萝卜中一未知dsRNA全长cDNA克隆及其序列

 采用双链RNA ( double-stranded RNA, dsRNA) 分析法, 发现杭州市近郊的部分萝卜植株及其子代含有约118 kbp的dsRNA; 在聚丙烯酰胺凝胶上可分离为大小接近的dsRNA1和dsRNA2。以dsRNA1为模板, 采用单引物扩增法获得其全长cDNA; 克隆测序确认为1 866 nt; 预测其最大开放阅读框ORF(Open Reading Frame) 为574个氨基酸, 与双分病毒属( Partitivirus) 中部分病毒的RdRp (RNA dependent RNA polymerase) 氨基酸序列具有一定的同源性。推测此dsRNA可能为萝卜黄边病毒( radish yellow edge virus, RYEV) 感染所致。     

Effect of RNA interference on HIF-2 in the renal cancer cell line 786-0

AIM: To study the effect of RNA interference on hypoxia-inducible factor-2 (HIF-2) in the renal cell cancer in vitro and in vivo. METHODS: HIF-2 RNAi was synthesized and inserted into RNA interference eukaryotic expression vector which was confirmed by sequencing. The vector was transfected into the renal cancer cell 786-0 and positive clone was selected by using G418. The HIF-2 expression was detected by RT-PCR and Western blotting method. The growth of cells was measured by MTT method. Nude mouse xenograft assays were also done. RESULTS: Compared with empty vector group and control group, the amounts of HIF-2 mRNA and protein expression were lower in the HIF-2 RNAi group, the difference was significant (P<0.01). No significant difference between empty vector group and control group was observed. The cell growth in the HIF-2 RNAi group become slower. Compared with control group, the growth of tumor was slower in the RNAi group in the nude mice (P<0.01). CONCLUSION: HIF-2 RNAi inhibits the expression of 786-0 and cell growth, and slows the growth of tumor in the nude mice. The result provides new application for biological therapy in the renal cell cancer.     

Effects of lentivirus-mediated c-met RNAi on biological behaviors of human papillary thyroid cancer K1 cells

AIM: To evaluate the expression of c-Met in papillary thyroid cancer (PTC) by constructing lentiviral vectors for RNA interference (RNAi) of c-met gene and detecting its silencing effect on the biological behaviors of human papillary thyroid cancer cell line K1 cells. METHODS: Immunohistochemical assay was performed to detect the expression of c-Met protein in 35 cases of PTC and 25 cases of benign thyroid disease. Lentiviral vector for RNA of c-met gene was constructed and the silencing effect was detected by RT-PCR and Western blotting. The colony-forming ability, cell cycle, migration and invasion of K1 cells were measured by colony-forming assay, flow cytometry, wound-healing observation and Transwell experiment, respectively. In vivo tumorigenicity assay was performed to analyze in vivo proliferation of K1 cells in a xenograft model. RESULTS: The expression of c-Met in PTC was significantly higher than that in benign thyroid tissues. Lentiviral RNAi vectors targeting c-met gene were successfully constructed, and they efficiently inhibited the expression of c-met at mRNA and protein levels. Transfection of c-met lentiviral RNAi vectors inhibited the colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells. CONCLUSION: Lentivirus-mediated c-met RNAi efficiently inhibits colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells.     

Construction of a lentiviral vector for RNA interference of human EGFL7 gene and its stable expression in human laryngeal carcinoma cells

AIM：To investigate the role of epidermal growth factor-like domain 7 (EGFL7) in the pathogenesis and progress of laryngeal carcinoma via constructing a lentiviral expression vector for RNA interference (RNAi) of human EGFL7 gene and assessing the gene-silencing effect of the vector in human laryngeal epidermoid carcinoma (HEp-2) cells. METHODS：Specific RNAi target sequences were designed focused on human EGFL7 gene sequence. The double-stranded oligonucleotides were cloned into the pcDNA6.2-GW/EmGFP-miR plasmid after synthesis and annealing. A positive clone was subcloned into the pLenti6.3-MCS/V5-DEST vector after sequence analysis. The recombinant lentivirus was harvested from 293T cells co-transfected with the positive recombinant plasmid and lentiviral packing materials. HEp-2 cells were infected with the recombinant lentivirus and the cells with stable EGFL7 knockdown were screened by blasticidin selection. EGFL7 mRNA expression in the cells was determined by real-time fluorescence quantitative PCR. RESULTS：A recombinant lentiviral vector expressing short hairpin RNAs (shRNAs) against EGFL7 gene was obtained and confirmed by DNA sequencing. The virus titer was 5×1011 TU/L, and the silencing efficiency was 97%. CONCLUSION：A lentiviral vector targeting human EGFL7 gene, capable of stable EGFL7 gene knockdown in HEp-2 cells, has been successfully constructed, which provides a basis for further study of the relationship between human laryngeal carcinoma and EGFL7 protein.     

原核表达的PRSV HC-Pro 基因同源dsRNA 诱导番木瓜抗性的研究

 利用番木瓜环斑病毒（PRSV）HC-Pro 基因3′端824 bp 区段,通过OZ-LIC 法构建了含有PDK 内含子的发夹RNA 编码结构,并选用pSP73 和M-Jm109LacY 分别作为宿主载体和宿主菌,构建了高效的同源dsRNA 原核表达工程菌M-Jm109LacY/pSP73-RNAi-H824,经IPTG 诱导表达的dsRNA 不被DNase I 和RNase A 降解,稳定性较好。采用喷洒dsRNA 粗制品的方式对番木瓜植株进行保护性处理和治疗性处理,症状观察及ELISA、Real-time RT-PCR 分析结果表明,保护性处理能有效诱发对番木瓜环斑病毒的抗性（发病率低,发病时间晚）;治疗性处理（植株已接种PRSV 25 d）能在处理初期引起番木瓜环斑病毒积累量发生短暂降低。     

番茄生长素响应因子基因SlARF12在果实发育过程中的功能分析

以番茄‘Micro-Tom’为材料,研究了生长素响应因子基因SlARF12 （序列号：HM565127.1）在果实发育过程的生物学功能。实时定量PCR 检测表明,SlARF12在花蕾中表达量逐渐降低,而在授粉后的子房中显著高于去雄后未授粉的子房。利用RNA干扰（RNAi）技术抑制SlARF12表达,对番茄植株营养生长与花的发育没有显著影响,但SlARF12 RNAi果实显著大于野生型与空载转化植株的果实,且开花前去雄未授粉不能形成单性结实的果实。利用半薄切片观察转基因番茄果实早期发育细胞学特性发现,转化植株的果实果皮细胞显著大于对照果实细胞,但是两者果皮细胞层数没有显著差异。基因表达分析发现在SlARF12 RNAi的子房与幼果中细胞分化相关基因CycB1.1和CDKB2.1等的表达水平同对照相比下降,而SlPEC等细胞膨大基因表达量显著高于对照。所以抑制SlARF12可增强果实中细胞膨大相关基因的表达,从而促进果实膨大。以上结果表明,SlARF12可负调控番茄果实膨大但不参与坐果启动调控过程,显示了生长素通过ARF信号精细调控果实发育的各个阶段。     

Effect of N-cadherin knock-down on tumor formation of EC9706 cells in nude mice

AIM: To explore the effect of N-cadherin knock-down on the biological behavior of EC9706 cells in vivo.METHODS: The control vector pEGFP-MSCVneo and recombinant retroviral vector pMSCVneo/N-cadherin plasmids were transfected into esophageal squamous cell carcinoma(ESCC) cell line EC9706 according to the manufacturer's instructions. Stable EC9706 cell clones were selected using selection medium containing G418. Untreated EC9706 cells, control vector-transfected EC9706 cells and N-cadherin RNAi-transfected EC9706 cells were inoculated subcutaneously into the right flank of BALB/c mice (5 for each group), respectively. When tumors became palpable, the diameters of the tumors were measured with a caliper each week after subcutaneous implantation, and the volume (mm³) and weight (g) of the tumors were also calculated. Immunohistochemistry and Western blotting were employed to examine the expression levels of E-cadherin, N-cadherin and MMP-9 in the tumor tissues. The cell apoptosis was analyzed by TUNEL method.RESULTS: Compared with untreated group and control vector group, there was an obvious decrease in the volumes and weights of the tumors in N-cadherin RNAi group (P<0.05). No difference of E-cadherin expression in the 3 groups was observed. However, the expression of N-cadherin and MMP-9 in N-cadherin RNAi group was apparently reduced, and the positive number of cell apoptosis was obviously increased in N-cadherin RNAi group (106.81±6.47) as compared with that in untreated group (51.55±4.68) and control vector group (54.17±5.26). CONCLUSION: N-cadherin knock-down inhibits the tumor formation of EC9706 cells in nude mice by decreasing MMP-9 expression, resulting in less degradation of ECM and less aggression of the cancer cells. N-cadherin is an important factor in the progression and metastasis of ESCC,and may serve as a potential molecular target for biotherapy of ESCC.     

百合无症病毒CP基因的克隆及其RNA i载体的构建

 以感染百合无症病毒(LSV) 的百合叶片总RNA为模板, 利用反转录聚合酶链式反应(RT2PCR) 扩增出了876 bp的LSV CP基因, 经Blast比对发现该片段与GenBank上发表的LSV CP基因序列高度同源, 同源率98% , 由该序列推导出的氨基酸序列相似性达到98%。应用Gateway技术将扩增的片段通过BP反应连接到入门载体pDONR201, 并进行序列测定, 再通过LR反应将目的片段插入到RNAi植物表达载体pH7GW IWG2 ( Ⅱ) , 成功构建了适合农杆菌介导的百合转化的RNAi载体。     

Effects of BCL-3 gene silencing on the proliferation and drug sensitivity of human colorectal cancer cell line RKO with high expression of BCL-3

AIM：To construct lentiviral vectors for RNA interference (RNAi) of BCL-3 gene, and to detect the changes of biological behaviors and drug sensitivity of colorectal cancer cells after BCL-3 gene silencing. METHODS：The expression of BCL-3 in five human colorectal cancer cell lines was detected by RT-PCR and Western blotting. Lentiviral vectors for RNAi of BCL-3 gene were constructed and transfected into the human colorectal cancer cell line with high expression of BCL-3, and then the silencing effect was detected by Western blotting. After BCL-3 gene silencing, the change of cell proliferation was detected by MTT assay and soft agar colony formation assay, and the change of drug sensitivity was detected by MTT assay. RESULTS：BCL-3 was highly expressed in human colorectal cancer cell line RKO. Lentiviral vectors for RNAi of BCL-3 gene were successfully constructed, and Western blotting showed that BCL-3-shRNA2 could efficiently inhibit the expression of BCL-3 protein in RKO cells. After BCL-3 gene silencing, the proliferation ability and colony formation rate of RKO cells were decreased, and the median inhibitory concentration of oxaliplatin for RKO cells also decreased significantly. CONCLUSION: Inhibition of BCL-3 gene expression decreases the proliferation ability of human colorectal cell line RKO with high expression of BCL-3, and enhances the sensitivity of RKO cells to oxaliplatin.     

Effect of RNA interference on Polo-like kinase-1 in A549 cells

AIM: To investigate whether RNA interference(RNAi) induced by small interference RNA(siRNA) could suppress Polo-like kinase-1 (Plk 1) expression and its effects in A549 cells.METHODS: A recombinant plasmid containing siRNA targeting Plk1 (psiRNA-hH1-Plk1) was transfected into A549 cells with Lipofectamine 2000.Expressions of Plk1,cyclin B1 and p53 protein were detected by Western blotting.Cell proliferation was evaluated by direct cell counting,while cell cycle and apoptosis were examined by flow cytometry,and expression of α-tubulin was detected by immunofluorescence.RESULTS: The results demonstrated that sequence specific siRNA targeting Plk1 was capable of suppressing Plk1 expression,and reflecting in lower kinase activity in A549 cells.The level of Plk1 protein was reduced by at least 70% after 48 h of psiRNA-hH1-Plk1 treatment relative to controls.Expressions of cyclin B1 and p53 were increased greatly after Plk1 depletion,and cells showed absence of microtubule polymerization and spindle abnormalities in staining for α-tubulin.Growth inhibition,G₂/M arrest and apoptosis were observed in psiRNA-hH1-Plk1 transfected group.CONCLUSION: All these data suggest that siRNA targeted against human Plk1 may be a valuable tool in cancer therapy.     

Effects of RNA interference of FABP5 on subcutaneous tumor of human hepatocellular carcinoma cell line HepG 2 transplanted in nude mice

AIM: To investigate the effect of recombinant lentiviral vector for RNA interference (RNAi) on the expression of fatty acid-binding protein 5 (FABP5) gene in hepatocellular carcinoma HepG2 cells and tumor formation in nude mice.METHODS: RNAi lentiviral vector was used in the experiment. Human hepatocellular carcinoma HepG2 cells were divided into 3 groups:the HepG2 cells in experimental group were transfected with the recombinant lentivirirus vector LV-shRNA-FABP5, the cells in negative control group were transfected with a control lentiviral vector LV-shRNA-NC, and the cells in normal control group were without any treatment. The nude mice were randomly divided into 3 groups. The growth of the transplanted tumor cells in the nude mice was observed. The tumor growth curve, volume and weight were determined 4 weeks after the cell inoculation. The expression of FABP5 was detected by real-time PCR, Western blot and immunohistochemical staining.RESULTS: Transfection of the lentiviral vector FABP5-shRNA obviously reduced FABP5 expression in the HepG2 cells. Tumor formation was all positive in the 3 groups of the nude mice inoculated with the tumor cells. Compared with normal control group and negative control group, the tumor growth slowed significantly in experimental group with smaller volume and weight. FABP5 expression in the transplanted tumor tissues was significantly down-regulated at mRNA and protein levels in experimental group as compared with normal control group and negative control group.CONCLUSION: RNAi-induced down-regulation of FABP5 effectively inhibits the growth of transplanted hepatocellular carcinoma, suggesting that FABP5 gene may be an effective target for gene therapy in treating liver cancer.     

Grafting and RNA transport via phloem tissue in horticultural plants

Grafting is a cultivation method that exploits a cooperative relationship between partner plants possessing different genomes. It is most commonly used for the propagation and cultivation of trees, shrubs, and fruit vegetables. In addition, as represented by florigen (flowering hormone) experiments, grafting has been utilized in the field of plant physiology to clarify the mechanism of long-distance transport by which signals arising in organs that perceive an environmental change are transmitted to response organs. Recent analytical technology has revealed that some specific RNA molecules are also transported through phloem tissue as genetic information to execute coordinated organ growth and development. Therefore, it is anticipated that the RNA transport system could be applied for the improvement of cultivars of various horticultural crops, if the mechanism were controllable by artificial means.     

日本梨活体花柱内花粉RNA的降解

 用³² P标记的花粉进行自花与异花授粉后, 提取含有花粉³² P-RNA的授粉花柱RNA测定放射性活度, 并将授粉花柱RNA样品进行非变性琼脂糖电泳。结果表明, 授粉后12～48 h, 自花授粉花柱RNA 中花粉³² P-RNA只占异花授粉花柱RNA中的40.7%～25.3% , 自花授粉花柱中花粉完整RNA的量特异性地减少, 表明自花花粉RNA在花柱中被特异性地降解。     

萝卜中一种新dsRNA的全长cDNA克隆及序列分析

 从一点红萝卜品种（Raphanus sativus-root cv. Yidianhong）叶片中提取dsRNA,应用SPAT方法对各dsRNA条带进行cDNA克隆并测序,除获得先前报道的5条序列（RasR1-RasR5）以外,还得到一条新的全长序列,将其定名为RasR 6（EU285027）。序列分析结果表明：RasR 6全长为1 778 bp,其正义链编码1个由502个氨基酸组成、分子量约为55.1 kDa的蛋白质。该序列与前人报道的双分病毒科5个病毒序列具有相似性,且它们均编码双分病毒科病毒的外壳蛋白(CP,Capsid protein)。核苷酸序列比对结果显示：RasR 6与同时来源于萝卜的RasR 1和RasR 2的5'UTR序列高度同源,且其3'末端具有poly A结构,而与RasR 3、RasR 4和RasR 5的UTR则没有明显的相似性。因此,我们推测RasR 6与RasR 1、RasR 2同属于双分病毒RasV 1（Raphanus sativus virus 1）,它可能与RasR 2共同编码该病毒的CP或作为RasV 1的卫星RNA存在。     

RNAi沉默BcMF3基因对菜薹花粉发育的影响

根据从白菜(Brassica campestris ssp. chinensis var. communis) 核雄性不育两用系中分离到编码果胶甲酯酶( PME) 的雄性不育相关基因BcMF3 cDNA序列设计引物, 从白菜花蕾cDNA中扩增出一短一长两个片段, 分别反向和正向连接至双元载体pB I12 l中, 得到了RNAi (RNA interference) 植物表达载体pB I2B3R, 并导入农杆菌LBA4404菌株中; 通过组织培养途径转化菜薹(B. campestris ssp. chinensis var.parachinensis) , 分子检测证明B cM F3片段单拷贝整合到转基因菜薹的基因组中; 50%转基因菜薹植株的花粉畸形, 花粉离体萌发率为32。3% , 出现畸形花粉植株的花药PME活性降低了13。5%。这一结果从转基因植株后代的表型和酶活性上证明, 采用RNAi技术沉默BcMF3基因, 可能通过影响PME活性而引起转基因菜薹植株部分花粉的败育, 从而证明BcMF3基因在普通白菜和菜薹等植物的花粉发育中起着重要作用。     

RNA interference inhibits the secretion of IL-1α in mice spleen lymphocytes

AIM:To decide the effect that selected siRNA degrades mRNA of IL-1α specifically and suppression of its expression after connected with target site with homology complementary sequence. METHODS:Synthesized DNA expression box aimed directly at target site through PCR reaction in vivo was purified, and transfected into lymphocytes stimulated by LPS. siRNA was transcribed by cellular endogenous RNA polymerase Ⅲ and then evoke the degradation of target mRNA. After 48 hours of transfection, the cell culture supernatant was collected and the concentration of IL-1α was assayed using ELISA. RESULTS:Compared with blank-control and negative- control, selected sequence decreased the expression of IL-1α. Rate of the suppression was about 15%. CONCLUSION:RNAi technology produces specific interference effect in mouse spleen lymphocytes in original culture and inhibits the excretion of IL-1α.     

Effects of MAGEA3 inhibition by shRNA on apoptosis in human hepatocellular cancer cells

AIM:To investigate the inhibitory effect of vector-based RNA interference ( RNAi) on the expression of melanoma associated antigen A3 (MAGEA3) protein in hepatocellular carcinoma cells and on apotposis of hepatocellular carcinoma cells. METHODS:A vector for transcribing specific small hairpin RNA ( shRNA) targeting MAGEA3 gene was constructed ,introduced into hepatocellular carcinoma MEL-ED1 cells by Lipofectamine 2000. The MAGEA3 protein and mRNA expression levels of MEL-ED1 cells were detected by Western blotting and RT-PCR, respectively. The cell apoptosis was studied by DNA fragmentation, electron microscopy ，TUNEL assay, and annexin V/PI staining. RESULTS:The vector of RNA interference was successfully constructed and MAGEA3 expression was descreased significantly in MEL-ED1 cells. After the shRNA expression vector was transfected into the MEL-ED1 cells, the expression of MAGEA3 gene was inhibited significantly ( by 90% ). DNA fragmentation，electron microscopy and TUNEL assay showed classic apoptosis characters in the MEL-ED1 cells transfected with pSilencer-MAGEA3 plasmid with an apoptosis rate of 21.41% ±1.98%, significantly higher than those in the negative control group transfected with pSilencer-neo and in the non-transfected group (both P<0.01). CONCLUSION:The specific small hairpin RNA targeting MAGEA3 mRNA can inhibit the expression of MAGEA3 and cause apoptosis of hepatocellular carcinoma cells , which suggests inhibitory effect of MAGEA3 on apoptosis in cancer and provides an experimental basis for treating human tumors with RNAi.