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.

Annexin A2 silencing enhances apoptosis of human umbilical vein endothelial cells in vitro

Objective:To study the effects of inhibited Annexin A2(ANXA2) on human umbilical vein endothelial cells(HUVECs) in vitro.Methods:Short hairpin RNA(shRNA) targeting ANXA2 was designed and cloned into double marked lentvirial vector GV248 for RNAi to generate the recombinant expression plasmids,which were stably transfected into HUVECs.The protein and mRNA expression levels of ANXA2 were analyzed by western blotting and realtime polymerase chain reaction,respectively.Cell proliferation(cell counting kit-8 assay),apoptosis(flow cytometry analysis),the expression(western blotting) and the activity of easpases(enzyme-linked immunosorbent assay) were used to assess the effects of silencing ANXA2 on HUVECs in vitro.Results:The plasmids to express ANXA2-specific shRNA were constructed and were infected into HUVEC resulting in the stably transfected experimental(ANXA2-shRNA),control(eontrol-shRNA) and mock(no plasmid) cell lines,which were verified with western blot and real-time PCR.HUVEC/ANXA2-shRNA showed an inhibition rate 91.89%of ANXA2 expression compared to the mock HUVEC.ANXA2 silencing cell strain obviously presented a lower cell proliferation activity compared to the control and mock HUVECs,with an inhibition rate 82.35%on day 7 in vitro.FACS analysis indicated that the HUVEC/ANXA2-shRNA cells undergoing apoptosis increased by 102.61%compared to the mock HUVECs(P<0.01).Moreover,the activity levels of caspase-3,caspase-8 and caspase-9in HUVEC/ANXA2-shRNA cells were increased and the activated cleaved caspase-3,cleaved Caspase-8 and cleaved caspase-9 were upregulated evidently compared with that of the control and mock HUVECs by 56.29%,89.59%and 144.58%(P<0.01).Conclusions:shRNAmediated silencing of ANX A2 could not only be able to suppress HUVECs:proliferation but to upregulate the enzyme activity of easpases,which bring to an increase of cell apoptosis This work suggested that ANX A2 may represent a useful target of future molecular therapies.

Effect of siRNA on Wisp-1 gene expression,proliferation,migration and adhesion of mouse hepatocellular carcinoma cells

Objective:To study the inhibition effect of siRNA on the expression of Wisp-1 gene in Hca-F of mouse hepatocellular carcinoma cells strain and also its effect on the proliferation,migration and adhesion of hepatocellular carcinoma cells.Methods:Three expression vectors of siRNA were constructed.Lipo2000 was employed to transfect Hca-F cells and Western blot was used to detect the inhibition effect of siRNA on the expression of Wisp-1 gene.Afterwards,CCK8 was adopted to detect the effect of Wisp-1 siRNA on the proliferation of Hca-F cells;Annexin V-FTTC/PI double staining flow cytometry was used to detect the effect of Wisp-1 siRNA on the apoptosis of Hca-F cells;Transwell was used to detect the effect of Wisp-1 siRNA on the migration of Hca-F cells.The in vitro cell adhesion kit was used to detect of Wisp-1 siRNA on the change in the components of extracellular matrix to which Hca-F cells adhered.Western blot was used to detect the activation of protein kinase B(AKT)/glycogen synthase kinase-3(3pathway and the expression of downstream target protein p53 and matrix metal!oproteinases-2.Results:The siRNA showed interference effect on the expression of Wisp-1 gene.Compared with the control group,after being transfected to cells,Wisp-1 siRNA could significantly inhibit the proliferation,migration and adhesion of Hca-F cells and also promote the cell apoptosis,which was related to the down-regulated phosphorylation of AKT and glycogen synthase kinase-3β and the expression of p53 and matrix metalIoproteinases-2(P<0.05).Conclusions:The inhibition of Wisp-1 expression can reduce the proliferation,migration and adhesion of mouse hepatocellular carcinoma cells,which is related to the AKT/ glycogen synthase kinase-3 β pathway.Wisp-1 gene may be the potential target to cure the hepatocellular carcinoma.

中科院上海生化与细胞所发明安全高效的RNAi新技术

2015年10月12日,国际学术期刊Nature Communications在线发表了中国科学院上海生命科学研究院生物化学与细胞生物学研究所国家蛋白质科学中心(上海)吴立刚研究组的最新研究成果'Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient

Inhibiting the expression of CD28 costimulatory molecule on human lymphocytes by special siRNA

Background The B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity.Methods According to CD28 gene sequence, we designed and synthysized three different siRNAs ( siRNA-1,siRNA-2, siRNA-3 ) containing 21 bases using SilencerTM siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay.Results Three siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22. 10% ± 1.63% ,73.50% ± 1.02% and 42.90% ± 0.89% respectively compared with the control ( P < 0. 001 ). Neither of the groups transfected only with siRNA or lipo showed marked reduction in CD28 expression (3.15% ± 0.75% and 4. 55% ±0. 80% ) (P >0. 05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07% ± 0. 96% ) (P > 0. 05 ). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P < 0. 001 ). MTT assay and trypan blue dye exclusion assay demonstrated that the viable cell rations of transfected lymphocytes were significantly reduced in siRNA-1, siRNA-2 and siRNA-3 groups at 48-hour post transfection (P <0.01 ). The control groups showed no marked reduction in cell viability ( P > 0.05 ).Conclusions Three different siRNAs were synthesized and transfected into lymphocytes. They could reduce the expression of CD28 and the CD28 mRNA level, siRNA-2 was the most efficient. The cell viability reduced correspondingly. Therefore, the silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade. This result show that siRNA may be useful for further study on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT).