ABSTRACT
Objective To investigate the effect of silencing Notch1 gene by RNA interference on the proliferation,apoptosis and Akt/mTOR signaling pathway in clear renal cell carcinoma.Methods The optimal segment targeting Notch1 gene was designed and transfected into 786-O cells by Lipofectamine TM2000.The Notch1 mRNA and protein were detected by RT-PCR and Western blot.The proliferation rate of 786-O cells was evaluated by MTT and the variation of apoptosis was measured by TUNEL.The protein expression level of apoptosis-related protein Bcl-2,caspase-3,caspase-9,and signaling pathway protein Akt,p-Akt,p-mTOR,p-P70S6K were detected by Western blott.Results Notchl mRNA and protein was markedly suppressed by the siRNA targeting Notch1.Treated with 0,40,60,80,100 and 120 nmol/L of Notch1 siRNA for 24 hours,cell proliferation rates were (98.51 ± 1.33) ％,(87.34 ± 2.26) ％,(64.72 ± 3.24)％,(57.68 ±3.32)％,(31.91 ± 1.85)％ and (19.27 ±2.73)％,and the difference was statistically significant (P ＜ 0.01).Treated with 0,40,80,and 120 nmol/L of Notchl siRNA for 24 hours,apoptosis rates were (7.6 ± 3.8) ％,(21.5 ± 4.8) ％,(32.3 ± 3.5) ％,and (46.3 ± 4.7％),the difference was statistically significant (P ＜ 0.05).Decreased expression of Akt signaling pathway proteins p-Akt,p-mTOR,p-70S6K and apoptosis-related protein Bcl-2,procaspase-3 was detected,but no change in the total protein of Akt.Conclusions Depletion of Notch1 gene could inhibit cell growth and induce apoptosis in 786-O cell line.It inhibits Akt/mTOR signaling pathway by dephosphorylated.
ABSTRACT
ObjectiveTo investigate the effect of intrathecal injection of TRPV3-siRNA lentivirus on bone cancer pain(BCP) behaviors in rats.Methods40 female SD rats successfully received intrathecal catheter implantation and without motor dysfunction were randomly divided into 4 groups (n=10 in each group):Sham group (S),BCP group (B),negative control lentivirus group (C) and TRPV3-siRNA lentivirus group (T).Group B,C and T were induced bone cancer pain by intra-right-femur inoculation of Wallker 256 cells,while rats in group S were injected of inactivated cell.Rats in group T were intrathecally treated with 5 μl TRPV3-siRNA lentivirus while rats in group C received 5 μl negative lentivirus on 1~6 d after surgery.All the rats received pain behaviors including paw withdrawal thermal latency(PWTL) and paw withdrawal mechanical threshold (PWMT) at 1 d before BCP and 1,3,6,9,12,15,18 and 21 d after BCP.L4~L6 spinal cords were reserved for RT-PCR and Western Blot.ResultsCompared with group S,PWTL and PWMT of group B were decreased (P0.05).The results of RT-PCR and Western blot demonstrated that the expression of TPPV3 in group T was decreased compared with that in group C(P<0.05).ConclusionIntrathecal injection of TRPV3-siRNA lentivirus can inhibit the expression of TRPV3 and thus alleviate symptom of PWTL,but not PWMT.
ABSTRACT
Hydrogen sulphide is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in macrophages. To determine the role of H2S and macrophages in sepsis, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of sepsis. Cecal ligation puncture (CLP)-induced sepsis is characterized by increased levels of myeloperoxidase (MPO) activity, morphological changes in liver and pro-inflammatory cytokines and chemokines in the liver and lung. SiRNA treatment attenuated inflammation in the liver and lungs of mice following CLP-induced sepsis. Liver MPO activity increased in CLP-induced sepsis and treatment with siRNA significantly reduced this. Similarly, lung MPO activity increased following induction of sepsis with CLP while siRNA treatment significantly reduced MPO activity. Liver and lung cytokine and chemokine levels in CLP-induced sepsis reduced following treatment with siRNA. These findings show a crucial pro-inflammatory role for H2S synthesized by CSE in macrophages in sepsis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition.
ABSTRACT
Purpose To explore the effect of EMS1-siRNA on the growth,invasion and migration of human gastric cancer cell line MGC803.Methods It used the Colony formation assay to determine the abilities of proliferation,and flow cytometry analysis to asses cell cycle distribution and apoptosis,transwell invasion and migration experiment to determine the ability of cell invasion and migration after knockdown the expression of EMS1 in MGC803.Results These results suggested that EMS1 gene down-regulated have no affect on cell cycle and cell apoptosis,but the ability of colony formation depressed and migration lowerd obviously (P ＜ 0.05).Conclusion The results shows EMS1 gene is related to proliferation and migration of tumor.
ABSTRACT
AIM:To investigate the expression of Grb2-associated binding protein 2 (Gab2) in human osteo-sarcoma cells and its relationship with the invasion and metastases of human osteosarcoma cells.METHODS: The tech-nique of small RNA interference was used to transfect human osteosarcoma U2-OS cell lines.Western blotting and RT-PCR were used to detect the protein and mRNA expression of Gab2 in transfected U2-OS cells.After transfection, through chem-otaxis and invasion assays in vitro, the cell migration and invasion abilities were detected.RESULTS:After transfection, the expression of Gab2 at mRNA and protein levels in Gab2 siRNA transfected cells ( SiGab2/U2-OS) was lower than that in scrambled siRNA transfected cells ( Scr/U2-OS ) and U2-OS cells.After stimulation with epidermal growth factor ( EGF) at concentration of 10 μg/L, the migration SiGab2/U2-OS cells was significantly less than Scr/U2-OS cells and U2-OS cells ( P<0.01 ) .The number of invasion cells of SiGab2/U2-OS group was significantly lower than the other 2 control groups ( P<0.01) .CONCLUSION:Inhibition of Gab2 expression obviously attenuates the migration and invasion abilities of human osteosarcoma U2-OS cell line.
ABSTRACT
AlM: To silent hypoxia inducible factor-1α ( HlF-1α) gene in malignant melanoma of the choroid cell by small interference RNA ( siRNA ) and investigate its effect on the expression of matrix metalloproteinase-2 ( MMP-2 ) in the choroid cell line human uveal melanoma cell (OCM-1) in hypoxia environment.METHODS:OCM-1 cells cultured on culture flask were divided into normal group and hypoxia group. Hypoxia group were divided into five groups: simple hypoxic group, and interference group, and negative control group, and positive control group, and liposome group. Normal group cells were cultured on DMEM culture flask with 10% FBS, 100U/mL penicillin, 100μg/mL streptomycin as well as high concentration of glucose. The cells were maintained at 37℃ in a humidified 5% CO2 incubator. Cells in good condition were selected for experiment. For hypoxia group, chemical hypoxia inducer CoCl2 was added into nutrient medium at the concentration of 100μmol/L to simulate hypoxia microenvironment. We designed and synthesised siRNA ( siRNA + negative control+positive control ) , the target sequences of the HlF-1α to transfect hypoxic malignant melanoma of the choroid cell. SiRNA including HlF-1α siRNA, β-actin siRNA and negative control group synthesized in vitro transfected hypoxic OCM - 1 cell through Lipofectamine2000. The expression of HlF-1α, MMP-2 gene and the protein were detected by RT-PCR and Western blot. RESULTS: Compared with the normal group, the expression of HlF-1α mRNA was not obviously changed (P>0. 05), but the expression of HlF-1α protein and MMP- 2 mRNA protein was significantly higher ( P0. 05).CONCLUSlON: Hypoxia status may upregulate the HlF-1α in OCM-1 cells by increasing the expression of protein. Hypoxia can also inactivate MMP-2, resulting in upregulation of MMP-2 RNA and the expression of its protein. The expression of HlF-1α and MMP-2 mRNA can be down-upregulated by transfecting OCM-1 with HlF-1α siRNA.
ABSTRACT
To screen the siRNAs (small interference RNA sequences ) which specifically inhibit the gene expression of TLR2 in human monocyte-derived macrophage , and discuss their prospects on the treatment of HIV at the level of molecular immunology.Methods:We obtained the mRNA sequences of human TLR 2 gene from NCBI gene bank ,then designed three siRNAs by siDESIGNTM software.The siRNA targeting human housekeeping gene GAPDH was used as positive control.The fluorescent labeling missense siRNA sequences (NC-FAM ) was used as negative control.We collected fresh peripheral blood from healthy volunteers and isolated mononuclear cells from the blood samples.The human mononuclear macrophages were then purified from mononuclear cells by utilizing adherence method.Cationic liposome reagent Lipofectamine 2000 was used to mediate siRNAs into the human mononuclear macrophages.The levels of TLR2 mRNA expression of siRNA-transfected monocyte-derived macrophage were determined by q-PCR.Expression of TLR2 protein was determined by Western blot.Results: At 72 h after transfection ,we found that the expression of GAPDH mRNA and protein in positive control group decreased significantly.Also found there existed significant differences between each siRNA group (F=41.957,P<0.001).Compared with negative control ,the relative expression of TLR2 mRNA in all siRNAs groups decreased significantly (P<0.05 ) , and the inhibition rates were 46%, 43%, 43% by three miRNAs respectively.Western blot showed that the expression of TLR 2 protein in siRNAs groups decreased significantly compared with that of control (P<0.05 ).Conclusion: The designed siRNAs in this study could inhibit the expression of TLR 2 gene in human monocyte-derived macrophage ,indicating that mediation of TLR-2 expression by siRNA might be a novel strategy for HIV treatment from the per-spective of molecular immunology.
ABSTRACT
Objective To explore the effect of the gene silencing of phosphatidic acid-preferring phospholipase A1 (PA-PLA1) on insulin secretion in mouse insulin-secreting cell line MIN6. Methods The siRNA expression vector of mouse PA-PLA1 gene targeting was constructed using mouse PA-PLA1 mRNA sequence available in GenBank, and MIN6 cells were transfected with the vector. Fluorescence quantitative PCR and Western-blotwere applied to screen efficient RNAi-vector. After transfection with obtained efficient RNAi-vectors for 48 hours, glucose-stimulated insulin secretion experiments were conducted, and the changes of insulin secretion were examined. Results Four siRNA expression vectors of mouse PA-PLA1 gene targeting were confirmed to be successfully constructed by the analyses of enzyme cleavage and sequencing. The results of fluorescence quantitative PCR and Western blot analyses indicated that the siRNA expression vectorpGPU6-PA-PLA1-1885was the most effective RNAi-vector in the four vectors. The expression levels of the PA-PLA1 mRNA and protein of the MIN6 cells transfectedwith pGPU6-PA-PLA1-1885 decreased to 46.3% and 33.9% of that of the control, respectively, and meanwhile the insulin secretion levels of the cells decreased to 65.0% of that of the control (P < 0.05). Conclusion The gene silencing of phosphatidic acid-preferring phospholipase A1 might decrease insulin secretion in MIN6 cells.
ABSTRACT
Objective To investigate the effect of long hairpin RNA ( lhRNA) expression vector targeting HBV X gene ( HBx) on replication of hepatitis B virus ( HBV) and gene expression.Methods Four kinds of small interference RNAs ( siRNAs) were synthesized and lhRNA expression vectors targeting HBx were constructed.Four siRNA oligonucleotides and two lhRNA expression vectors were transfected into HepG2.2.15 cells.HBsAg, HBV DNA in culture supernatants and HBx mRNA in HepG2.2.15 cells were detected by time-resolved immunofluorometric assay, real-time quantitative PCR, and reverse transcription PCR, respectively.Negative sequence group or empty vector group was taken as the control.Independent-samples t test was performed to evaluate the inhibition effect on replication of HBV and gene expression. Results Compared with the negative control, HBsAg, HBV DNA level in culture supernatants and HBx mRNA in HepG2.2.15 cells were significantly decreased after siRNA-1 and siRNA-4 transfected at high concentrations (60 nmol/L or 90 nmol/L) (P<0.05), especially the HBsAg and HBV DNA levels in the siRNA-1 transfection group, which were significantly decreased at 24, 48 and 72 h after transfection ( P<0.05 or P <0.01 ) . Two lhRNA expression vectors ( pMD-HBxlh1 and pMD-HBxlh4 ) were successfully constructed and transfected into HepG2.2.15 cells, HBsAg and HBV DNA level in transfected cells was significantly lower than those in negative control (P<0.05).Conclusion The novel siRNA-1 is confirmed to target HBx gene and lhRNA expression vector targeting HBx can effectively inhibit the replication of HBV and expression of HBV gene.
ABSTRACT
Dengue virus infection has become a global threat affecting around 100 countries in the world. Currently, there is no licensed antiviral agent available against dengue. Thus, there is a strong need to develop therapeutic strategies that can tackle this life threatening disease. RNA interference is an important and effective gene silencing process which degrades targeted RNA by a sequence specific process. Several studies have been conducted during the last decade to evaluate the efficiency of siRNA in inhibiting dengue virus replication. This review summarizes siRNAs as a therapeutic approach against dengue virus serotypes and concludes that siRNAs against virus and host genes can be next generation treatment of dengue virus infection.
Subject(s)
Animals , Humans , Dengue , Therapeutics , Dengue Virus , Genetics , RNA Interference , RNA, Small Interfering , Genetics , Therapeutic UsesABSTRACT
Background Arresting the overexpression of vascular endothelial growth factor (VEGF) will be a new approach to the inhibition of neovascularization.RNA interference (RNAi) can inhibit the expression of specific gene,and its application in eye has little interference to other gene expression.Objective This study was to investigate the effect of small interference RNA (siRNA) targeting VEGF on the expression of VEGF and retinal neovascularization in oxygen-induced retinopathy (OIR) model.Methods psi-HITM/enhanced green fluorescent protein (EGFP)/VEGF siRNA was designed and prepared in vitro.Mouse endothelioma (EOMA) were cultured in DMEM without antibiotic and divided into 5 groups.The cells were incubated in DMEM only in the blank control group;while 1 μl of LipofectamineTM 2000 + psi-HITM/EGFP,1 μl LipofectamineTM 2000 + 40,50 or 60 nmol/L of psi-HITM/EGFP/VEGF siRNA was added into DMEM in the negative control group and siRNA groups,respectively.The expression of VEGF mRNA and protein was detected by real time PCR (RT-PCR) and Western blot.The optimal effective concentration of VEGF siRNA was assessed.OIR models were established in 48 7-day-old C57BL/6J mice by raising them at an oxygen concentration of (75±2) ％ for 5 days and then to normal air.The mice were randomized into the model group,null vector group and VEGF siRNA group.1 μl of a mixture of psi-HITM/EGFP or VEGF siRNA (60 nmol/L) and LipofectamineTM 2000 was intravitreally injected,respectively,in the null vector group and VEGF siRNA group.The normal mice were used as the normal control group.Expression of VEGF mRNA and protein in the mouse retinas was detected by RT-PCR and Western blot,respectively,and FITC-dextran stretched retinal preparation was examined to evaluate the neovascularization,and retinal sections were examined to quantify the number of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM).Results The in vitro transfection test showed that the expression of VEGF mRNA and protein in the EOMA cells was significantly different among the blank control group,negative control group and 40,50,60 nmol/L VEGF siRNA groups (F =148.890,P ＜ 0.001; F =306.960,P ＜ 0.001),and the expression of VEGF mRNA was lower in different concentrations of VEGF siRNA groups than that in the blank control group (t=73.950,119.890,156.480,all at P＜0.001).Also,the expression of VEGF protein was less in different concentrations of VEGF siRNA groups than that in the blank control group (t =15.452,23.344,42.119,all at P＜0.001).The optimal inhibitory concentration of VEGF siRNA was 60 nmol/L.In vivo study determined that compared to the model group and null vector group,the non-perfusion zones and neovascular net in the retina were decreased,and the number of vascular endothelial cell nuclei extending beyond the ILM was less in the VEGF siRNA group.The relative expression level of VEGF mRNA in the retinas was 1.23±0.18,4.02±0.16,3.98±0.19 and 1.98±0.12 in the normal control group,model group,null vector group and VEGF siRNA group,respectively,with a significant difference among them (F=369.780,P＜0.001),and the relative expression levels of VEGF mRNA in the model group and null vector group were higher than that in the normal control group (t=37.880,37.336,both P＜0.001),and the expression of VEGF mRNA in the VEGF siRNA group declined by 50.8％ (t=10.183,P＜0.001).The difference in the expression levels of VEGF protein also was assayed among the various groups (F=408.980,P＜0.001),and VEGF level in the retina was lowered by 68.0％ in the VEGF siRNA group compared to the model group (t =11.473,P＜0.001).However,VEGF level in the VEGF siRNA group remained at a high level in comparison with the normal control group (t =2.422,P＜0.001).Conclusions Intravitreal injection of VEGF siRNA can attenuate retinal neovascularization by effectively downregulate the expression VEGF mRNA and protein in the retina.
ABSTRACT
Objective To explore the effect of hyperoxia on A549 cells suppressed with surfactant protein A (SP-A) suppressed by small interference RNA(SiRNA)-mediated gene silencing,and discuss the function of SP-A in hyperoxic lung injury.Methods A549 cells were gained by serial sub cultivation in vitro and randomly divided into 2 groups,silenced of SP-A group and the control group.A549 cells were transfected with synthetic SP-A sequence-specific SiRNA by Lipofectamine 2000,continuously exposed to hyperoxia(950 mL/L 02,50 mL/L CO2).After exposure to hyperoxia for 48 hours,72 hours and 96 hours,total protein and culture supernatant were gained.SP-A protein was detected by Western-blot,the capacity of proliferation was detected by methyl thiazolyl tetrazolium,and thiobarbituric acid colorimetric method was used to detect the malondialdehyde (MDA) in culture supernatant.Results Sequence-specific SiRNA targeted SP-A2 and significantly down-regulated its expression in A549 cells.Compared with the control group in hyperoxia,the expression of SP-A significantly decreased after 48 hours,72 hours in the silenced group (all P ＜ 0.05),and the capacity of proliferation in A549 cells silenced by SP-A were obviously decreased after 48 hours,72 hours and 96 hours(all P ＜0.05).But there was no significant difference in the MDA in culture supernatant between 2 groups(all P ＞ 0.05).Conclusions The capacity of resisting hyperoxia decreased in A549 cells silenced by SP-A,which indicates that SP-A can protect hyperoxic lung injury.
ABSTRACT
Objective To investigate the effects of a proliferation-inducing ligand(APRIL)gene silencing by small interfering RNA(siRNA)on cell cycle and proliferation of colon carcinoma SW480 cells.Methods The siRNA plasmid vector targeting APRIL gene,named as siRNA-APRIL,was transfected into SW480 cells,transfected with scrambled vector as a nontargeting control and nontransfected group as another control.APRIL mRNA and protein expression were examined by real-time PCR and Western blot,respectively.Cell proliferation activity was analyzed by cell counting kit-8(CK-8),cell cycle was detected by flow cytometry,and p21 together with p27,two important regulatory genes in cell cycle,were measured by RTPCR.Results Compared with nontargeting control and nontransfected control,APRIL expression was inhibited significantly at both mRNA and protein level by siRNA-APRIL being transfected in SW480 cells(P ＜0.05).Cell proliferation ability was drastically repressed after siRNA-APRIL being transfected at 48 h,72 h and 96 h(P ＜ 0.05).After transfected 48 h,the percent of Go/G1 phase cell was significantly increased,S and G2/M phase cell were significantly decreased,the number of cell in apoptosis was increased and the expression of p21 and p27 mRNA were up-regulated(P ＜ 0.05).There was no significant difference when compared the two control groups each other(P ＞ 0.05).Conclusion siRNA-APRIL can effectively knockdown the expression of APRIL gene in SW480 cells,moreover,it can inhibit the cell proliferation and induce G0/G1 phase cell cycle arrest,which occurrence may involve in upregulation the mRNA expression of p21 and p27.
ABSTRACT
Objective To investigate the transfection efficiency of combination of ultrasound (US) microbubbles (MBs) mediated liposome small interference RNA (siRNA) to human and rat retinal pigment epithelium (RPE) cells. Methods Human and rat RPE cells and siRNA were incubated in 24-well plates (2×10~5/well and 1×10~5/well, respectively). The cells were devided into 5 groups: siRNA+US, siRNA+MBs+US, siRNA+L (liposome), siRNA+L+US, siRNA+L+US+MBs. After 12 h, transfection efficiency was observed with fluorescence microscopy and flow cytometry. Results US or ultrasound targeted microbubbles destruction without liposome-mediated could not promote siRNA transfection efficiency to human and rat RPE cells. The transfection efficiency of human and rat RPE cells significantly decreased in the siRNA+L+US+MBs group, but increased in siRNA+L+US group. Conclusion Ultrasonic irradiation can promote lipid-mediated siRNA transfected human RPE cells.
ABSTRACT
Aim According to various target sites of HLA-E mRNA,to design and synthesize 3 pieces of HLA-E siRNA chain,to compare quantitatively their efficiency of silencing gene in BEL-7402 with HLA-E(+)in order to select the dominant siRNA.Methods The hepatocarcinomal BEL-7402 cells,induced by 5×10~5 IU·L~(-1) IFN-γ,expressed HLA-E(+) and was pured by flow cytometry selecting as target cells for research.3 pieces of specific siRNA(A,B,C group)were designed and chemically synthesized,then the concentration of which(0.1 mmol·L~(-1))was respectively transfected through Lipofectamin 2000 into target cells.After 48 h,the gene silent effect on HLA-E gene in A,B and C groups was quantitatively observed by cytoimmunofluorence,flow cytometery,Western blot and real-time PCR,as well as on NK cytotoxicity to target cells tested by NK killing rate.Results Compared with those of control or non-specific siRNA group,HLA-E antigen,protein product,HLA-E mRNA and HLA-E molecule on cell surface were statistically down-regulated in A,B,and C group(P<0.01),whose were silenced more (above 90%) in B or C group than in A group (P<0.01).The NK killing rate in A,B and C groups was dominantly improved(P<0.01),which in B or C group was higher than in A group (P<0.01).Conclusion The targeted siRNA can specifically and high-efficiently silence HLA-E expression in hepatocarcinomal cells,and may keep them from immunoescape through non-classic HLAⅠ pathway to imply new strategy for hepatocarcinomal gene-immunotherapy.
ABSTRACT
Objective To study the inhibition of tumor necrosis factor-alpha(TNF-α)cytokine expression of BV-2 cells induced by hypoxia-reoxygenation injury by siRNA targeting TLR4 gene via the RNAi mechanisms.Method BV-2 mouse microglial cell line was cultured in six-well plates and randomly divided into group N(nor-mal group),group H(hypoxia-reoxygenation),group T(hypoxia-reoxygenation+TLR4-siRNA transfected group),group C(hypoxia-reoxygenation+pEGFP-H1/control-siRNA transfected group)and group B(hypox-ia-reoxygenation+pEGFP-H1 transfected group).Group H,group T,group C and group B were cultured in hy-poxia condition for 3 h followed by reoxygenation for 24 h.The plasma was transfected into BV-2 cells mediated by lipofectamine 2000.The efficiency of transfection were detected by flow cytometry to observe the expression of EGFP.RT-PCR method was used to detect the level of mRNA of TLR4 or NF-кB p65.Westem blot methed was used to test the expression of TLR4 protein.and ELISA was used to test the level of TNF-α in the supernatants.Analysis of variances was used for statistical analysis.Results The expression of EGFP gene waa;(67.58±7.16)% after transfection by flow cytometry analysis.Compared to group N,the TLR4 mRNA,NF-кB p65 mR-NA,TLR4 protein level and the TNF-α quantity in group H,group T,group C and group B increased after the hy-poxia-reoxygenation treatment(P＜0.01).While the expression of the TLR4 mRNA,NF-кB p65 mRNA,TLR4 protein level and the TNF-α quantity in the group T down-regulated compared to group H,group C and group B(P＜0.01).And there were no changes in group C,group B and group H about observation index(P＞0.05).Conclusions The siRNA targeting TLR4 mRNA could inhibit the inflammatory reaction released by BV-2 cells in-duced by hypoxia-reoxygenation stimulation.
ABSTRACT
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 of APP gene.
ABSTRACT
RNA interference (RNAi) is a process to inhibit specific gene expression via the degradation of the target mRNA induced by the double strand RNA. The use of RNAi in mammals as a tool to study gene function has rapidly developed in recent years. Here we described the novel progress and applications of the vector for mediating RNAi in mammals.
ABSTRACT
Objective: To investigate the role of Bcl-XL siRNA in sensitizing ovary cancer cells for TRAIL-induced apoptosis and the underlying mechanisms.Methods: Western blotting analysis was performed to confirm whether Bcl-XL siRNA could effectively down-regulate Bcl-XL protein after SKOV3 cells were transfected with Bcl-XL siRNA and then the cells were treated with TRAIL.Flow cytometry analysis and cell counting were used for assessment of apoptotic rate and survival rate,respectively.Western blotting analysis was performed to determine the changes of apoptosis-related protein in SKOV3 cells.Results: Compared with control group,Bcl-XL siRNA transfection effectively down-regulated the expression of Bcl-XL protein and suppressed the growth of SKOV3 cells;the suppression peaked at 96 h after transfection,being 43.9% that of the control group(P
ABSTRACT
Objective To test the antiproliferative and proapoptotic effect of Survivin small interfering RNA(siRNA) expression vector on lung adenocarcinoma cell A549 in vitro.Methods The Survivin-siRNA expression vector was constructed and conformed by sequencing.The inhibitory effect of Survivin-siRNA was tested by fluorescent quantitative reverse transcription polymerase chain reaction(FQ RT-PCR),Western blot and immunohistochemistry.The cell proliferation and apoptotic rate were assayed by tetrazolium bromide(MTT)colorimetry and flow cytometry.Results Survivin-siRNA expression vector was constructed and transfected into A549 cell.It effectively reduced mRNA and protein level of Survivin.Immunohistochemistry also showed lower expression of Survivin.The A549 cells transfected with Survivin-siRNA had a lower cellular growth rate than that of control group(P