AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).ME...AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).METHODShRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.RESULTSPGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.CONCLUSIONBlocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.展开更多
To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, th...To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.展开更多
Summary: Alzheimer's disease (AD) is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was repo...Summary: Alzheimer's disease (AD) is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was reported in AD previously. This study aimed to investigate whether sphK1 could exacerbate the accumulation of amyloid protein (Aβ) and sharpen the learning and memory ability of the animal model of AD using siRNA interference. An adenovirus vector expressing small interfering RNA (siRNA) against the sphK1 gene (sphKl-siRNA) was designed, and the effects of sphKl-siRNA on the APP/PS1 mouse four weeks after treatment with sphKl-siRNA hippocampal injection were examined. SphK1 protein expression was confirmed by using Western blotting and ceramide content coupled with SIP secretion was evaluated by enzyme-linked immunosorbent assay (ELISA). Aβ load was detected by immunohistochemical staining and ELISA. Morris water maze was adopted to test the learning and memory ability of the APP/PS 1 mice. A significant difference in the expression of sphK1 protein and mRNA was observed between the siRNA group and the control group. Aβ load in transfected mice was accelerated in vivo, with significant aggravation of the learn- ing and memory ability. The sphKl gene modulation in the All load and the learning and memory ability in the animal model of AD may be important for the treatment of AD.展开更多
Objective This study aims to investigate the feasibility of knockdown of Survivin gene with small interfering RNA and to observe the apoptosis in gliomas which is influenced by siRNA. Methods Survivin specific siRNA o...Objective This study aims to investigate the feasibility of knockdown of Survivin gene with small interfering RNA and to observe the apoptosis in gliomas which is influenced by siRNA. Methods Survivin specific siRNA oligonucleotides were designed and synthesized artificially. This siRNA展开更多
In many ways,cancer cells are different from healthy cells.A lot of tactical nano-based drug delivery systems are based on the difference between cancer and healthy cells.Currently,nanotechnology-based delivery system...In many ways,cancer cells are different from healthy cells.A lot of tactical nano-based drug delivery systems are based on the difference between cancer and healthy cells.Currently,nanotechnology-based delivery systems are the most promising tool to deliver DNA-based products to cancer cells.This review aims to highlight the latest development in the lipids and polymeric nanocarrier for siRNA delivery to the cancer cells.It also provides the necessary information about siRNA development and its mechanism of action.Overall,this review gives us a clear picture of lipid and polymer-based drug delivery systems,which in the future could form the base to translate the basic siRNA biology into siRNA-based cancer therapies.展开更多
Background The immunologic response to allergens mediated by T lymphocytes is an incipient key element in the pathogenesis of asthma, and Thl/Th2 balance is regarded as the core of asthma pathogenesis. Notch is a sing...Background The immunologic response to allergens mediated by T lymphocytes is an incipient key element in the pathogenesis of asthma, and Thl/Th2 balance is regarded as the core of asthma pathogenesis. Notch is a single-pass transmembrane receptor protein that regulates differentiation, proliferation and apoptosis in a broad range of cells. It is considered that the Notch signal pathway works in every stage of T cell development and differentiation. Whether the pathway of asthma pathogenesis is related to Notch1 remains unknown. This study is aimed to investigate whether the pathway of asthma pathogenesis is related to Notch1 by examining the effect of knockdown of the Notch1 gene by small interfering RNA on T cell differentiation. Methods An OVA-induced asthma mouse model was established. The expression of Notch1 in the tissue and T cells of the lung from asthmatic mice was detected by RT-PCR and Western blotting. The expression of Notch1 and cytokine interleukin (IL)-4 and interferon (IFN)-γ in activated lung T cells was detected by RT-PCR and enzyme-linked immunosorbent assay after blocking Notch1 by small interfering RNA. Results The mRNA and protein expression of Notch1 increased significantly both in the lung tissue and lung T cells of asthmatic mice (both P 〈0.05). IL-4 decreased and IFN-y increased significantly in active lung T cells after Notch1 was blocked by Notchl-specific small interfering RNA (IL-4: (2.51±0.51) pg/ml vs 0.64±0.27) pg/ml protein; IFN-γ: (21.72±4.24) pg/ml vs (39.79±4.09) pg/ml protein, P 〈0.05). Conclusion This study demonstrated that the Notch1 signal might play a role in the pathogenesis of asthma by its involvement in Thl/Th2 differentiation.展开更多
Background:Psoriasis is a common chronic inflammatory skin disease with 2%to 3%prevalence worldwide and a heavy social-psychological burden for patients and their families.As the exact pathogenesis of psoriasis is sti...Background:Psoriasis is a common chronic inflammatory skin disease with 2%to 3%prevalence worldwide and a heavy social-psychological burden for patients and their families.As the exact pathogenesis of psoriasis is still unknown,the current treatment is far from satisfactory.Thus,there is an urgent need to find a more effective therapy for this disease.Keratin 17(K17),a type I intermediate filament,is overexpressed in the psoriatic epidermis and plays a critical pathogenic role by stimulating T cells in psoriasis.Therefore,we hypothesized that inhibiting K17 may be a potential therapeutic approach for psoriasis.This study aimed to investigate the therapeutic effect of K17-specific small interfering RNA(siRNA)on mice with imiquimod(IMQ)-induced psoriasis-like dermatitis.Methods:Eight-week-old female BALB/c mice were administered a 5%IMQ cream on both ears to produce psoriatic dermatitis.On day 3,K17 siRNA was mixed with an emulsion matrix and applied topically to the left ears of the mice after IMQ application every day for 7 days.The right ears of the mice were treated in parallel with negative control(NC)siRNA.Inflammation was evaluated by gross ear thickness,histopathology,the infiltration of inflammatory cells(CD3+T cells and neutrophils)using immunofluorescence,and the expression of cytokine production using real-time quantitative polymerase chain reaction.The obtained data were statistically evaluated by unpaired t-tests and a one-way analysis of variance.Results:The severity of IMQ-induced dermatitis on K17 siRNA-treated mice ears was significantly lower than that on NC siRNA-treated mice ears,as evidenced by the alleviated ear inflammation phenotype,including decreased ear thickness,infiltration of inflammatory cells(CD3+T cells and neutrophils),and inflammatory cytokine/chemokine expression levels(interleukin 17[IL-17],IL-22,IL-23,C-X-C motif chemokine ligand 1,and C-C motif chemokine ligand 20)(P<0.05 vs.the Blank or NC siRNA groups).Compared to the NC siRNA treatment,the K17 siRNA treatment resulted in increased K1 and K10 expression,which are characteristic of keratinocyte differentiation(vs.NC siRNA,K17 siRNA1 group:K1,t=4.782,P=0.0050;K10,t=3.365,P=0.0120;K17 siRNA2 group:K1,t=4.104,P=0.0093;K10,t=4.168,P=0.0042;siRNA Mix group:K1,t=3.065,P=0.0221;K10,t=10.83,P<0.0001),and decreased K16 expression,which is characteristic of keratinocyte proliferation(vs.NC siRNA,K17 siRNA1 group:t=4.156,P=0.0043;K17 siRNA2 group:t=2.834,P=0.0253;siRNA Mix group:t=2.734,P=0.0250).Conclusions:Inhibition of K17 expression by its specific siRNA significantly alleviated inflammation in mice with IMQ-induced psoriasis-like dermatitis.Thus,gene therapy targeting K17 may be a potential treatment approach for psoriasis.展开更多
We report the development of a small interfering RNA (siRNA) delivery vector based on cationic perfluorocarbon nanoemulsions. We have prepared perfluorodecalin (PFD) emulsions with a positive surface charge provid...We report the development of a small interfering RNA (siRNA) delivery vector based on cationic perfluorocarbon nanoemulsions. We have prepared perfluorodecalin (PFD) emulsions with a positive surface charge provided by a fluorinated poly(ethylenimine) (F-PEI). The fluorinated emulsion (F-PEI@PFD) reduced cytotoxicity of F-PEI and demonstrated effective binding with siRNAs to form nanosized emulsion polyplexes. The prepared emulsion polyplexes enhanced cellular uptake and improved endosomal escape of the siRNA. In addition to increased reporter gene silencing in multiple cancer cell lines, when compared with control F-PEI and PEI polyplexes, the siR_NA emulsion polyplexes showed an excellent resistance to serum deactivation and maintained high activity, even in high-serum conditions. The F-PEI@PFD emulsion polyplexes carrying an siRNA to silence the expression of Bcl2 gene induced apoptosis and inhibited tumor growth in a melanoma mouse model in vivo and showed potential for in vivo ultrasound imaging. This study demonstrates the potential of F-PEI@PFD emulsions as a multifunctional theranostic nanoplatform for safe siRNA delivery, with integrated ultrasound imaging functionality.展开更多
OBJECTIVE: To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application...OBJECTIVE: To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases. METHODS: Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells. RESULTS: Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect. CONCLUSION: siRNA may be effective in inhibiting SARS-associated coronavirus replication.展开更多
Background RNA interference (RNAi) is a potential cure for amyotrophic lateral sclerosis (ALS) caused by dominant, gain-of-function superoxide dismutase 1 (SOD1) mutations. The success of such therapy relies on ...Background RNA interference (RNAi) is a potential cure for amyotrophic lateral sclerosis (ALS) caused by dominant, gain-of-function superoxide dismutase 1 (SOD1) mutations. The success of such therapy relies on the functional small interfering RNAs (siRNAs) that can effectively deliver RNAi. This study aimed to design the functional siRNAs targeting ALS-associated mutant alleles. Methods A modified dual luciferase system containing human SOD1 mRNA target was established to quantify siRNA efficacy. Coupled with validated siRNAs identified in the literature, we analyzed the rationale of siRNA design and subsequently developed an asymmetry rule-based strategy for designing siRNA. We then further tested the effectiveness of this design strategy in converting a naturally symmetric siRNA into functional siRNAs with favorable asymmetry for gene silencing of SOD1 alleles. Results The efficacies of siRNAs could vary tremendously by one base-pair position change. Functional siRNAs could target the whole span of SOD1 mRNA coding sequence as well as non-coding region. While there is no distinguishable pattern of the distribution of nucleobases in these validated siRNAs, the high percent of GC count at the last two positions of siRNAs (P18 and P19) indicated a strong effect of asymmetry rule. Introducing a mismatch at position 1 of the 5' of antisense strand of siRNA successfully converted the inactive siRNA into functional siRNAs that silence SOD1 with desired efficacy. Conclusions Asymmetry rule-based strategy that incorporates a mismatch into siRNA most consistently enhances RNAi efficacy and guarantees producing functional siRNAs that successfully silence ALS-associated SOD1 mutant alleles regardless target positions. This strategy could also be useful to design siRNAs for silencing other disease-associated dominant, gain-of-function mutant genes.展开更多
This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labele...This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labeled dsRNA oligmers were used to determine the transfection efficiency in cultured astrocytes. TREK-1-specific siRNA duplexes (siT1, siT2, siT3) at the optimal concentration were transfected into cultured astrocytes, and the most efficient siRNA was identified by the method of immunocytochemical staining and Western blotting. The proliferation of astrocytes tranfected with TREK-1-targeting siRNA under hypoxia condition was measured by fluorescence-activated cell sorting (FACS). The results showed that TREK-1 was expressed in cultured astrocytes. The dsRNA oligmers targeting TREK-1 could be transfected efficiently in cultured astrocytes and down-regulate the expression of TREK-1 in astrocytes. Moreover, the down-regulation of TREK-1 in astrocytes contributed to the proliferation of astrocytes under hypoxia condition as determined by cell cycle analysis. It was concluded that siRNA is a powerful technique that can be used to knockdown the expression of TREK-1 in astrocytes, which helps further investigate the function of TREK-1 channel in astrocytes under physicological and pathological condition.展开更多
Small RNAs in Penicillium digitatum were identified and analyzed via transcriptome sequencing on the BGISEQ-500 platform. A total of 15 predicted miRNAs and 10718 novel siRNAs were found. Their length distribution, se...Small RNAs in Penicillium digitatum were identified and analyzed via transcriptome sequencing on the BGISEQ-500 platform. A total of 15 predicted miRNAs and 10718 novel siRNAs were found. Their length distribution, sequence, predicted construction, base bias, expression levels and potential targets were determined as well. Through pathway and KEGG enrichment analysis, the miRNA target genes were mostly involved in carbohydrate metabolism, transport and catabolism, translation and amino acid metabolism. The target genes involved in aflatoxin biosynthesis and proteasome had a higher rich factor value. The results will provide a theoretical foundation for understanding the developmental and pathogenic mechanisms of P. digitatum at the transcriptional level.展开更多
How to effectively transform the pro-oncogenic tumor microenvironments(TME)surrounding a tumor into an anti-tumoral never fails to attract people to study.Small interfering RNA(siRNA)is considered one of the most note...How to effectively transform the pro-oncogenic tumor microenvironments(TME)surrounding a tumor into an anti-tumoral never fails to attract people to study.Small interfering RNA(siRNA)is considered one of the most noteworthy research directions that can regulate gene expression following a process known as RNA interference(RNAi).The research about siRNA delivery targeting tumor cells and TME has been on the rise in recent years.Using siRNA drugs to silence critical proteins in TME was one of the most efficient solutions.However,the manufacture of a siRNA delivery system faces three major obstacles,i.e.,appropriate cargo protection,accurately targeted delivery,and site-specific cargo release.In the following review,we summarized the pharmacological actions of siRNA drugs in remolding TME.In addition,the delivery strategies of siRNA drugs and combination therapy with siRNA drugs to remodel TME are thoroughly discussed.In the meanwhile,the most recent advancements in the development of all clinically investigated and commercialized siRNA delivery technologies are also presented.Ultimately,we propose that nanoparticle drug delivery siRNA may be the future research focus of oncogene therapy.This summary offers a thorough analysis and roadmap for general readers working in the field.展开更多
Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ische...Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy.The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored.However,the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated.In this study,we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function.Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats.Following transplantation of human placental chorionic plate-derived mesenchymal stem cells,interleukin-3 expression was downregulated.To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy,we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA.We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown.Furthermore,interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy.The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy,and this effect was mediated by interleukin-3-dependent neurological function.展开更多
Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivatio...Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA (siRNA)-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.展开更多
Spinal dorsal horn N-Methyl-D-aspartic acid receptor 2B (NR2B) overexpression plays an important role in the production and maintenance of neuropathic pain. Because small interfering RNA (siRNA) can inhibit NR2B e...Spinal dorsal horn N-Methyl-D-aspartic acid receptor 2B (NR2B) overexpression plays an important role in the production and maintenance of neuropathic pain. Because small interfering RNA (siRNA) can inhibit NR2B expression, siRNA may provide a novel approach to treat neuropathic pain and possibly nerve injury. However, an efficient and safe vector for NR2B siRNA has not been discovered. This study shows that a water soluble lipopolymer (WSLP) comprised of low molecular weight polyethyleneimine (PEI) and cholesterol can deliver siRNA targeting NR2B for the treatment of neuropathic pain. Results show that intrathecal injection of WSLP/siRNA complexes for 3 days inhibit NR2B gene expression with reductions in mRNA and protein levels by 59% and 54%, respectively, compared with control rats (P 〈 0.01). Injection of WSLP complexed with scrambled siRNA, or PEI with siRNA did not show this inhibitory effect. Moreover, injection of WSLP/siRNA complexes significantly relieved neuropathic pain at 3, 7, 12, and 21 days, while injection of WSLP with scrambled siRNA or PEI with siRNA did not. These results demonstrate that WSLP can efficiently deliver siRNA targeting NR2B in vivo and relieve neuropathic pain.展开更多
Large amounts of aberrantly spliced mRNA from the β^654 allele was present in erythroid cells, which might impair the erythropoiesis. A therapeutic strategy for β-thalassemia was explored by knocking down the aberra...Large amounts of aberrantly spliced mRNA from the β^654 allele was present in erythroid cells, which might impair the erythropoiesis. A therapeutic strategy for β-thalassemia was explored by knocking down the aberrantly spliced mRNA of β-globin. Lentiviral vector with siRNA fragment targets on the specific portion of β^654-globin aberrantly spliced pre-mRNA was constructed. In HeLa β^654 cells, the siRNA vector could reduce approximately 60% of aberrantly spliced mRNA, which was assessed by RT-PCR and qRT-PCR. Furthermore, a disease model of β^654 thalassemia mice with lentiviral-mediated siRNA was produced by subzonal injection (named Hβi-Hbb^th-4/Hbb^+ transgenic mice). Our results showed that the hemotological parameters were improved in Hβi-Hbb^th-4/Hbb^+ transgenic mice. This study provides a potential way for β^654-thalassemia therapy by knocking down the aberrantly spliced β-globin mRNA, whilst supporting that the aberrantly spliced β-globin mRNA may aggravate the disease.展开更多
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 S...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.展开更多
Cyclin dependent kinases (cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegen-erative diseases, we packed r...Cyclin dependent kinases (cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegen-erative diseases, we packed recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA (rAAV-EGFP-U6-cdc2-siRNA) was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful. rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.展开更多
A previous study by our group found that inhibition of nischarin promotes neurite outgrowth and neuronal regeneration in Neuro-2 a cells and primary cortical neurons.In recent years,more and more studies have shown th...A previous study by our group found that inhibition of nischarin promotes neurite outgrowth and neuronal regeneration in Neuro-2 a cells and primary cortical neurons.In recent years,more and more studies have shown that nanomaterials have good prospects in treatment of spinal cord injury.We proposed that small interfering RNA targeting nischarin(Nis-si RNA) delivered by polyethyleneimine-alginate(PEIALG) nanoparticles promoted motor function recovery in rats with spinal cord injury.Direct microinjection of 5 μL PEI-ALG/Nis-si RNA into the spinal cord lesion area of spinal cord injury rats was performed.From day 7 after surgery,Basso,Beattie and Bresnahan score was significantly higher in rats from the PEI-ALG/Nis-si RNA group compared with the spinal cord injury group and PEI-ALG/Control-si RNA group.On day 21 after injection,hematoxylin-eosin staining showed that the necrotic area was reduced in the PEI-ALG/Nis-si RNA group.Immunohistochemistry and western blot assay results confirmed successful inhibition of nischarin expression and increased protein expression of growth-associated protein-43 in the PEI-ALG/Nis-si RNA group.These findings suggest that a complex of PEI-ALG nanoparticles and Nis-si RNA effectively suppresses nischarin expression,induces expression of growth-associated protein-43,and accelerates motor function recovery after spinal cord injury.展开更多
基金Supported by National Natural Science Fundation of China(No.81000387)
文摘AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).METHODShRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.RESULTSPGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.CONCLUSIONBlocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.
基金Supported by the Ministry of Agricultural Nuarture of New Varieties Genetically Modified Organisms Significant Special Funding (2008ZX08007-002)
文摘To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.
基金supported by the National Natural Science Foundation of China (No. 81070879)
文摘Summary: Alzheimer's disease (AD) is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was reported in AD previously. This study aimed to investigate whether sphK1 could exacerbate the accumulation of amyloid protein (Aβ) and sharpen the learning and memory ability of the animal model of AD using siRNA interference. An adenovirus vector expressing small interfering RNA (siRNA) against the sphK1 gene (sphKl-siRNA) was designed, and the effects of sphKl-siRNA on the APP/PS1 mouse four weeks after treatment with sphKl-siRNA hippocampal injection were examined. SphK1 protein expression was confirmed by using Western blotting and ceramide content coupled with SIP secretion was evaluated by enzyme-linked immunosorbent assay (ELISA). Aβ load was detected by immunohistochemical staining and ELISA. Morris water maze was adopted to test the learning and memory ability of the APP/PS 1 mice. A significant difference in the expression of sphK1 protein and mRNA was observed between the siRNA group and the control group. Aβ load in transfected mice was accelerated in vivo, with significant aggravation of the learn- ing and memory ability. The sphKl gene modulation in the All load and the learning and memory ability in the animal model of AD may be important for the treatment of AD.
文摘Objective This study aims to investigate the feasibility of knockdown of Survivin gene with small interfering RNA and to observe the apoptosis in gliomas which is influenced by siRNA. Methods Survivin specific siRNA oligonucleotides were designed and synthesized artificially. This siRNA
文摘In many ways,cancer cells are different from healthy cells.A lot of tactical nano-based drug delivery systems are based on the difference between cancer and healthy cells.Currently,nanotechnology-based delivery systems are the most promising tool to deliver DNA-based products to cancer cells.This review aims to highlight the latest development in the lipids and polymeric nanocarrier for siRNA delivery to the cancer cells.It also provides the necessary information about siRNA development and its mechanism of action.Overall,this review gives us a clear picture of lipid and polymer-based drug delivery systems,which in the future could form the base to translate the basic siRNA biology into siRNA-based cancer therapies.
基金This study was supported by the grants from the Major Program of Shanghai Education Committee Foundation (No. 09ZZ114) and the National Natural Science Foundation of China (No. 30971302).
文摘Background The immunologic response to allergens mediated by T lymphocytes is an incipient key element in the pathogenesis of asthma, and Thl/Th2 balance is regarded as the core of asthma pathogenesis. Notch is a single-pass transmembrane receptor protein that regulates differentiation, proliferation and apoptosis in a broad range of cells. It is considered that the Notch signal pathway works in every stage of T cell development and differentiation. Whether the pathway of asthma pathogenesis is related to Notch1 remains unknown. This study is aimed to investigate whether the pathway of asthma pathogenesis is related to Notch1 by examining the effect of knockdown of the Notch1 gene by small interfering RNA on T cell differentiation. Methods An OVA-induced asthma mouse model was established. The expression of Notch1 in the tissue and T cells of the lung from asthmatic mice was detected by RT-PCR and Western blotting. The expression of Notch1 and cytokine interleukin (IL)-4 and interferon (IFN)-γ in activated lung T cells was detected by RT-PCR and enzyme-linked immunosorbent assay after blocking Notch1 by small interfering RNA. Results The mRNA and protein expression of Notch1 increased significantly both in the lung tissue and lung T cells of asthmatic mice (both P 〈0.05). IL-4 decreased and IFN-y increased significantly in active lung T cells after Notch1 was blocked by Notchl-specific small interfering RNA (IL-4: (2.51±0.51) pg/ml vs 0.64±0.27) pg/ml protein; IFN-γ: (21.72±4.24) pg/ml vs (39.79±4.09) pg/ml protein, P 〈0.05). Conclusion This study demonstrated that the Notch1 signal might play a role in the pathogenesis of asthma by its involvement in Thl/Th2 differentiation.
基金National Natural Science Foundation of China(Nos.81803125 and 81903192)。
文摘Background:Psoriasis is a common chronic inflammatory skin disease with 2%to 3%prevalence worldwide and a heavy social-psychological burden for patients and their families.As the exact pathogenesis of psoriasis is still unknown,the current treatment is far from satisfactory.Thus,there is an urgent need to find a more effective therapy for this disease.Keratin 17(K17),a type I intermediate filament,is overexpressed in the psoriatic epidermis and plays a critical pathogenic role by stimulating T cells in psoriasis.Therefore,we hypothesized that inhibiting K17 may be a potential therapeutic approach for psoriasis.This study aimed to investigate the therapeutic effect of K17-specific small interfering RNA(siRNA)on mice with imiquimod(IMQ)-induced psoriasis-like dermatitis.Methods:Eight-week-old female BALB/c mice were administered a 5%IMQ cream on both ears to produce psoriatic dermatitis.On day 3,K17 siRNA was mixed with an emulsion matrix and applied topically to the left ears of the mice after IMQ application every day for 7 days.The right ears of the mice were treated in parallel with negative control(NC)siRNA.Inflammation was evaluated by gross ear thickness,histopathology,the infiltration of inflammatory cells(CD3+T cells and neutrophils)using immunofluorescence,and the expression of cytokine production using real-time quantitative polymerase chain reaction.The obtained data were statistically evaluated by unpaired t-tests and a one-way analysis of variance.Results:The severity of IMQ-induced dermatitis on K17 siRNA-treated mice ears was significantly lower than that on NC siRNA-treated mice ears,as evidenced by the alleviated ear inflammation phenotype,including decreased ear thickness,infiltration of inflammatory cells(CD3+T cells and neutrophils),and inflammatory cytokine/chemokine expression levels(interleukin 17[IL-17],IL-22,IL-23,C-X-C motif chemokine ligand 1,and C-C motif chemokine ligand 20)(P<0.05 vs.the Blank or NC siRNA groups).Compared to the NC siRNA treatment,the K17 siRNA treatment resulted in increased K1 and K10 expression,which are characteristic of keratinocyte differentiation(vs.NC siRNA,K17 siRNA1 group:K1,t=4.782,P=0.0050;K10,t=3.365,P=0.0120;K17 siRNA2 group:K1,t=4.104,P=0.0093;K10,t=4.168,P=0.0042;siRNA Mix group:K1,t=3.065,P=0.0221;K10,t=10.83,P<0.0001),and decreased K16 expression,which is characteristic of keratinocyte proliferation(vs.NC siRNA,K17 siRNA1 group:t=4.156,P=0.0043;K17 siRNA2 group:t=2.834,P=0.0253;siRNA Mix group:t=2.734,P=0.0250).Conclusions:Inhibition of K17 expression by its specific siRNA significantly alleviated inflammation in mice with IMQ-induced psoriasis-like dermatitis.Thus,gene therapy targeting K17 may be a potential treatment approach for psoriasis.
基金This work was financially supported by the National Science and Technology Major Project (No. 2017YFA0205400), the Changjiang Scholar program, the National Natural Science Foundation of China (Nos. 81373983 and 81573377), China Postdoctoral Science Foundation (No. 2016M601923), and Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX17_0671).
文摘We report the development of a small interfering RNA (siRNA) delivery vector based on cationic perfluorocarbon nanoemulsions. We have prepared perfluorodecalin (PFD) emulsions with a positive surface charge provided by a fluorinated poly(ethylenimine) (F-PEI). The fluorinated emulsion (F-PEI@PFD) reduced cytotoxicity of F-PEI and demonstrated effective binding with siRNAs to form nanosized emulsion polyplexes. The prepared emulsion polyplexes enhanced cellular uptake and improved endosomal escape of the siRNA. In addition to increased reporter gene silencing in multiple cancer cell lines, when compared with control F-PEI and PEI polyplexes, the siR_NA emulsion polyplexes showed an excellent resistance to serum deactivation and maintained high activity, even in high-serum conditions. The F-PEI@PFD emulsion polyplexes carrying an siRNA to silence the expression of Bcl2 gene induced apoptosis and inhibited tumor growth in a melanoma mouse model in vivo and showed potential for in vivo ultrasound imaging. This study demonstrates the potential of F-PEI@PFD emulsions as a multifunctional theranostic nanoplatform for safe siRNA delivery, with integrated ultrasound imaging functionality.
文摘OBJECTIVE: To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases. METHODS: Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells. RESULTS: Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect. CONCLUSION: siRNA may be effective in inhibiting SARS-associated coronavirus replication.
文摘Background RNA interference (RNAi) is a potential cure for amyotrophic lateral sclerosis (ALS) caused by dominant, gain-of-function superoxide dismutase 1 (SOD1) mutations. The success of such therapy relies on the functional small interfering RNAs (siRNAs) that can effectively deliver RNAi. This study aimed to design the functional siRNAs targeting ALS-associated mutant alleles. Methods A modified dual luciferase system containing human SOD1 mRNA target was established to quantify siRNA efficacy. Coupled with validated siRNAs identified in the literature, we analyzed the rationale of siRNA design and subsequently developed an asymmetry rule-based strategy for designing siRNA. We then further tested the effectiveness of this design strategy in converting a naturally symmetric siRNA into functional siRNAs with favorable asymmetry for gene silencing of SOD1 alleles. Results The efficacies of siRNAs could vary tremendously by one base-pair position change. Functional siRNAs could target the whole span of SOD1 mRNA coding sequence as well as non-coding region. While there is no distinguishable pattern of the distribution of nucleobases in these validated siRNAs, the high percent of GC count at the last two positions of siRNAs (P18 and P19) indicated a strong effect of asymmetry rule. Introducing a mismatch at position 1 of the 5' of antisense strand of siRNA successfully converted the inactive siRNA into functional siRNAs that silence SOD1 with desired efficacy. Conclusions Asymmetry rule-based strategy that incorporates a mismatch into siRNA most consistently enhances RNAi efficacy and guarantees producing functional siRNAs that successfully silence ALS-associated SOD1 mutant alleles regardless target positions. This strategy could also be useful to design siRNAs for silencing other disease-associated dominant, gain-of-function mutant genes.
基金supported by grants from the National Natural Science Foundation of China (No.30971007)the Natural Science Foundation of Hubei Province for Outstanding Young Scholars (No.2010CDA103)the National Basic Research Program(No.2011CB504403)
文摘This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labeled dsRNA oligmers were used to determine the transfection efficiency in cultured astrocytes. TREK-1-specific siRNA duplexes (siT1, siT2, siT3) at the optimal concentration were transfected into cultured astrocytes, and the most efficient siRNA was identified by the method of immunocytochemical staining and Western blotting. The proliferation of astrocytes tranfected with TREK-1-targeting siRNA under hypoxia condition was measured by fluorescence-activated cell sorting (FACS). The results showed that TREK-1 was expressed in cultured astrocytes. The dsRNA oligmers targeting TREK-1 could be transfected efficiently in cultured astrocytes and down-regulate the expression of TREK-1 in astrocytes. Moreover, the down-regulation of TREK-1 in astrocytes contributed to the proliferation of astrocytes under hypoxia condition as determined by cell cycle analysis. It was concluded that siRNA is a powerful technique that can be used to knockdown the expression of TREK-1 in astrocytes, which helps further investigate the function of TREK-1 channel in astrocytes under physicological and pathological condition.
文摘Small RNAs in Penicillium digitatum were identified and analyzed via transcriptome sequencing on the BGISEQ-500 platform. A total of 15 predicted miRNAs and 10718 novel siRNAs were found. Their length distribution, sequence, predicted construction, base bias, expression levels and potential targets were determined as well. Through pathway and KEGG enrichment analysis, the miRNA target genes were mostly involved in carbohydrate metabolism, transport and catabolism, translation and amino acid metabolism. The target genes involved in aflatoxin biosynthesis and proteasome had a higher rich factor value. The results will provide a theoretical foundation for understanding the developmental and pathogenic mechanisms of P. digitatum at the transcriptional level.
基金supported by the Natural Science Foundation of China(U2230123,81870683,82121003 and 82201234)CAMS Innovation Fund for Medical Sciences(2019-I2M-5-032)the Department of Science and Technology of Sichuan Province,China(22ZYZYTS0159,2022YFS0606,2023YFS0125,2023YFS0131,2023NSFSC0033,and 22ZYZYTS0151)
文摘How to effectively transform the pro-oncogenic tumor microenvironments(TME)surrounding a tumor into an anti-tumoral never fails to attract people to study.Small interfering RNA(siRNA)is considered one of the most noteworthy research directions that can regulate gene expression following a process known as RNA interference(RNAi).The research about siRNA delivery targeting tumor cells and TME has been on the rise in recent years.Using siRNA drugs to silence critical proteins in TME was one of the most efficient solutions.However,the manufacture of a siRNA delivery system faces three major obstacles,i.e.,appropriate cargo protection,accurately targeted delivery,and site-specific cargo release.In the following review,we summarized the pharmacological actions of siRNA drugs in remolding TME.In addition,the delivery strategies of siRNA drugs and combination therapy with siRNA drugs to remodel TME are thoroughly discussed.In the meanwhile,the most recent advancements in the development of all clinically investigated and commercialized siRNA delivery technologies are also presented.Ultimately,we propose that nanoparticle drug delivery siRNA may be the future research focus of oncogene therapy.This summary offers a thorough analysis and roadmap for general readers working in the field.
基金supported by the National Natural Science Foundation of China,No.82001604Guizhou Provincial Higher Education Science and Technology Innovation Team,No.[2023]072+1 种基金Guizhou Province Distinguished Young Scientific and Technological Talent Program,No.YQK[2023]040Guizhou Provincial Basic Research Program(Natural Science),No.ZK[2021]-368(all to LXiong),and Zunyi City Innovative Talent Team Training Plan,No.[2022]-2.
文摘Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy.The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored.However,the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated.In this study,we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function.Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats.Following transplantation of human placental chorionic plate-derived mesenchymal stem cells,interleukin-3 expression was downregulated.To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy,we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA.We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown.Furthermore,interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy.The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy,and this effect was mediated by interleukin-3-dependent neurological function.
基金supported by the National Natural Science Foundation of China,No.30970995the Postgraduate Science Research Innovation Project of Higher Learning University of Jiangsu Province in China,No.CXLX11_0735
文摘Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA (siRNA)-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.
基金the Natural Science Foundation of Guangdong Province,No.07000059the Science and Technology Development Program of Guangzhou,No.2010Y1-C301the Science and Technology Development Program of Guangdong Province,No.2010B031600123
文摘Spinal dorsal horn N-Methyl-D-aspartic acid receptor 2B (NR2B) overexpression plays an important role in the production and maintenance of neuropathic pain. Because small interfering RNA (siRNA) can inhibit NR2B expression, siRNA may provide a novel approach to treat neuropathic pain and possibly nerve injury. However, an efficient and safe vector for NR2B siRNA has not been discovered. This study shows that a water soluble lipopolymer (WSLP) comprised of low molecular weight polyethyleneimine (PEI) and cholesterol can deliver siRNA targeting NR2B for the treatment of neuropathic pain. Results show that intrathecal injection of WSLP/siRNA complexes for 3 days inhibit NR2B gene expression with reductions in mRNA and protein levels by 59% and 54%, respectively, compared with control rats (P 〈 0.01). Injection of WSLP complexed with scrambled siRNA, or PEI with siRNA did not show this inhibitory effect. Moreover, injection of WSLP/siRNA complexes significantly relieved neuropathic pain at 3, 7, 12, and 21 days, while injection of WSLP with scrambled siRNA or PEI with siRNA did not. These results demonstrate that WSLP can efficiently deliver siRNA targeting NR2B in vivo and relieve neuropathic pain.
基金the National Basic Research Program of China (973 Program) (No 2004CB518806)the National High-tech R&D Program (863 Program) (No 2007AA021206)+1 种基金the National Natural Science Foundation of China (No 30571777)the Chinese National and Shanghai Leading Academic Disci-pline Project (No B204)
文摘Large amounts of aberrantly spliced mRNA from the β^654 allele was present in erythroid cells, which might impair the erythropoiesis. A therapeutic strategy for β-thalassemia was explored by knocking down the aberrantly spliced mRNA of β-globin. Lentiviral vector with siRNA fragment targets on the specific portion of β^654-globin aberrantly spliced pre-mRNA was constructed. In HeLa β^654 cells, the siRNA vector could reduce approximately 60% of aberrantly spliced mRNA, which was assessed by RT-PCR and qRT-PCR. Furthermore, a disease model of β^654 thalassemia mice with lentiviral-mediated siRNA was produced by subzonal injection (named Hβi-Hbb^th-4/Hbb^+ transgenic mice). Our results showed that the hemotological parameters were improved in Hβi-Hbb^th-4/Hbb^+ transgenic mice. This study provides a potential way for β^654-thalassemia therapy by knocking down the aberrantly spliced β-globin mRNA, whilst supporting that the aberrantly spliced β-globin mRNA may aggravate the disease.
基金supported by National Natural Science Foundation of China(No.81441126)
文摘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.
基金supported by grants from the National Natural Sciences Foundation of China (No. 30400141, No. 30670737).
文摘Cyclin dependent kinases (cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegen-erative diseases, we packed recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA (rAAV-EGFP-U6-cdc2-siRNA) was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful. rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.
基金supported by the Natural Science Foundation of Zhejiang Province of China,No.LY15H250001 and LY14H090002the National Natural Science Foundation of China,No.81000535 and 81402872+1 种基金the Medical Science and Technology Project Foundation of Zhejiang Province of China,No.2014KYA166the Science and Technology Innovation Talents Development Plan Foundation for High School Students in Zhejiang Province of China,No.2014R401186
文摘A previous study by our group found that inhibition of nischarin promotes neurite outgrowth and neuronal regeneration in Neuro-2 a cells and primary cortical neurons.In recent years,more and more studies have shown that nanomaterials have good prospects in treatment of spinal cord injury.We proposed that small interfering RNA targeting nischarin(Nis-si RNA) delivered by polyethyleneimine-alginate(PEIALG) nanoparticles promoted motor function recovery in rats with spinal cord injury.Direct microinjection of 5 μL PEI-ALG/Nis-si RNA into the spinal cord lesion area of spinal cord injury rats was performed.From day 7 after surgery,Basso,Beattie and Bresnahan score was significantly higher in rats from the PEI-ALG/Nis-si RNA group compared with the spinal cord injury group and PEI-ALG/Control-si RNA group.On day 21 after injection,hematoxylin-eosin staining showed that the necrotic area was reduced in the PEI-ALG/Nis-si RNA group.Immunohistochemistry and western blot assay results confirmed successful inhibition of nischarin expression and increased protein expression of growth-associated protein-43 in the PEI-ALG/Nis-si RNA group.These findings suggest that a complex of PEI-ALG nanoparticles and Nis-si RNA effectively suppresses nischarin expression,induces expression of growth-associated protein-43,and accelerates motor function recovery after spinal cord injury.