靶向Plk1的siRNA对肝癌细胞凋亡的影响

目的:观察靶向作用于Plk1的siRNA对肝癌细胞系BCL-7402细胞中Plk1基因和p53基因表达及细胞凋亡的影响,探求靶向该基因的治疗在肝癌基因治疗中的可行性及效应.方法:设计合成两对靶向作用于Plk1基因的双链siRNA序列,分别命名为siRNA1和siRNA2,应用脂质体法将其转入BCL-7402细胞中.实验...     

靶向Skp2基因siRNA对大肠癌细胞生物学特性的影响

目的:设计和构建Skp2特异性的siRNA真核表达载体,观察Skp2siRNA重组体对人大肠癌HCT116细胞株生物学特性的影响.方法:人工合成Skp2基因干扰序列并定向插入到RNAi真核表达载体pRNAT-U6.1/Neo,通过测序鉴定.用脂质体介导的基因转染方法转入HCT116细胞,经G418筛选出稳定转染的细胞系,半定量RT-PCR方法检测靶基因的表达.流式细胞技术分析细胞周期分布,MTT比色法检测细胞的生长抑制率.结果:测序表明Skp2干扰序列完全正确.RTPCR结果显示2条Skp2siRNA真核表达质粒均能有效抑制核干细胞因子mRNA的转录表达( P<0.05).MTT比色法检测显示,与阴性对照组及未转染组细胞比较,干扰组细胞增殖率明显下降(29.21%±1.40%,30.10%±1.50% vs49.05%±4.50%,53.03%±4.92%,均P<0.05).流式细胞术检测结果显示转染后G0/G1期细胞数占生长周期细胞数的比例增多,即表现为G1期阻滞,S期细胞的比例降低.结论:成功构建了S k p 2 干扰真核表达载体,siRNA重组体能有效抑制人大肠癌细胞Skp2mRNA表达,细胞增殖能力减弱,为大肠癌基因治疗的可行性提供了有力的证据.     

siRNA抑制survivin基因及其对人肝癌细胞株HepG2细胞凋亡的影响

目的观察特异小干扰RNA（small interfering RNA,siRNA）抑制survivin基因表达对人肝癌细胞株HepG2细胞凋亡的影响。方法构建survivin-siRNA真核表达载体,利用脂质体转染人肝癌HepG2细胞,采用反转录聚合酶链反应（RT-PCR）技术检测survivin基因mRNA表达水平。针对转染后不同的时间点采用四甲基偶氮唑蓝（MTT）法检测各组细胞的增殖以及HepG2细胞的凋亡情况。结果构建的survivin-siRNA转染组survivin蛋白表达较对照组明显下调,差异有显著性（P〈0.01）,其增殖能力较对照组显著下降（P〈0.001）;而阳性对照组及未转染组对survivin表达及mRNA抑制作用不明显（P=0.219）。结论所构建的针对人survivin-siRNA可显著抑制survivin的表达及HepG2细胞凋亡。     

腺病毒介导的ezrin抑制对肝癌细胞生物学行为的影响

膜-细胞骨架连接蛋白ezrin是一种与细胞骨架重排,细胞变形运动有关的分子,近年来被报道与肿瘤转移有关[1].我们以前期研究[2-3]筛选出的有效小干扰RNA序列为基础,构建以ezrin为靶点的腺病毒小发夹RNA(shRNA)载体,使ezrin在细胞内获得稳定持续的下调,从而为深入研究膜-骨架连接蛋白ezrin在肿瘤细胞运动转移方面的作用及其相关的分子信号转导奠定了基础.     

HCCR-2干扰对HepG2肝癌细胞生物学行为的影响

人子宫颈癌基因HCCR是韩国科学家Ko等Ⅲ发现的一种与人类多种肿瘤有关的癌基因。HCCR可分为HCCR－1、HCCR－2两种亚型。HCCR－1基因由9个外显子构成,编码360个氨基酸,其蛋白相对分子质量为42000。HCCR－2比HCCR－1少1个外显子,其蛋白由304个氨基酸组成,相对分子质量为36000。研究发现,HCCR－2较HCCR－1具有更强的致癌能力。     

电场对细胞生物学行为的影响

电场的生物学效应对胚胎发育、组织损伤修复、肿瘤发生有一定作用。本文综述电场对细胞生物学行为的影响及其机制的研究进展。     

电场对细胞生物学行为的影响

电场的生物学效应对胚胎发育、组织损伤修复、肿瘤发生有一定作用.本文综述电场对细胞生物学行为的影响及其机制的研究进展.     

siRNA targeting VEGF inhibits hepatocellular carcinoma growth and tumor angiogenesis in vivo

BACKGROUND/AIMS: We have investigated whether siRNA targeted against VEGF inhibits functional properties of endothelial cells in vitro and HCC tumor growth and blood vessel formation in vivo. METHODS: The influence of siRNA-VEGF on endothelial cell proliferation, apoptosis and tube formation were analyzed in vitro. Antitumoral effects were examined in an orthotopic tumor model after ex vivo transfer or intraperitoneal treatment of siRNA, respectively. Intratumoral microvessel density was assessed by CD31 staining. RESULTS: VEGF expression was inhibited in Hepa129 by 70% and in SVEC4-10 by 48% within two days after transfection. In vitro, endothelial cell proliferation and tube formation was reduced by 23% and 38%, respectively. Interference with VEGF signaling was demonstrated by reduced pAKT in hepatoma cells. Tumor growth was inhibited by ex vivo transfer or intraperitoneal application of siRNA-VEGF by 83% or 63% in orthotopic tumors within 14 days. VEGF protein was reduced in both models by 29% and 44%. Microvessel density dropped to 34% for tumors from ex vivo transfected cells and 39% for systemic treated tumors. CONCLUSIONS: The results show that VEGF knockdown can be associated with reduced endothelial cell proliferation and tube formation in vitro and decreased tumor growth and microvessel density in vivo.     

以Survivin为靶点的反义寡核苷酸在肝癌研究中的应用进展

Survivin是目前恶性肿瘤基因治疗研究中的新靶点.采用反义寡核苷酸等治疗方法可以抑制Survivin的表达,从而诱导肿瘤细胞的凋亡、抑制细胞增殖并增强对化疗药物的敏感性.近年来,已有学者将其用于肝癌的研究,为肝癌的治疗提供了新的方向.     

Induction of hepatocellular carcinoma by in vivo gene targeting

The distinct phenotypic and prognostic subclasses of human hepatocellular carcinoma (HCC) are difficult to reproduce in animal experiments. Here we have used in vivo gene targeting to insert an enhancer-promoter element at an imprinted chromosome 12 locus in mice, thereby converting ～1 in 20,000 normal hepatocytes into a focus of HCC with a single genetic modification. A 300-kb chromosomal domain containing multiple mRNAs, snoRNAs, and microRNAs was activated surrounding the integration site. An identical domain was activated at the syntenic locus in a specific molecular subclass of spontaneous human HCCs with a similar histological phenotype, which was associated with partial loss of DNA methylation. These findings demonstrate the accuracy of in vivo gene targeting in modeling human cancer and suggest future applications in studying various tumors in diverse animal species. In addition, similar insertion events produced by randomly integrating vectors could be a concern for liver-directed human gene therapy.     

Effect of shRNA targeting survivin on ovarian cancer

Purpose

This study investigated the effect of shRNA targeting survivin on cultured ovarian cancer cells and on a murine ovarian cancer xenograft.

Methods

An RNAi plasmid for survivin was transfected into SKOV3 cells, and the effect of shRNA targeting survivin on the expression of survivin was determined. Transmission electron microscopy (TEM), flow cytometry, and TUNEL staining were used to assess apoptosis. The MTT assay was used to measure cell growth and changes in cisplatin sensitivity. SKOV3 cells were injected into nude mice, and the effect of shRNA targeting the survivin gene on tumor growth was assessed.

Results

SKOV3 cells transfected with an RNAi plasmid against survivin had increased apoptosis and slower growth. At the molecular level, these cells also had lower expression of survivin. Nude mice inoculated with SKOV3 cells developed cancers, and treatment with shRNA targeting survivin markedly inhibited the growth of these cancers with no obvious side effects.

Conclusions

Our studies of SKOV3 cells and ovarian cancer xenografts in nude mice indicate that shRNA targeting survivin has potential for the treatment of ovarian cancer.     

Effects of miR-340 on hepatocellular carcinoma by targeting the DcR3 gene

In hepatocellular carcinoma (HCC), miR-340 plays a vital role in the regulation of tumor occurrence and deterioration, while DcR3 gene is involved in cancer cell proliferation and apoptosis. This study analyzed miR-340 in the serum of patients with HCC and healthy controls. Then, miR-340, DcR3, TGF-β1 and Smad2 expression were measured in HCC tissues and adjacent parts. Relationship between miR-340 and DcR3 was verified. Effects of miR-340 on human HepG2 cell proliferation and apoptosis were explored. miR-340, DcR3, TGF-β1, Smad2 mRNA and protein expression were also determined after miR-340 transfection. Compared with the control, miR-340 was significantly lower in the serum of the HCC patients (p < 0.01). miR-340 was lower in HCC tissues than in adjacent; however, DcR3, TGF-β1 and Smad2 were higher (p < 0.01). Furthermore, luciferase activity was significantly lower in the cells co-transfected with miR-340 mimics and DcR3-3′UTR-WT (p < 0.01), indicating that DcR3 was a target gene of miR-340. Moreover, decreased expression in DcR3, TGF-β1 and Smad2 was detected after miR-340 overexpression (p < 0.01), thus promoting apoptosis and blocking the proliferation of human HepG2 cells (p < 0.05). Furthermore, overexpression of DcR3 could activate the TGF-β1/Smad2 signal transduction pathway and increase the phosphorylation of Smad2. In conclusion, miR-340 plays a suppressive role in HCC development by targeting DcR3 and silencing the TGF-β1/Smad2 signaling pathway.     

Survivin靶向RNA干扰对人结肠癌HCT116细胞生长影响的研究

目的设计和构建survivin特异性的siRNA真核表达载体,观察survivin siRNA重组体对人大肠癌HCT116细胞株生长的影响,为大肠癌的基因治疗提供理论依据。方法针对survivin mRNA序列设计合成编码siRNA的DNA模板,构建2个survivin RNAi真核表达载体;实验分为survivin siRNA重组质粒转染组、空载体组和HCT116组,转染大肠癌细胞HCT116细胞,采用RT-PCR法检测survivin mRNA的表达,观察重组质粒对转染的HCT116细胞survivin基因表达的影响;用四甲基偶氮唑盐（MTT）法测量细胞生长情况;用流式细胞术检测细胞凋亡情况。结果测序表明survivin干扰序列完全正确,RT-PCR结果显示2条survivin siRNA真核表达质粒均能有效抑制survivin mRNA的转录表达。MTT比色法检测显示,与阴性对照组及未转染组细胞比较,干扰组细胞增殖率明显下降（P〈0．05）。流式细胞术检测的转染重组质粒组细胞凋亡率高于脂质体对照组和空质粒对照组（P〈0．05）。结论成功构建了survivin干扰真核表达载体,siRNA重组体能有效抑制人大肠癌细胞survivin mRNA表达,并抑制结肠癌细胞生长,促进其凋亡。     

Effects of p16 gene on biological behaviours in hepatocellular carcinoma cells

AIM: To investigate the effects of p16 gene on biological behaviours in hepatocellular carcinoma cells. METHODS: HCC cell lines SNU-449 and HepG2.2.15 were infected respectively by a replication defective, recombinant retrovirus capable of producing a high level of p16 protein expression (pCLXSN-p16). G418 resistant stable P16 protein expression cell lines were selected. And the biological behaviours of the p16 gene transfected HCC cells were observed. RESULTS: Initial in vitro experiments in HCC cell line SNU-449 with loss of p16 protein expression demonstrated the pCLXSN-p16 treatment significantly inhibited cell growth. But there was no treatment effect when the pCLXSN-p16 was used in another HCC cell line HepG2.2.15 which has positive p16 protein expression. Subsequent study in a nude mouse model demonstrated that the p16 gene transfected SNU-449 had a lower succeeding rate in the first time establishment of tumors and grew more slowly in the nude mice when compared with non-transfected SNU-449. Moreover, the nude mice inoculated with transfected SNU-449 had a longer surviving time than those inoculated with non-transfected SNU-449. CONCLUSION: Our results show that the p16INK4a gene transfer can inhibit the proliferation and reduce the invasion ability of hepatocellular carcinoma.     

A novel HBV antisense RNA gene delivery system targeting hepatocellular carcinoma

AIM: To construct a novel HBV antisense RNA delivery system targeting hapatocellular carcinoma and study its inhibitory effect in vitro and in vivo.METHODS: GE7,a 16-peptide specific to EGFR, and HA20,a homologue of N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized and conjugated with polylysin. The above conjugates were organized into the pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system, named AFP-enhancing 4-element complex. Hepatocelluar carcinoma HepG2.2.15 cells was used to assay the in vitro inhibition of the complex on HBV. Expression of HBV antigen was assayed by ELISA.BALB/c nude mice bearing HepG2.2.15 cells were injected with AFP-enhancing 4-element complex. The expression of HBV antisense RNA was examined by RT-PCR and the size of tumor in nude mice were measured.RESULTS: The AFP-enhancing 4-element complex was constructed and DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1:1.The expression of HBsAg and HBeAg of HepG2.2.15 cells was greatly decreased after being transfected by AFP-enhancing 4-element complex. The inhibitory rates were 33.4 % and 58.5 % respectively. RT-PCR showed HBV antisense RNA expressed specifically in liver tumor cells of tumor-bearing nude mice. After 4 injections of AFP-enhancing 4-element complex containing 0.2 μg DNA, the diameter of the tumor was 0.995 cm&#177;0.35,which was significantly smaller than that of the control groups (2.215 cm&#177;025, P&lt;0.05).CONCLUSION: AFP-enhancing 4-element complex could deliver HBV antisense RNA targeting on hepatocarcinoma and inhibit both HBV and liver tumor cells in vitro and in vivo.