INHIBITION OF PROLIFERATION OF HUMAN BREAST CANCER MCF-7 CELLS BY SMALL INTERFERENCE RNA AGAINST LRP16 GENE

Objective: Our previous studies have firstly demonstrated that 17β -E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA) strategy, by which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviral vector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot. Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cells proliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results: The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells. Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.

RNA INTERFERENCE OF ANNEXIN II GENE IN PC3 CELLS BY USING SMALL INTERFERENCE RNA SYNTHESIZED WITH IN VITRO TRANSCRIPTION

Objective To silence annexin Ⅱ gene expression by using small interference RNA （siRNA） in prostate cancer cell line PC3. Methods For in vitro transcription, four sequences of 29-nucleotide DNA template oligonucleotides were designed, and one pair of the sequences were complementary to annexin Ⅱ gene. The other pair was negative control. The 8 nucleotides at the 3＇ end of each oligonucleotide were complementary to the T7 Promoter Primer. The sense and anti-sense siRNA templates were transcribed by T7 RNA polymerase and the resulting RNA transcripts were hybridized to create dsRNA. The siRNA was transfected into prostate cancer cell PC3. For assaying the efficiency of siRNA, confocal microscopy, Northern blotting, and Western blotting were employed to examine the expression of annexin Ⅱ protein and its mRNA. ^3H thymidine was used to measure DNA synthesis. Results The siRNA sequence specific to annexin Ⅱ gene was capable of inhibiting the expression of annexin Ⅱ protein and its mRNA. And cellular DNA synthesis was significantly reduced in siRNA transfected cells.Conclusions The protocol for the synthesis of siRNA by T7 RNA polymerase is feasible. Annexin Ⅱ might be involved in DNA synthesis.

miRNA对褐飞虱鞘脂质代谢基因表达的影响及沉默NlSPT1和Nl SMase4的small RNA分析

【背景】鞘脂质是第二大类膜脂,作为信号转导物质参与细胞生长发育、繁殖及凋亡等过程。鞘脂质代谢酶调控鞘脂质代谢以维持生物体内代谢的稳态。【目的】以重要水稻害虫褐飞虱(Nilaparvata lugens)为研究对象,通过RNA干扰(RNA interference,RNAi)技术检测微小RNA(microRNA,miRNA)生物合成通路核心组分NlAgo1、NlDicer1和NlDrosha沉默后褐飞虱鞘脂质代谢通路相关基因的相对转录水平,结合small RNA测序分析沉默丝氨酸棕榈酰转移酶1(serine palmitoyltransferase 1,SPT1)和鞘磷脂酶4(sphingomyelinase 4,SMase4)基因的差异miRNA,探究miRNA在褐飞虱鞘脂质代谢中的作用,为害虫防治提供新的分子靶标。【方法】利用RNAi技术,分别对羽化后第1天(1 PAE,post adult eclosion)的雌成虫NlAgo1、NlDicer1和NlDrosha进行dsRNA注射,以ds GFP为对照;分别解剖羽化后第5天的卵巢组织,以β-actin作为内参基因,采用实时荧光定量PCR(qRT-PCR)方法检测NlAgo1、NlDicer1和NlDrosha沉默后鞘脂质代谢通路相关基因的表达量变化;根据已有的小RNA文库联合miRNA-靶基因预测软件对可能调控NlSPT1和NlSMase4表达的miRNA进行预测;通过small RNA测序技术对沉默NlSPT1和NlSMase4的差异miRNA进行鉴定和靶基因富集性分析。【结果】与对照组相比,沉默NlAgo1、NlDicer1或NlDrosha显著上调卵巢中NlSPT1和NlSMase4等鞘脂质代谢通路相关基因的表达;靶基因预测结果显示,有6条miRNA能与NlSPT1结合,13条miRNA能与NlSMase4结合;沉默NlSPT1和NlSMase4的差异miRNA的靶基因显著富集在细胞核和蛋白质结合等生物学过程以及内吞作用、内质网加工、MAPK信号通路、TOR信号通路、凋亡、脂质代谢等代谢通路。【结论】NlAgo1、NlDicer1和NlDrosha依赖性的miRNA通过影响鞘脂质代谢相关基因的表达影响鞘脂质代谢。NlSPT1和NlSMase4沉默引起褐飞虱卵巢miRNA表达水平的改变。研究结果可为基于鞘脂质代谢基因为靶标的害虫防治提供理论依据。

Effects of RNA interference targeting transforming growth factor-beta 1 on immune hepatic fibrosis induced by Concanavalin A in mice

BACKGROUND: Previous studies have shown that transforming growth factor-beta 1 (TGF-beta 1) is the most potent means of stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells. Thus, TGF-beta 1 could be a target for treating hepatic fibrosis. This study aimed to investigate the inhibitory effects of specific TGF-beta 1 small interference RNA (siRNA) on immune hepatic fibrosis induced by Concanavalin A (Con A) in mice. METHODS: Three short hairpin RNAs targeting different positions of TGF-beta 1 were designed and cloned to the plasmid pGenesil-1 to obtain three recombinant expression vectors (pGenesil-TGF-beta 1-ml, pGenesil-TGF-beta 1-m2 and pGenesil-TGF-beta 1-m3). Thirty male Kunming mice were randomly divided into 6 groups: normal, model, control, and three treatment groups. The immune hepatic fibrosis models were constructed by injecting Con A via the tail vein at 8 mg/kg per week for 6 weeks. At weeks 2, 4 and 6, pGenesil-TGF-beta 1-ml, pGenesil-TGF-beta 1-m2 or pGenesi1-TGF-beta 1-m3 was injected by a hydrodynamics-based transfection method via the tail vein at 0.8 ml/10 g within 24 hours after injection of Con A in each of the three treatment groups. The mice in the control group were injected with control plasmid pGenesil-HK at the same dose. All mice were sacrificed at week 7. The levels of hydroxyproline in liver tissue were determined by biochemistry. Liver histopathology was assessed by Van Gieson staining. The expression levels and localization of TGF-beta 1, Smad3, and Smad7 in liver tissue were detected by immunohistochemistry. The expression of TGF-beta 1, Smad3, Smad7 and alpha-smooth muscle actin (alpha-SMA) mRNAs in the liver were assessed by semi-quantitative RT-PCR. RESULTS: The levels of hydroxyproline in the liver tissue of the treatment groups were lower than those of the model group (P<0.01). Histopathologic assay showed that liver fibrogenesis was clearly improved in the treatment groups compared with the model group. The expression levels of TGF-beta 1 and Smad3 of liver tissue were also markedly lower in the treatment groups than in the model group (P<0.01), while the levels of Smad7 were higher in the treatment groups than in the model group (P<0.01). RT-PCR further showed that the expression of TGF-beta 1, Smad3 and alpha-SMA mRNA was significantly inhibited in the treatment groups compared with the model group, while the levels of Smad7 were increased. There was no difference in the above parameters among the three treatment groups or between the control and model groups (P>0.05), but the inhibitory effect of pGenesil-TGF-beta 1-ml was the highest among the treatment groups. CONCLUSIONS: Specific siRNA targeting of TGF-beta 1 markedly inhibited the fibrogenesis of immune hepatic fibrosis induced by Con A in mice. The anti-fibrosis mechanisms of siRNAs may be associated with the down-regulation of TGF-beta 1, Smad3 and alpha-SMA expression and up-regulation of Smad7 expression in liver tissue, which resulted in suppressing the activation of hepatic stellate cells. (Hepatobiliary Pancreat Dis Int 2009; 8: 300-308)

Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells

AIM： To observe the inhibition of hepatitis B virus （HBV） replication and expression in HepG2.2.15 cells by combination of small interfering RNAs （siRNAs）. METHODS： Recombinant plasmid psiI-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay （ELISA）. Intracellular viral DNA and covalently closed circular DNA （cccDNA） were quantified by real-time polymerase chain reaction （PCR）. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR （RT-PCR）. RESULTS： siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantlycombination of siRNAs significantly suppressed HBV cccDNA amplification. CONCLUSION： Combination of siRNAs mediates a stronger inhibition on viral replication and antigenexpression in HepG2.2.15 cells, especially on cccDNA amplification.

KNOCKDOWN OF SURVIVIN EXPRESSION BY SMALL INTERFERING RNA SUPPRESSES PROLIFERATION OF TWO HUMAN CANCER CELL LINES

Objective To construct an expression vector of small interfering RNA （siRNA） against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast cancer cell line MCF-7. Methods Constructed an expression vector of siRNA against survivin and transfected it into PC-2 and MCF-7 cells using lipofectamine^TM 2000. The expression of survivin was detected by semi-quanfifive RT-PCR and immunohistochemistry, and its effects on proliferation of PC-2 and MCF-7 cells were detected by MTT assay. Results The introduction of sequence-specific siRNA could efficiently suppress survivin expression at both mRNA and protein levels in the two cancer cell lines. In PC-2 cells, the expression inhibition rates were 81.25% at mRNA level and 74.24% at protein level In MCF-7 cells, the expression inhibition rates were 64.91% at mRNA level and 79. 72% at protein level The proliferation of PC-2 and MCF-7 cells was also suppressed, and24 and 48 hours after the cells were reseeded, the proliferation inhibition rates of PC-2 cells were 28. 00% and 33. 38%, and that of MCF-7 cells were 31.58% and 33.02%, respectively. Conclusions The expression vector of siRNA against survivin can block survivin expression in PC-2 and MCF-7 cells efficiently and specifically. Down regulation of survivin expression can suppress proliferation of PC-2 and MCF-7 cells. Survivin RNAi may have potential value in gene therapy of human cancers.

Effects of RNA Interference Combined with Ultrasonic Irradiation and SonoV ue Microbubbles on Expression of STAT3 Gene in Keratinocytes of Psoriatic Lesions

The most effective sequence of small interfering RNA（si RNA） silencing STAT3 of psoriatic keratinocytes（KCs） was screened out,and the effects of the most effective si RNA combined with ultrasonic irradiation and Sono Vue microbubbles on the expression of STAT3 of KCs and the dose-and time-response were investigated.Three chemically-synthetic si RNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs,and the effects on STAT3 expression were detected,then the most effective si RNA was selected for the subsequent experiments.The negative controls of siR NA（si RNA-NC） labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles were transfected into KCs,then the optimal parameters of ultrasonic irradiation were determined.The most effective si RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and Sono Vue microbubbles was transfected into KCs,and the dose-and time-response of RNA interference was determined.The effect of RNA interference by the most effective si RNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles（LUS group） was compared with that only carried by Lipofectamine 3000（L group）.The results showed that si RNA-3 achieved the highest silencing efficacy.0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation.The si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles could effectively knock down the STAT3 expression at m RNA and protein levels in dose-and time-dependent manners determined at 100 nmol/L with maximum downregulation on m RNA at 48 h,and on protein at 72 h after transfection.The LUS group achieved the highest silencing efficacy.It was concluded that si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoV ue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs,and the optimized transfection condition and the sequence of si RNA-3 could serve for further research on gene therapy of psoriasis.

Inhibition of HOXB7 Gene Expression in Melanoma Cells by Small Interfering RNA

Objective： HOXB7 gene is a kind of transcription regulator over-expressed in malignant melanoma （MM） cell lines. It can specifically up-regulate the expression of angiogenic factors and tumor growth factors such as bFGF, GROa, VEGF and induce angiogenesis in melanoma, resulting in the proliferation and metastasis of tumor cells. We designed and synthesized HOXB7 specific siRNA to study its interfering effect on the expressions of HOXB7 and bFGF genes in melanoma A375 cell line and the biologic characteristics of A375 cells. Methods： Three synthesized siRNA with different sequences were separately transfected into A375 cells by lipofecter 2000. The expression of HOXB7 and bFGF mRNA in transfected cells was detected by RT-PCR 24 and 48 hours after transduction. The expression of bFGF protein in the transfected cells were detected by flowcytometry 48 hours after transfection. MTT assay was used to analyze the cell proliferation rate of siRNA transfected cells. Based on the in vitro experiment results, one effective siRNA sequence was selected for the construction of in vivo siRNA expression vector. Then, a malignant melanoma animal model was established. The siRNA expression plasmid was injected into the tumor foci and its influence on the growth and angiogenesis of tumor was observed. Results： The mRNA expressions of both HOXB7 and bFGF genes in the A375 cells reduced significantly 24 and 48 hour after transfection of siRNA. Expression level of the protein of angiogenic factor bFGF induced by HOXB7 gene in siRNA transfected cells was significantly lower than that in control cells 48 hours after transduction. Cell proliferation was also suppressed in siRNA transfected cells. Two of the three siRNA strands showed prominent interference effect. The in vivo study indicated that the tumor size and the microvessel density in the tumor both reduced after injection of HOXB7siRNA plasmid. Conclusion： Down-regulation of HOXB7 gene expression can effectively reduce the expression of angiogenic factor bFGF and the proliferation of MM cells. Besides, the growth and angiogenesis of MM tumor were also inhibited.

Induction of Silencing Effect of Swedish Mutant Amyloid Precursor Protein by RNA interference

Summary： Over-expression of APP and Swedish mutation could cause some familial early onset AD. In this study, a primary screening was conducted of effective small interference RNAs （siRNAs） targeted wild type APP （APPwt） and Swedish mutant APP （APPswe）. One siRNA targeting APPwt and the other siRNA targeting APPswe were designed, All these siRNAs were endogenously expressed by siRNAs expressing plasmids, COS-7 cells were transiently co-transfected with APP-GFP recombinant plasmids and siRNA expression vector, The silencing effect of each siRNA was quantitatively assessed by the level of expression of green fluorescent protein （GFP）. It was found that the siRNAs silenced APPwt and APPswe to different degrees, siRNA directed against APPswe was more effective in suppressing the expression of fusion gene of APPswe than that of APPwt. The silencing effect of siRNA directed against APPswe indicating allele-specific silencing property of the siRNAs. Therefore, siRNAs directed against APP play an important role both in the therapeutic study of Alzheimer disease and functional exploration ofAPP gene.

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor,and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.

Application and challenges of RNA interference in reversing multidrug resistance of tumor cells

RNA interference,widely regarded as a key mechanism for cells to regulate gene expression,is a natural gene silencing phenomenon.It can be used as the gene knockdown to reverse the multidrug resistance of tumor cells and has been applied in the field of biomedicine,exhibiting huge potential in drug target identification,optimization of drug targets,multidrug resistance,etc.This paper first introduces the mechanism of RNA interference and the formation mechanism of multidrug resistance of tumor cells,on the basis of which it reviews the application and challenges of RNA interference technology in reversing multidrug resistance.Additionally,the development of the siRNA delivery system is illustrated.

EGFR siRNA序列的筛选及其对HepG2细胞活性的影响

目的构建EGFR shRNA重组真核表达载体,并筛选抑制效果最好的EGFR shRNA序列。方法应用在线工具设计人EGFR siRNA序列,采用限制性内切酶BamHI和HindIII切割真核表达质粒pcDNA3.1(+),构建三种人EGFR的siRNA干扰序列载体(psilencer 4.1-CMV neo-EGFR siRNA)。将重组质粒载体转化大肠杆菌DH5α感受态并筛选阳性克隆,通过DNA测序鉴定重组质粒。应用脂质体lipo2000将三种人EGFR siRNA干扰序列载体转染到HepG2细胞,通过荧光显微镜观察转染效率,实时定量PCR检测EGFR mRNA表达水平,MTT法检测细胞活性。结果Psilencer 4.1-CMV neo-EGFR siRNA重组质粒被成功克隆。EGFR shRNA-1、EGFR shRNA-2和EGFR shRNA-3敲低EGFR mRNA的效率分别是80%、60%和70%以上。shRNA-2和shRNA-3使细胞活性分别下降50%(P<0.05),但shRNA-1对细胞活性无明显影响(P>0.05)。结论重组psilencer 4.1-CMV neo-EGFR siRNA质粒可下调肝癌细胞株EGFR表达水平和细胞活性。EGFR shRNA-3较EGFR shRNA-1和shRNA-2对HepG2细胞的抑制作用更显著。

Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro

AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).

siRNA纳米递送系统在类风湿性关节炎治疗中的研究进展

类风湿性关节炎是一种自身免疫性疾病,其病因复杂,目前尚未有很好的治疗方法,长期服用抗风湿药物也带来诸多副作用。RNA干扰技术是指通过外源性或内源性的双链RNA在体内诱导靶基因m RNA产生特异性降解,进而引起不同水平的基因沉默,具有高效、高特异性、低毒等优点,在生物医药领域有着巨大潜力。小干扰RNA(si RNA)作为RNA干扰技术的重要效应分子,在治疗类风湿关节炎方面具有巨大潜力。本文综述了siRNA递送系统用于治疗类风湿关节炎的最新研究进展,阐明了基于siRNA纳米给药系统治疗类风湿关节炎的优势,并展望了未来siRNA递送的发展方向。

LncRNAuc.48+对CGRP介导的三叉神经痛的作用

目的探究长链非编码核糖核酸uc.48+(long non-coding RNA,lncRNA uc.48+)如何影响三叉神经痛(trigeminal neuralgia,TN)大鼠三叉神经节(trigeminal ganglion,TG)中降钙素基因相关肽(calcitonin gene related peptide,CGRP)以及其潜在的作用机制。方法慢性压迫性损伤大鼠眶下神经(chronic compression injury to the infraorbital nerve,CCI-ION)建立大鼠TN动物模型。成模后,经眶下孔局部注射uc.48+小干扰敲低lncRNAuc.48+,以及向正常大鼠转染uc.48+质粒过表达lncRNAuc.48+。通过行为学来观察各组大鼠面部机械痛阈值(mechanical pain threshold,MWT),结合qPCR、蛋白印迹等方法观察大鼠TG中CGRP的含量及变化。ELISA法观察1L-1β的变动状况。结果接受uc.48+小干扰处理的TN大鼠机械痛敏阈值明显上升;TG中CGRP的蛋白、mRNA水平明显下降(P<0.01),1L-1β的水平也降低(P<0.01);此外,与正常大鼠相比,正常大鼠转染uc.48+质粒后,其机械痛敏阈值明显下降,TG中CGRP的蛋白、mRNA水平明显上升(P<0.01),1L-1β的水平明显增加(P<0.01)。结论TN大鼠敲除uc.48+显著减轻疼痛,而过表达uc.48+则加剧了TN的疼痛传导。uc.48+小干扰对TN有一定的抑制作用,该机制可能是通过减少神经病理痛大鼠TG中CGRP的表达,抑制初级感觉神经节对三叉神经病理痛信息的传播,进而减轻三叉神经病理痛对机械性疼痛的敏感性。

RNA干涉分子机制研究进展

RNA干涉 (RNAinterference ,RNAi)是生物体内的一种通过双链RNA (dsRNA)来抵抗病毒入侵和抑制转座子活动的自然机制 .双链RNA与同源mRNA互补结合而使特定基因失活 ,这一过程已经在包括拟南芥、线虫和真菌等多种模式生物中得到揭示 .近来研究表明 ,2 1～ 2 5nt的小干涉RNA (smallinterferenceRNA ,siRNA)可介导哺乳动物细胞特异性基因沉默 .RNAi具有高效性和高度特异性 。

RNA干扰技术在哺乳动物中的应用

RNA干扰 (RNAi)是生物界普遍存在的一种抵御外来基因和病毒感染的进化保守机制 .RNAi是由双链RNA触发的转录后基因沉默机制 ,具有序列特异性 ,在哺乳动物细胞中 ,RNAi由 2 1～ 2 3个核苷酸组成的双链RNA引发 .小干扰RNA (siRNA)可以在体外合成或通过表达载体在哺乳动物细胞内合成 .由于RNAi技术具有快速、简单和特异性强等特点 ,在基因功能研究。

利用siRNA阻抑survivin基因表达诱导乳腺癌细胞系MCF-7细胞凋亡的研究(英文)

背景与目的:survivin属IAP基因家族成员,表达于多种肿瘤组织中,能促使细胞逃避凋亡,并促进细胞的异常有丝分裂。本研究旨在探讨敲除survivin基因后,癌细胞的增殖和凋亡情况。方法:利用RNAi阻抑人乳腺癌细胞内survivin基因的表达,RT-PCR和Westernblot法分析survivin基因mRNA和蛋白的表达,MTT法检测细胞生长增殖抑制率,流式细胞术检测细胞凋亡率。结果:survivinsiRNA转染组,survivin基因表达水平与未转染组比较下调了64%。随着siRNA浓度的增加,细胞增殖抑制率逐渐增高,200nmol/L剂量组细胞增殖抑制率最高,可达60.9%。不同浓度的siRNA可不同程度地诱导细胞凋亡,200nmol/L剂量组凋亡率最高,可达29.0%。结论:survivinsiRNA有可能成为治疗乳腺癌的新药物。

猪繁殖与呼吸综合征病毒ORF2～4特异siRNA的筛选及其抑制病毒复制效果的研究

RNA干扰（RNAi）技术是基因功能研究的有效工具,为了了解猪繁殖与呼吸综合征病毒（PRRSV）次要结构蛋白GP2、GP3、GP4在病毒复制中的作用,针对各自的编码基因ORF2、ORF3、ORF4分别选取4个小干扰RNA（siRNA）位点（共12个）,构建相应的短发夹RNA（shRNA）表达载体,转染MARC-145细胞后,通过荧光定量PCR和病毒滴度检测干扰效果。筛选了可以减少GP2、GP3、GP4相应基因mRNA含量的ORF2、ORF3、ORF4特异shRNA表达载体,病毒效价滴定表明shRNA表达载体处理细胞可以减少GP2、GP3、GP4相应基因mRNA含量,细胞培养上清中的病毒滴度比对照低184~4.65倍。

质粒介导靶向c-myc的siRNA对MCF-7乳腺癌细胞增殖的影响

目的观察靶向原癌基因c-myc的siRNA对MCF-7人乳腺癌细胞c-myc/c-Myc表达及细胞增殖的影响。方法以原癌基因c-myc mRNA 589-609位碱基为靶序列构建靶向c-myc的siRNA真核表达质粒p-Mat01-1及其错配质粒p-Mis09-1,空质粒pEGFP-C1为对照,用脂质体Lipo2000包裹转染MCF-7细胞。采用RT-PCR、Western blot检测c-myc mRNA及蛋白表达,MTT法检测MCF-7细胞增殖。结果与pEGFP-C1及p-Mis09-1比较,转染p-Mat01-1能够特异性抑制MCF-7细胞c-myc mRNA(24h:P<0.01)及蛋白(5d:P<0.01)表达,显著降低MCF-7细胞增殖能力(3d:P<0.05,5、7d:P<0.01)。结论采用siRNA表达质粒技术能够有效抑制MCF-7乳腺癌细胞c-myc/c-Myc表达,初步证实下调c-myc/c-Myc表达能够抑制MCF-7细胞增殖,为RNA干扰技术在乳腺癌生物治疗中的应用打下一定实验基础。