靶向survivin shRNA在肿瘤治疗中的研究进展

生存素在细胞凋亡抑制中起重要作用,越来越多的研究者将其作为肿瘤基因治疗的理想靶点。应用RNA干扰技术靶向抑制生存素的表达,从而抑制肿瘤细胞增殖、促进肿瘤细胞凋亡、抑制肿瘤血管生成、增强肿瘤细胞对放化疗的敏感性。     

逆转录病毒载体介导shRNA对人胚胎肾细胞中p53基因的沉默作用

目的：检测逆转录病毒载体介导的小发夹环RNA（small hairpin RNA,shRNA）对人胚胎肾细胞（HEK293）中p53基因的干扰作用。方法：将人H₁启动子由XholⅠ和EcoRⅠ酶切位点插入CMV启动子上游,人CD₄基因替代匀霉素抗性基因,作为检测标志。针对人野生型p53基因设计的shRNA被连接在H₁启动子的下游,构建含有shRNA的逆转录病毒载体LTRH₁-p53,即RNAi表达载体;利用流式细胞术检测逆转录病毒感染HEK293细胞72 h后细胞内P53蛋白水平的变化。结果：成功构建LTRH₁-p53载体,该载体感染HEK293细胞后72 h,与感染空病毒载体LTRH₁组相比,P53蛋白表达降至空病毒载体LTRH₁组的69％（P<0.01）。结论：LTRH₁-p53表达载体成功使p53基因沉默,shRNA表达载体的使用为基因功能研究提供有力工具。     

Interference of lysine-specific demethylase 1 inhibits cellular invasion and proliferation in vivo in gastric cancer MKN-28 cells

BackgroundLysine-specific demethylase 1(LSD1), the first identified histone demethylase, plays an important role in the epigenetic regulation of gene activation and repression. Up-regulated LSD1expression has been reported in several malignant tumors.Our aim, therefore, was to better understand the mechanisms underlying the upregulation of LSD1 in gastric cancer.MethodsWe used lentiviral shRNA to knockdown LSD1 in the gastric cancer MKN-28 cell line. Cell proliferation was measured by MTT assay while cell apoptosis was assessed by Annexin V-FITC/PI double staining flow cytometry. The invasive potential of gastric cancer cells was determined by matrigel invasion assay. Protein expression was detected by Western blot. In vivo, the effect of knocking down LSD1 on tumor growth and protein expression in gastric cancer cells in nude mice was investigated.ResultsLSD1 knockdown in MKN-28 cell lines resulted in increasing the activity of cisplatin in vitro and the inhibition of cancer cell proliferation and invasion, and induced cell apoptosis. The expression of TGF-β1, VEGF, Bcl-2, β-catenin, p-ERK and p-Smad 2/3 proteins was inhibited in LSD1 knockdown cells. Moreover, in an in vivo model of gastric cancer, LSD1 knockdown suppressed tumor growth and protein expression.ConclusionLSD1 knockdown affected the fuction of gastric cancer MKN-28 cell line. LSD1 may be a latent target in the diagnosis and therapy of gastric cancer.     

A Novel KCNJ13 Nonsense Mutation and Loss of Kir7.1 Channel Function Causes Leber Congenital Amaurosis (LCA16)

Mutations in the KCNJ13 gene that encodes the inwardly rectifying potassium channel Kir7.1 cause snowflake vitreoretinal degeneration (SVD) and leber congenital amaurosis (LCA). Kir7.1 controls the microenvironment between the photoreceptors and the retinal pigment epithelium (RPE) and also contributes to the function of other organs such as uterus and brain. Heterologous expressions of the mutant channel have suggested a dominant‐negative loss of Kir7.1 function in SVD, but parallel studies in LCA16 have been lacking. Herein, we report the identification of a novel nonsense mutation in the second exon of the KCNJ13 gene that leads to a premature stop codon in association with LCA16. We have determined that the mutation results in a severe truncation of the Kir7.1 C‐terminus, alters protein localization, and disrupts potassium currents. Coexpression of the mutant and wild‐type channel has no negative influence on the wild‐type channel function, consistent with the normal clinical phenotype of carrier individuals. By suppressing Kir7.1 function in mice, we were able to reproduce the severe LCA electroretinogram phenotype. Thus, we have extended the observation that Kir7.1 mutations are associated with vision disorders to include novel insights into the molecular mechanism of disease pathobiology in LCA16.     

RNA interference targeting enhancer of polycomb1 exerts anti-tumor effects in lung cancer

Background and aim: Lung cancer is one of leading malignant tumor worldwide with a high mortality rate. A new therapy target, enhancer of polycomb1 (EPC1) knocked down by short hairpin RNA (shRNA) interference technology, for lung cancer was established to investigate its effects on lung cancer in present study. Methods: RNA interference technology was applied to down-regulate the expression of EPC1 by specific-shRNA with lentivirus vector in neoplastic human alveolar basal epithelial cells (A549 cells). The survival rate and apoptosis were respectively measured by MTT and Flow Cytometry to evaluate the effects of shRNA EPC1 on cells. Mice xenografts of HCT116 cells with shRNA EPC1 were also established to assess the effect on tumor growth. The levels of AKT and p65 were detected by western blotting. Results: The down-regulation of EPC1 by specific-shRNA with lentivirus vector was significantly decreased the survival rate and apoptosis of A549 cells, and the tumors in EPC1 shRNA transfection group had a significant lower size and weight compared with the ones with control shRNA. The protein expression of p-AKT and p65 was reduced by EPC1 shRNA in both in vitro and in vivo experiments. Conclusion: Silencing EPC1 by shRNA technology had the inhibition effects on cell proliferation and tumor growth in lung cancer, which provided a new potential target for treatment of cancers.     

Adenovirus expressing dual c-Met-specific shRNA exhibits potent antitumor effect through autophagic cell death accompanied by senescence-like phenotypes in glioblastoma cells

c-Met, a cognate receptor tyrosine kinase of hepatocyte growth factor, is overexpressed and/or mutated in number of tumors. Therefore, abrogation of c-Met signaling may serve as potential therapeutic targets. In this study, we generated Ads expressing single shRNA specific to c-Met (shMet) (dl/shMet4 and dl/shMet5) or dual shRNAs specific to c-Met (dl/shMet4+5); and examined the therapeutic potential of these newly engineered Ads in targeting c-Met, and delineated their mechanism of action in vitro and in vivo. Ads expressing shMet induced knock-down in c-Met, and phenotypically resulted in autophagy-like features including appearance of membranousvacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein1 light chain 3 to autophagosomes. Ads expressing shMet also suppressed Akt phosphorylation and increased number of senescence-related gene products including SM22, TGase II, and PAI-1. These changes resulted in inhibition of cell proliferation and G₂/M arrest of U343 cells. In vivo, intratumoral injection with dl/shMet4+5 resulted in a significant reduction of tumor growth with corresponding increasing overall survival. Histopathological analysis of these treated tumors revealed that Atg5 was highly up-regulated, indicating the therapeutic induction of autophagy. In sum, these results reveal that autophagic cell death induced by shMet-expressing Ads provide a novel strategy for targeting c-Met-expressing tumors through non-apoptotic mechanism of cell death.