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Objective:To treat mice with Alzheimer's disease （AD） with β-catenin RNA interference （RNAi） Huangjingwan （HW）， so as to explore the neuroprotective signal mechanism of its prevention and treatment of AD. Method:A total of 81 male Kunming mice were randomly divided into normal control group， sham model control group， AD model group， Donepezil group， HW+scrambled group， HW+RNAi group， HW group， with 8 mice in each of donepezil group and HW group， and 13 mice in each of other groups. The AD models were established through injection with D-galactose and scopolamine in the last 5 groups for 5 consecutive weeks. On the 1st day of the 4th week after modeling， 0.75 μL PEI-LMW/β-catenin siRNAs nano-complex was injected into the right lateral ventricle of each mouse in for one time to treat with β-catenin RNAi in mice brains of the HW+RNAi group. The 0.75 μL complex was injected into the right lateral ventricle of each mouse for one time as for β-catenin interference control of the HW+scrambled group. The 0.75 μL normal saline was injected into the right lateral ventricle of each mouse in one time of the sham control group. Two weeks after intracerebroventricular injection， β-catenin RNAi was confirmed to be successful， and Donepezil （6.5×10-4 g·kg-1） was intragastrically administered to each mouse of donepezil group. HW （2.5 g·kg-1） was intragastrically administered to each mouse of HW group， HW+RNAi group and HW+scrambled group. Normal saline （0.5 mL·d-1） was intragastrically administered to each mouse of the sham control group. All gastric perfusion lasted for 4 weeks. At the end of gavage， the difference in learning and memory ability of mice was evaluated by platform jumping test. Nissl staining was used to count the number of neurons in s1Tr area of cerebral cortex and CA1 and CA3 areas of hippocampus of each mouse in each group. The mRNA expressions of Wnt1， DVL2， GSK-3β， β-catenin and CyclinD1 in mice brain of each group were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Western blot was used to detect the expressions of Wnt1， DVL2， GSK-3β， β-catenin and CyclinD1 in mice brain of each group. Result:The expression of β-catenin could be significantly inhibited through the injection with PEI-LMW/β-catenin siRNAs nano-complex into the lateral ventricle of AD mice， and nearly no β-catenin expression could be detected， which successfully achieved gene silencing. Compared with the normal control group， mice in AD model group showed that the learning and memory performance decreased significantly， the number of jumping errors increased （P<0.01）， the number of neurons in S1Tr area of cerebral cortex and CA1， CA3 areas of hippocampus decreased significantly （P<0.01）， the mRNA and protein expressions of Wnt1， DVL2， β-catenin， CyclinD1 in brain decreased significantly （P<0.01）， while the mRNA and protein expressions of GSK-3β increased significantly （P<0.01）. Compared with the AD model group， mice in HW group showed that the learning and memory performance increased significantly， the number of jumping errors decreased， the number of neurons in S1Tr area of cerebral cortex and CA1， CA3 areas of hippocampus increased significantly， the mRNA and protein expressions of Wnt1， DVL2， β-catenin， CyclinD1 in brain increased significantly， while the mRNA and protein expression of GSK-3β decreased significantly （P<0.01）. Compared with the HW group， mice in HW+RNAi group showed that the learning and memory performance decreased significantly， the number of jumping errors increased significantly （P<0.01）， the number of neurons in S1Tr area of cerebral cortex and CA1， CA3 areas of hippocampus decreased significantly （P<0.01）， there was no significant change in mRNA and protein expressions of Wnt1， DVL2， GSK-3β in the brain， and the mRNA and protein expressions of β-catenin， CyclinD1 decreased significantly （P<0.01）. Conclusion:HW can treat and prevent AD by activating Wnt/β-catenin signal pathway.
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Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism whereintranscripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present in the cell will besubjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance andcross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might haveevolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA)molecules of 21–23 nucleotides length act as homology guides for triggering the systemic degradation of transcriptshomologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouringchalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has beenachieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying cosuppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of genesilencing has led to the development of several alternative strategies for inducing gene silencing in a precise andcontrolled way. This has paved the way for using PTGS as one ofthe chief functional genomicstools in plants and hashelped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides,opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencingthe deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in anew era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined thebasics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, ascase studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenicmale sterile lines developed based on orfH522 gene from sunflower PET1-CMS source, (b) as a tool to suppress theproduction of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistancein sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify theconcept and utility of gene silencing in crop plants.
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The delta-12 fatty acid desaturase (Δ¹² FAD or FAD2) is a key enzyme that catalyzes oleic acid to linoleic acid by dehydrogenation at Δ¹² position of fatty acid carbon chain. In peanut, reduction or loss of FAD2 activity could enhance the relative content of oleic acid in kernels, and improve the quality and oxidation stability of peanut kernels and products. RNA interference (RNAi) technology could lead to non-expression or down-regulated expression of AhFAD2 gene. We constructed two RNA interference expression vectors with the inverted repeat sequence of partial AhFAD2 gene, which were driven separately by cauliflower mosaic virus (CaMV) 35S promoter or soybean agglutinin lectin seed-specific promoter. Homozygous transgenic lines carrying the two constructs stably in genetics were developed by peanut genetic transformation. There were no significant differences between the transgenic lines and the control through investigating the main agronomic traits. We analyzed the transcriptional level expression of AhFAD2 gene in transgenic lines and the control by real-time fluorescence quantitative PCR (qRT-PCR). The results suggested that the target genes of transgenic lines were likely suppressed by RNA interference, but showed different transcriptional levels in different peanut transgenic lines. Compared with untransformed lines, the resulting down-regulation of AhFAD2 gene resulted in a 15.09% or 36.40% increase in oleic acid content in the seeds of transformed HY23 and FH1 lines respectively, and the content of linoleic acid decreased by 16.19% or 29.81%, correspondingly, the ratio of oleic acid and linoleic acid (O/L) improved by 38.02%, 98.10%. The oleic acid content had significant differences between the two transformation constructs, and also among different transgenic lines. Moreover, the inhibition effect of RNAi was more obvious in the transgenic lines with FH1 as the receptor, and with transformation structure driven by seed specific promoter. The suppressed expression of AhFAD2 gene enabled the development of peanut fatty acid, which indicated that RNA interference would be a reliable technique for the genetic modification of peanut seed quality and the potential for improvement of other traits as well.
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OBJECTIVE To explore the role of gecko crude peptides (GCPs) in the proliferation, apoptosis, migration and lymphangiogenesis of human hepatocellular carcinoma cells (HepG2) and human lymphaticendothelial cells (HLECs) in vitro. METHODS The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the anti- proliferative effect of GCPs and siRNA-VEGF-C on HepG2 cells, Hoechst 33258 staining and flow cytometry were performed to analyze cycle and apoptosis. The migration and invasion ability of cells were assayed by transwell chamber experiment and wound-healing assay. The protein and mRNA expressions of vascular endo?thelial growth factor-C (VEGF-C) and CXC chemokine receptor-4 (CXCR4) were detected by q-PCR, immunofluorescence, Western blot. The protein expressions of the extracellular signal regulated kinase (ERKI/2), c-Jun N-terminal kinase (JNK), p38-mitogen activated protein kinases (p38 MAPK), serine/threonine kinase (Akt) and phosphatidylinositol- 3- kinase (PI3K) were detected by western blot. The anti-lymphangiogenesis effect of GCPs on the HLECs was analyzed using an in vitro tube-formation assay. The protein and mRNA expressions of vascular endothelial growth factor receptor-3 (VEGFR-3) and stromal cell-derived factor-1 (SDF-1) were detected by q-PCR, Western blot. RESULTS GCPs and siRNA-VEGF-C inhibited HepG2 proliferation, invasion and migration, and the most obvious inhibitory effect was both synergistic effects. Thus, GCPs suppressed HLECs proliferation, migration and tube-like structure formationin a dose- dependent manner, and had inhibitory effect of tumor- induced lymphangiogenesis in vitro. Additionally, we found that GCPs and siRNA- VEGF- C decreased the expressions of MMP-2, MMP-9, VEGF-C, CXCR4, phospho-ERK1/2, phospho-P38, phospho-JNK and PI3K in HepG2 cells. Moreover, GCPs had a dose-dependent depressive effecton the expressions of VEGFR- 3, SDF- 1 in HLECs. CONCLUSION The low expression of VEGF- C mediated by siRNA-VEGF-C and GCPs inhibit tumor proliferation, invasion and migrationby suppressing the MAPK signaling pathway through reduced levels of VEGF-C, and GCPs inhibit tumor lymphangiogenesis by suppressing the CXCR4/SDF-1 signaling pathway through suppressed VEGF-C/VEGFR-3.
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Objective To study the structural features and characteristics of a novel gene Schistosoma japonicum 79(Sj79), and observe its effect of RNA interference(RNAi),so as to provide the experimental basis for its further function study and mechanism study of anti reproductive development of schistosome. Methods The gene structure and characteristics of Sj79 were analyzed by bioinformatics methods. Then the expressions of Sj79 messenger RNA(mRNA)during the different develop?mental stages of schistosome were analyzed and the effects of RNAi silencing were observed by the soaking method. The tran?scriptional levels of Sj79 after RNAi were detected by real time PCR. Results The open reading frame of Sj79 contained 696 base pairs with an exon structure. The gene had obvious stage specificity,and its transcriptional level in mature female worms was the highest. After soaking for 3 d,the Sj79 mRNA level[(41.0 ± 12.3)%]in the siRNA?1 group with low dosage(20 nmol/L) was lower than that in the siRNA?NC group[(103.2 ± 14.4)%],the difference was statistically significant(t=3.28,P<0.05). When with high dosage(200 nmol/L ),both the Sj79 mRNA levels in the siRNA?1 group[(15.8 ± 10.9)%]and siRNA?2 group [(11.1 ± 8.8)%]were significantly lower than that in the siRNA?NC group[(100.1 ± 6.3)%](t=13.44,27.84,both P<0.01). After soaking for 7 d,only the Sj79 mRNA levels in the siRNA?1group[(43.4 ± 4.5)%]and siRNA?2 group[(62.5 ± 5.4)%]with low dosage were lower than that in the siRNA?NC group[(100.4 ± 5.2)%],and the differences had statistical sig?nificance(t=8.33,5.07,both P<0.01). Conclusion Through this study,we have improved the mRNA sequence and genom?ic information of Sj79 gene,and understood its structural features,as well as selected out two effect fragments siRNA?1and siR? NA?2 which will provide the basic evidences for the further study on egg laying interference of the female adult worm of schisto?some in vitro.
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Objective To construct the lentiviral vector of RNA interference(RNAi) for DEK,and to detect its effect on breast cancer cell growth.Methods The DEK siRNA was designed and constructed based on DEK sequence using a lentiviral vector.The lentivirul vector containing DEK siRNA was named PSIH-H1-DEK as confirmed by PCR and sequenceing.PSIH-H1-DEK was then packaged with accessory plasmids into lentivirus in 293T cells and selected for 2 weeks with puromycin ( puro ) before the mixed colonies stably expressing DEK siRNA were obtained and the DEK expression was detected by real time PCR( RT-PCR) and Western blotting.The effect of DEK siRNA on ZR75-1 cell growth was determined by cell counting kit.Results Western blot and RT-PCR showed that PSIH-H1-DEK siRNA could suppress DEK gene expression.Suppression of DEK could markedly inhibit the growth of ZR75-1 cells.Conclusion The lentivirus-mediated DEK siRNA is obtained,which will facilitate further research on DEK function in breast cancer development.
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[Abstratc] Objective The function of cIAP1 in the progression of ovarian cancer has not been clarified . This study is to explore the involvement of cIAP 1 in regulating biological behaviors of ovarian cancer cells by u-sing RNA interference(RNAi)technology.Mte hods The short hairpin RNA plasmid targeting cIAP1 was con-structed and transfected into Skov 3 cells.The levels of cIAP1 mRNA and protein were investigated by RT -PCR and Western Blot respectively .MTT assay and flow cytometry were used to evaluate cell proliferation and apopto-sis.R esults The rate of cIAP1 transfection was 74.7%performed by flow cytometric analysis .cIAP1 expression was significantly down -regulated at both mRNA and protein levels ,which resulted in a decrease of cell prolifera-tion and invasion capability in vitro .Conclusion This study implies that cIAP 1 might play an important role in the progression of ovarian cancer ,and it could be a potential target for therapeutic anti -cancer drugs .
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Objective To explore the effects of recombinant plasmids of pGPU6/GFP/NeoshRNA-CTGF (shRNA-CTGF) on the type Ⅰ collagen (COL-Ⅰ) protein expression in keloid,through RNA interference on connective tissue growth factor (CTGF) in vivo and in vitro.Methods Recombinant plasmids were designed and constructed by specific shRNA-CTGF; After transfeced human keloid fibroblast with shRNA-CTGF in vitro,RT-PCR was used to detect the CTGF mRNA level,and Western blot to detect the secretion of COL-Ⅰ.After transfected the keloid of nude mice with shRNA-CTGF in vivo,RT-PCR was used to detect the CTGF and COL-Ⅰ mRNA level,and Western blot was used to detect the protein expression of COL-Ⅰ.Results Recombinant plasmids of CTGF were successfully constructed,and the CTGF gene expression was significantly decreased in vivo and in vitro by 86.8％ and 54.1 ％,respectively; Down-regulation of CTGF in vitro significantly inhibited the mRNA and protein level of COL-Ⅰ by 76.8％ and 65.6％,respectively; Down-regulation of CTGF in vivo significantly reduced the COL-Ⅰ mRNA and protein level by 52.7％ and 48.0％,respectively.Conclusions CTGF gene expression is successfully down-regulated by the recombinant plasmid of shRNA-CTGF in vivo and in vitro.shRNA-CTGF significantly reduces the COL-Ⅰ protein level in keloid.It implies that CTGF gene is a potential target in the therapy of pathological scar.
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The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined.The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK).The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay.GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells,no GCRV-specific siRNA could be detected.Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene.These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway,unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.
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Objective To prove the remarkable inhibitive effect of multiple siRNAs targeting a proliferation-inducing ligand (APRIL) on the human colorectal cancer cell.Methods We constructed a multiple short hairpin RNA(shRNA) expression vector containing four shRNAs (pG4) as well as four single one (pGsh644,pGsh1451,pGsh1938,pGsh2231) against APRIL gene in SW480 cell,and then transfected them into the human colorectal cancer cell line by cationic liposome.Ultimately,SW480 were screened by EGFP to obtain expression cell lines.APRIL expression levels including mRNA and APRIL protein were detected after transfected with all different kinds of vectors.Results A multiple shRNA expression vector containing four shRNAs (pG4) and four single ones were successfully constructed.Four single vectors (pGsh644,pGsh1451,pGsh1938,pGsh2231) and the multiple siRNAs expression vector (pG4) all decreased the APRIL mRNA by 56.2％,49.5％ ;50.9％,49.2％ and 79.3％.And APRIL protein expression was also remarkably reduced,especially by multiple siRNAs expression vector(87.5％).Conclusion Multiple siRNAs expression vector produced a more significant knockdown effect of APRIL than the vectors containing only one APRIL shRNA.What we found suggested us using the vector containing multiple shRNA to silence the expression of APRIL might be exploited as a novel therapeutic strategy for tumors.
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To counteract the immune system in parasitic hosts, some viruses encode proteins to suppress the RNA interference (RNAi) effect. In this report, we established two RNAi systems to be easily observed with strong and obvious effect. The function of the P19 of tomato bushy stunt virus, which suppresses RNAi in mammal cells, was then studied using these two systems. Short hairpin RNAs targeting green fluorescence protein (pshRNA-GFP) and firefly luciferase (pshRNA-luc) were designed and inserted into a eukaryotic transcriptional vector pTZU6+1, respectively. The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19. The GFP expression level was assayed by fluorescence microscopy, Western blotting and RT-PCR. The luciferase expression level was analyzed by the dual-luciferase assay system. pshRNA designed in this study down-regulated the target gene specifically and efficiently, with a decrease of expression of both genes of about 70%, respectively. When P19 was introduced into the RNAi systems, the expression of both GFP and the luciferase were mostly recovered compared with the control groups. The RNAi systems of GFP and luciferase were constructed successfully, demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.
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Objective To investigate the effects of siRNAs targeted protein kinase C ?(PKC?) on the proliferation and apoptosis of A549 cell line.Methods Six PKC? siRNAs were designed and chemical synthesized. Candidate PKC? siRNA was transfected into A549 cells,PKC? mRNA level was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Then,the effects of more effective PKC? siRNAs on A549 were further investigated by using clone formation assay,Hoechst 33258 staining,propidium iodide (PI) staining,Annexin V-FITC and PI dual staining and western blot. Results Six candidate PKC? siRNAs were designed and named as No.1,No.2,No.3,No.4,No.5 and No.6 PKC? siRNA. Compared with controls,PKC? mRNA levels were all significantly downregulated and the cell proliferation was inhibited in A549 cells treated with candidate PKC? siRNAs except No.5 (P
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@#ObjectiveTo observe the growth of BT-325 human glioma cells after interfering volume-regulated chloride channel ClC-2 gene.MethodsTwo expression recombinant vectors of ClC-2 gene were designed and constructed. The primary plasmid, pSUPER.puro-shRNA, and the two recombinant plasmids, pSUPER.puro-shRNA-ClC-21 and pSUPER.puro-shRNA-ClC-22, were transfected into BT-325 cells by LipofectamineTM2000 (Groups: control, PP1 and PP2, respectively). The mRNA expression of ClC-2 gene was detected with reverse transcription polymerasse chain reaction (RT-PCR), the cellular survival was determined with MTT assay, and the cell cycle was measured with flow cytometry (FCM). ResultsClC-2 mRNA expression and the growth of the cells in PP1 and PP2 groups were significantly lower than that of control group. The cell cycle progression was blocked in G1 phase (PP1 and PP2 vs control,P<0.01). ConclusionThe growth of BT-325 human glioma cells is prevented by knockdown of ClC-2 gene expression, which may be one of the novel targets to inhibit growth of human malignant glioma cells.
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RNA interference(RNAi) is an high efficient,specific gene silencing.The discovery process of RNAi and its mechanism was reviewed.In the further,RNAi will play important roles in characterizing new genes in the metabolic pathways,improving plant nutritional value,enhancing plant resistance and creating new varieties.
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RNA interference (RNAi) is the sequence-specific gene silencing induced by double-stranded RNA (dsRNA). Being a highly specific and efficient knockdown technique, RNAi not only provides a powerful tool for functional genomics but also holds a promise for gene therapy. The key player in RNAi is small RNA (~22-nt) termed siRNA. Small RNAs are involved not only in RNAi but also in basic cellular processes, such as developmental control and heterochromatin formation. The interesting biology as well as the remarkable technical value has been drawing widespread attention to this exciting new field.
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Animals , Humans , Genetic Therapy/methods , Genomics , RNA InterferenceABSTRACT
Objective: To investigate the inhibitory effect of adenovirus-mediated VEGF-siRNA on transplanted osteo- sarcoma in nude mice.Methods: VEGF-siRNA gene was cloned into the genome of replication-deficient adenovirus to construct Ad-VEGF-siRNA;the latter was then used to infect osteosarcoma MG63 cell line in vitro;and the expression of VEGF gene was detected by RT-PCR.Osteosarcoma transplantation model was established in nude mice;VEGF expression in tumor tissue was analyzed and the inhibitory effect on tumor growth and lung metastasis were also observed.Results: The recombinant adenovirus vector Ad-VEGF-siRNA was successfully constructed.In vivo and in vitro experiment both showed that Ad-VEGF-siRNA significantly downregulated VEGF expression in MG63 cells and transplanted tumor tissue. It was found that Ad-VEGF-siRNA significantly inhibited transplanted osteosarcoma growth(P