells with LipofectamineTM2000. The down-regulations of β-catenin, c-myc and cyclinD1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay and soft agar colony formation assay. The effect of these two β-catenin shRNAs on cell cycle distribution and apoptosis was examined by flow cytometry. RESULTS: These two shRNA vectors targeted against β-catenin efficiently suppressed the expression of β-catenin and its down stream genes, c-myc and cyclinD1. The expression inhibition rates were around 40%-50% either at the mRNA or at the protein level. The shRNA-mediated gene silencing of β-catenin resulted in significant inhibition of cell growth both on the culture plates and in the soft agar. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 h post transfection due to gene silencing. CONCLUSION: These specific shRNAs targeted against β-catenin could have a gene silencing effect and block the WNT signaling pathway. They could inhibit cell growth, increase apoptosis, and induce cell cycle arrest in Colo205 cells. ShRNA interference against β-catenin is of potential value in gene therapy of colon cancer.     

ShRNA-mediated gene silencing of β-catenin inhibits growth of human colon cancer cells

AIM: To observe the gene silencing mediated by the specific shRNA targeted against β-catenin and its effect on cell proliferation and cycle distribution in the human colon cancer cell line Colo205.METHODS: Two shRNA plasmid vectors against β-catenin were constructed and transfected into Colo205 cells with LipofectamineTM2000. The down-regulations of β-catenin, c-myc and cyclinD1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay and soft agar colony formation assay. The effect of these two β-catenin shRNAs on cell cycle distribution and apoptosis was examined by flow cytometry.RESULTS: These two shRNA vectors targeted against β-catenin efficiently suppressed the expression of β-catenin and its down stream genes, c-myc and cyclinD1. The expression inhibition rates were around 40%-50% either at the mRNA or at the protein level.The shRNA-mediated gene silencing of β-catenin resulted in significant inhibition of cell growth both on the culture plates and in the soft agar. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 h post transfection due to gene silencing.CONCLUSION: These specific shRNAs targeted against β-catenin could have a gene silencing effect and block the WNT signaling pathway. They could inhibit cell growth, increase apoptosis, and induce cell cycle arrest in Colo205 cells. ShRNA interference against β-catenin is of potential value in gene therapy of colon cancer.     

蛋白酶体抑制剂MG132对结肠癌细胞lovo侵袭力的影响

目的探讨小剂量蛋白酶体抑制剂MG132对结肠癌细胞(lovo)侵袭能力的影响。方法采用不同浓度MG132不同时间点分别作用于lovo细胞,用四甲基偶氮盐比色法(MTT法)检测lovo细胞抑制率;设定MG132浓度为0.5μmol/L、1μmol/L,分别作用lovo细胞24 h,倒置显微镜观察细胞形态学变化,Transwell小室检测MG132对lovo细胞的穿透基底膜能力的影响。结果低剂量MG132对lovo细胞生长无明显抑制,而细胞侵袭力较未处理组明显减弱(P<0.05)。结论低剂量MG132可使结肠癌lovo细胞侵袭能力减弱。     

RNA interference-mediated gene silencing of vascular endothelial growth factor in colon cancer cells

AIM To inhibit the expression of vascular endothelial growth factor (VEGF) in colon cancer cell line by RNA interference (RNAi). METHODS Followed the service of E-RNAi, we designed and constructed two kinds of shRNA expression vectors aiming at the VEGF gene, then transfected them into colon cancer HT29 cells by lipofectamineTM 2000. The level of VEGF mRNA was investigated by RT-PCR and Northern blotting. The protein expression of VEGF was observed by immunofluoresence staining and Western blotting. RESULTS We got two kinds of VEGF specific shRNA expression vectors which could efficiently inhibit the expression of VEGF in HT29 cells. RT-PCR, Northern blotting, immunofluoresence staining and Western blotting showed that inhibition rate for VEGF expression was up to 42%, 89%, 73% and 82%, respectively. CONCLUSION The expression of VEGF can be inhibited by RNA interference in HT29 cells.