靶向人Pokemon基因的shRNA重组质粒的构建(英文)

Objective:The aim of this study was to establish the foundation for studying the role of pokemon gene in tumorigenesis and development by constructing recombinant plasmids that can express small interfering RNA(siRNA)targeting human Pokemon gene.Methods:Hairpin siRNA templates targeting Pokemon gene were synthesized and cloned into plasmid vector psiRNA-H1neo.Three vectors derived siRNAs(psiRNA1,2,3)and one mocking psiRNAc(as control)were con- structed.The recombinant Pokemon siRNA plasmids were constructed...     

Survivin shRNA重组慢病毒构建及对A549细胞增殖的影响

背景与目的 Survivin是凋亡抑制蛋白(inhibitor of apoptosis protein,IAP)家族的成员,在多种肿瘤组织中高度表达而在终末分化细胞中极少表达,因此可以作为癌症治疗的理想靶点.本研究旨在通过构建Survivin基因shRNA的慢病毒质粒并干扰肺癌细胞A549中Survivin的表达,分析其对细胞增殖的影响.方法 设计Survivin干扰靶序列,构建重组质粒;将pLL3.7-Survivin转染293T细胞后利用Hela细胞检测病毒的滴度并感染A549细胞,应用RTPCR和Western blot检测干扰效果;MTT与流式细胞术分析其对细胞增殖的影响.结果 本研究成功构建了重组质粒;重组质粒可抑制A549细胞中Survivin基因的表达;细胞受阻于G2/M期.结论 本研究构建的重组质粒可抑制Survivin基因的表达并影响细胞的增殖,其为研究RNAi介导的肺癌基因治疗打下基础.     

shRNA对A549细胞survivin基因表达及紫杉醇敏感性的影响

目的观察靶向survivin基因的短发夹RNA(shorthairpin RNA,shRNA)对人肺腺癌细胞A549生物学行为及紫杉醇敏感性的影响。方法将靶向survivin的基因片段插入载体后构建重组质粒,用转染试剂FuGENE(HD将其导入A549细胞,RT-PCR及Western blot分析转染前后survivin基因的表达情况,TUNEL法检测细胞凋亡情况,MTT法检测转染后A549细胞对紫杉醇的敏感性变化。结果成功构建重组质粒。与对照组相比,转染重组质粒后,survivin基因的表达明显降低,细胞凋亡率增加。转染前紫杉醇抑制A549细胞的IC50为转染后的11.9倍,两者比较,差异有显著性(P<0.05)。结论靶向survivin的shRNA能下调survivin基因表达,诱导细胞凋亡,增强A549细胞对紫杉醇的敏感性。     

Recombinant adenovirus-mediated shRNA silencing of midkine gene in BxPC-3 cells

Objective:To investigate the silencing effects of recombinant adenovirus Ad-shRNA-MK on midkine(MK) gene in pancreatic cancer cells. Methods:Ad-shRNA-MK was used to infect pancreatic cancer BxPC-3 cells. Assays were conducted for knockdown of the MK gene on the day of infection and on the 1a, 3rd, 5th, 7th, and 9th days post-infection by using immunocytochemistry, real-time RT-PCR, and Western blot analysis. Results:The adenoviral Ad-shRNA-PTN was constructed successfully, and infection was confirmed by electron microscopic observation. By using real-time RT-PCR, the inhibition rates of MK mRNA expression in the BxPC-3 cells were 20%, 80%, 55%, and 23% on the 1st, 3rd, 5th, and 7th days post-infection. Immunocytochemistry and Western blot analysis confirmed this effect at the gene product level. Conclusion:Efficient and specific knockdown of MK in pancreatic cancer cells by adenoviral Ad-shRNA-PTN is a potentially powerful tool for the study of gene therapy of pancreatic cancer nerve infiltration.     

P53-Derived peptides conjugation to PEI: an approach to producing versatile and highly efficient targeted gene delivery carriers into cancer cells

ABSTRACT

Objectives: Targeted delivery of cytotoxic drugs or therapeutic antisense RNAs into specific cells is a major bottleneck in cancer therapy. To overcome this problem and improve the specificity for cancer cells, we describe a new-targeted delivery system using p53-derived peptides, namely PNC 27 and PNC 28. These peptides target HDM-2 on the surface of cancer cells. HDM-2 is overexpressed on the surface of cancerous cells, but not present on the untransformed cells.

Methods: To determine HDM-2-expressing cells, we used immunocytochemistry and flow cytometry analysis on nine cell lines including MCF-7 and NIH-3t3. Conjugation of peptides to vectors was confirmed using reverse-phase high-pressure liquid chromatography (RP-HPLC). Physicochemical properties of vector/DNA complexes including particle size, surface charge and DNA condensation ability were determined. In transfection studies, three plasmids were used including luciferase, pEGFP and shRNA plasmid against Bcl-XL mRNA. The level of Bcl-XL expression was determined by real-time PCR and western blot techniques.

Results: The results of gene delivery and shRNA-based gene silencing studies indicated that conjugation of PNC peptides could enhance gene delivery efficiently with high-targeted activity exclusively into cancer cells.

Conclusion: Our results strongly indicated that this targeting system could be utilized as an efficient targeting method for most cancer cells.     

慢病毒载体在RNA干扰中的应用进展

小分子RNA干扰技术是一种新兴的特异性地阻断某些基因表达的研究手段,在肿瘤、病毒感染,遗传病及其它疾病的治疗中均有广泛应用。慢病毒载体是一类来源于重组逆转录病毒的载体,由于具有可以感染非分裂期与分裂期细胞,容纳大的外源性目的基因片段,免疫反应小等特点,现已成为转移目的基因进入哺乳动物细胞的理想载体。将慢病毒载体应用于RNA干扰当中,可以特异性抑制哺乳动物的各类细胞中基因的表达,成为基因功能研究和基因治疗的有力手段。本文将试图对近期慢病毒载体在RNA干扰当中的应用的最新进展进行综述。     

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.     

Inhibition of hepatitis B virus replication in 2.2.15 cells by expressed shRNA

Summary.  Hepatitis B virus (HBV) infection is a worldwide health problem. To determine whether RNA interference (RNAi) could inhibit ongoing HBV replication in 2.2.15 cells, we constructed shRNA-producing vector pU6P based on the mouse U6 RNA promoter and cloned 12 targeted sequences against HBV into the vector, resulting in a series of pU6-siHBV vectors. The recombinant vectors were transfected into 2.2.15 cells, HBsAg and HBeAg in cultured media were assayed using enzyme-linked immunosorbent assay at various days after transfection. The amount of HBV DNA in the culture medium was quantitated by real-time polymerase chain reaction. HBsAg and HBeAg expression were inhibited by 72.8 ± 5.4% ( P  = 0.00003) and 55.8 ± 6.2% ( P  = 0.000026), respectively, 4 days after transfection with pU6-siHBV5. The greatest inhibition of HBV DNA was decreased by approximately 1.9-fold ( P  = 0.013) on day 6 post transfection with pU6-siHBV11 compared with that of empty vector. No change was found for HBV protein expression and DNA replication on pU6-siGFP (negative control) transfected cells. Our data demonstrate that the transfection of HBV-targeted shRNA-producing vector in 2.2.15 cells could inhibit the HBV protein expression and HBV DNA replication specifically. RNAi may be considered as a potential antiviral approach for human HBV infection.     

核糖体蛋白S6 shRNA慢病毒载体的构建及其对肺腺癌A549细胞株增殖的影响

目的 探讨靶向抑制核糖体蛋白S6（rpS6）表达的短发夹RNA（short hairpin RNA, shRNA）慢病毒载体的构建方法及抑制rpS6表达对肺腺癌A549细胞株增殖的影响。方法 合成针对rpS6基因的双链寡核苷酸序列,插入质粒载体pGCsil-GFP,转化大肠杆菌细胞,测序鉴定插入片段;再通过293T细胞转染和慢病毒包装,收集浓缩病毒并感染肺腺癌A549细胞株。流式细胞技术分选强阳性表达绿色荧光蛋白(GFP)的细胞克隆,荧光定量PCR及Western blot检测rpS6基因的mRNA和蛋白干扰效率。体外利用CCK-8试剂盒定点检测细胞的光密度(OD)值,分析抑制rpS6表达对肺腺癌A549细胞株增殖能力的影响。结果 重组pGCsil-sh-rpS6-GFP质粒经测序鉴定示：插入序列与rpS6干扰序列完全符合,证实pGCsil-sh-rpS6-GFP质粒构建成功。sh-rpS6慢病毒稳定感染A549细胞株后,流式细胞技术分选GFP强阳性表达的细胞克隆比率为86.80%。荧光定量PCR与Western blot检测示： sh-rpS6组的mRNA和蛋白干扰效率分别为(79.72±6.83)%、（83.77±12.13)%。体外增殖实验示：与A549细胞相比,sh-rpS6组在各时间点的OD值较对照组均下降（均P<0.05）。结论 构建的sh-rpS6慢病毒载体能稳定、有效地抑制肺腺癌A549细胞株rpS6的表达,并有效减慢A549细胞株的增殖速度。