AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression in HepG2.2.15 cells by combination of small interfering RNAs (siRNAs). METHODS: Recombinant plasmid psiI-HBV was constructed a...AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression in HepG2.2.15 cells by combination of small interfering RNAs (siRNAs). METHODS: Recombinant plasmid psiI-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay (ELISA). Intracellular viral DNA and covalently closed circular DNA (cccDNA) were quantified by real-time polymerase chain reaction (PCR). HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR (RT-PCR). RESULTS: siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantlycombination of siRNAs significantly suppressed HBV cccDNA amplification. CONCLUSION: Combination of siRNAs mediates a stronger inhibition on viral replication and antigenexpression in HepG2.2.15 cells, especially on cccDNA amplification.展开更多
Hepatocellular carcinoma(HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective,esp...Hepatocellular carcinoma(HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective,especially for the advanced forms of the disease. In the last year,short double stranded RNA molecules termed small interfering RNAs(si RNAs) and micro interfering RNAs(mi RNA),emerged as interesting molecules with potential therapeutic value for HCC. The practical use of these molecules is however limited by the identification of optimal molecular targets and especially by the lack of effective and targeted HCC delivery systems. Here we focus our discussion on the most recent advances in the identification of si RNAs/mi RNAs molecular targets and on the development of suitable si RNA/mi RNAs delivery systems.展开更多
Objective To construct an expression vector of small interfering RNA (siRNA) against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast can...Objective To construct an expression vector of small interfering RNA (siRNA) against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast cancer cell line MCF-7. Methods Constructed an expression vector of siRNA against survivin and transfected it into PC-2 and MCF-7 cells using lipofectamine^TM 2000. The expression of survivin was detected by semi-quanfifive RT-PCR and immunohistochemistry, and its effects on proliferation of PC-2 and MCF-7 cells were detected by MTT assay. Results The introduction of sequence-specific siRNA could efficiently suppress survivin expression at both mRNA and protein levels in the two cancer cell lines. In PC-2 cells, the expression inhibition rates were 81.25% at mRNA level and 74.24% at protein level In MCF-7 cells, the expression inhibition rates were 64.91% at mRNA level and 79. 72% at protein level The proliferation of PC-2 and MCF-7 cells was also suppressed, and24 and 48 hours after the cells were reseeded, the proliferation inhibition rates of PC-2 cells were 28. 00% and 33. 38%, and that of MCF-7 cells were 31.58% and 33.02%, respectively. Conclusions The expression vector of siRNA against survivin can block survivin expression in PC-2 and MCF-7 cells efficiently and specifically. Down regulation of survivin expression can suppress proliferation of PC-2 and MCF-7 cells. Survivin RNAi may have potential value in gene therapy of human cancers.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
Objective: HOXB7 gene is a kind of transcription regulator over-expressed in malignant melanoma (MM) cell lines. It can specifically up-regulate the expression of angiogenic factors and tumor growth factors such as...Objective: HOXB7 gene is a kind of transcription regulator over-expressed in malignant melanoma (MM) cell lines. It can specifically up-regulate the expression of angiogenic factors and tumor growth factors such as bFGF, GROa, VEGF and induce angiogenesis in melanoma, resulting in the proliferation and metastasis of tumor cells. We designed and synthesized HOXB7 specific siRNA to study its interfering effect on the expressions of HOXB7 and bFGF genes in melanoma A375 cell line and the biologic characteristics of A375 cells. Methods: Three synthesized siRNA with different sequences were separately transfected into A375 cells by lipofecter 2000. The expression of HOXB7 and bFGF mRNA in transfected cells was detected by RT-PCR 24 and 48 hours after transduction. The expression of bFGF protein in the transfected cells were detected by flowcytometry 48 hours after transfection. MTT assay was used to analyze the cell proliferation rate of siRNA transfected cells. Based on the in vitro experiment results, one effective siRNA sequence was selected for the construction of in vivo siRNA expression vector. Then, a malignant melanoma animal model was established. The siRNA expression plasmid was injected into the tumor foci and its influence on the growth and angiogenesis of tumor was observed. Results: The mRNA expressions of both HOXB7 and bFGF genes in the A375 cells reduced significantly 24 and 48 hour after transfection of siRNA. Expression level of the protein of angiogenic factor bFGF induced by HOXB7 gene in siRNA transfected cells was significantly lower than that in control cells 48 hours after transduction. Cell proliferation was also suppressed in siRNA transfected cells. Two of the three siRNA strands showed prominent interference effect. The in vivo study indicated that the tumor size and the microvessel density in the tumor both reduced after injection of HOXB7siRNA plasmid. Conclusion: Down-regulation of HOXB7 gene expression can effectively reduce the expression of angiogenic factor bFGF and the proliferation of MM cells. Besides, the growth and angiogenesis of MM tumor were also inhibited.展开更多
OBJECTIVE To investigate whether small hairpin RNA (shRNA)targeting Bcl-2 mRNA could inhibit the growth of lymphomatransplanted subcutaneously in nude mice.METHODS Recombinant Bcl-2 shRNA expression vector withgreen f...OBJECTIVE To investigate whether small hairpin RNA (shRNA)targeting Bcl-2 mRNA could inhibit the growth of lymphomatransplanted subcutaneously in nude mice.METHODS Recombinant Bcl-2 shRNA expression vector withgreen fluorescence protein (GFP) gene was constructed andpreserved in our lab. We evaluated the antitumor effect of the Bcl-2shRNA in vivo which was the model of nude mice bearing Rajicells xenografts. Human Raji cells were injected subcutaneouslyinto nude mice to establish lymphoma models. When thediameters of tumor were above 0.5 cm after Raji cells injection,the mice bearing tumor were randomly divided into four groups:saline control group, negative shRNA group, plasmid vectorgroup, Bcl-2 shRNA group. The polyethylenimine (PEI) was usedto transfect shRNA into tumor. The mixed PEI and shRNA wasinjected into tumors. The growth and size of tumor were observed.Tissue was stained by H&E for its pathological morphology. Theexpression of Bcl-2 mRNA in the tumor mass was detected byreverse transcription polymerase chain reaction (RT-PCR).RESULTS A significant difference in median tumor weightwas observed in mice treated with Bcl-2 shRNA, compared withthose in the groups of negative shRNA or plasmid vector or salinesolution (P< 0.05). Pathological evaluation was completed in allexcised tumors from nude mice bearing Raji cells xenografts.The tumor tissue of the mice treated with Bcl-2 shRNA showedapoptosis, serious necrosis of the cells and inflammatory cellsinfiltration. There was no change in the morphology of cellsamong negative shRNA, plasmid vector and saline solution group.In the group of the Bcl-2 shRNA, the expression levels of Bcl-2mRNA of the tumor tissue were effectively inhibited (P < 0.05).CONCLUSION The shRNA targeting at the Bcl-2 mRNAcould inhibit the growth of human lymphoma transplantedsubcutaneously in nude mice.展开更多
Hepatocellular carcinoma(HCC) is the 5th most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorablesystemic side...Hepatocellular carcinoma(HCC) is the 5th most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorablesystemic side-effects of chemotherapeutic agents and susceptibility to the degradation of small interfering RNAs(si RNAs), which can knock down a specific gene involved in the disease, have hampered their clinical application. So, it could be beneficial to develop an efficient carrier for the stabilization and specific delivery of drugs and si RNA to cells. Targeted nanoparticles have gained considerable attention as an efficient drug and gene delivery system, which is due to their capability in achieving the highest accumulation of cytotoxic agents in tumor tissue, modifiable drug pharmacokinetic- and bio-distribution, improved effectiveness of treatment, and limited sideeffects. Recent studies have shed more light on the advantages of novel drug loaded carrier systems vs free drugs. Most of the animal studies have reported improvement in treatment efficacy and survival rate using novel carrier systems. Targeted delivery may be achieved passively or actively. In passive targeting, no ligand as homing device is used, while targeting is achieved by incorporating the therapeutic agent into a macromolecule or nanoparticle that passively reaches the target organ. However, in active targeting, the therapeutic agent or carrier system is conjugated to a tissue or cell-specific receptor which is overexpressed in a special malignancy using a ligand called a homing device. This review covers a broad spectrum of targeted nanoparticles as therapeutic and nonviral si RNA delivery systems, which are developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and their characteristics and opportunities for the clinical applications of drugs and therapeutic si RNA are discussed in this article. Asialoglycoprotein receptors, low-density lipoprotein, ganglioside GM1 cell surface ligand, epidermal growth factor receptor receptors, monoclonal antibodies, retinoic acid receptors, integrin receptors targeted by Arg-Gly-Asp peptide, folate, and transferrin receptors are the most widely studied cell surface receptors which are used for the site specific delivery of drugs and si RNA-based therapeutics in HCC and discussed in detail in this article.展开更多
RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequenc...RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0~60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.展开更多
Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving inc...Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving increased synthesis of matrix components and a failure of physiological mechanisms of matrix turnover. Activation of hepatic stellate cells(HSCs) remains a central event in fibrosis. HSCs are the main source of extracellular matrix(ECM). Transforming growth factor-beta(TGF-β), which is the fibrogenic master cytokine, can induce the activation of HSCs to produce a large amount of ECM, and is capable of inducing apoptosis of liver cells. RNA interference(RNAi) is a novel gene disruption technology. Studies have shown that small interfering RNA(si RNA) targeting TGF-β1 may inhibit the activation and proliferation of HSCs, suppress ECM synthesis and block liver fibrosis. TGF-β1 si RNA-mediated gene silencing therapy provides a new avenue for liver fibrosis. This review summarizes recent progresses in research on HSCs, TGF-β1 and TGF-β1 si RNA in liver fibrosis.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
RNA interference(RNAi),which causes the degradation of any RNA in a sequence specific manner,is a posttranscriptional gene silencing mechanism.Targeting the invariant chain(Ii)in DCs has been used as an approach to en...RNA interference(RNAi),which causes the degradation of any RNA in a sequence specific manner,is a posttranscriptional gene silencing mechanism.Targeting the invariant chain(Ii)in DCs has been used as an approach to enhance antitumor immunity.It is demonstrated in this article that transfection of H-2(K)DCs with siRNA specific for Ii gene can significantly knock down Ii.When exposed to TNF-alpha,immature DCs transfected with Ii siRNA can differentiate into mature DCs without reducing viability or IL-12p70 production.Ii siRNA-treated H-2(K)DCs exhibited an increased allostimulatory capacity in a lymphocyte proliferation assay.Furthermore,Ii siRNA-transfected H-2(K)DCs enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production,and much stronger cytotoxic activity was observed when DCs were co-transfected with Ii siRNA and an endogenous tumor antigen in vitro.Our findings indicate that silencing the Ii gene in DCs with siRNA may offer a potential approach to enhancing antitumor immunotherapy.展开更多
Small nucleolar RNAs(snoRNAs)represent a class of non-coding RNAs that play pivotal roles in post-transcriptional RNA processing and modification,thereby contributing significantly to the maintenance of cellular funct...Small nucleolar RNAs(snoRNAs)represent a class of non-coding RNAs that play pivotal roles in post-transcriptional RNA processing and modification,thereby contributing significantly to the maintenance of cellular functions related to protein synthesis.SnoRNAs have been discovered to possess the ability to influence cell fate and alter disease progression,holding immense potential in controlling human diseases.It is suggested that the dysregulation of snoRNAs in cancer exhibits differential expression across various cancer types,stages,metastasis,treatment response and/or prognosis in patients.On the other hand,colorectal cancer(CRC),a prevalent malignancy of the digestive system,is characterized by high incidence and mortality rates,ranking as the third most common cancer type.Recent research indicates that snoRNA dysregulation is associated with CRC,as snoRNA expression significantly differs between normal and cancerous conditions.Consequently,assessing snoRNA expression level and function holds promise for the prognosis and diagnosis of CRC.Nevertheless,current comprehension of the potential roles of snoRNAs in CRC remains limited.This review offers a comprehensive survey of the aberrant regulation of snoRNAs in CRC,providing valuable insights into the discovery of novel biomarkers,therapeutic targets,and potential tools for the diagnosis and treatment of CRC and furnishing critical cues for advancing research into CRC and the judicious selection of therapeutic targets.展开更多
Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and hap...Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype.Methods:Thirty-nine blood samples infected with P.knowlesi were collected in Sabah,Malaysian Borneo and Peninsular Malaysia.The S-type SSU rRNA gene was amplified using polymerase chain reaction,cloned into a vector,and sequenced.The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10.This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11.Results:Overall,the phylogenetic analysis showed no evidence of a geographical cluster of P.knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences.The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect.Haplotype sharing of the S-type SSU rRNA gene was observed between the P.knowlesi isolates in Sabah,Malaysian Borneo,but not between Sabah,Malaysian Borneo and Peninsular Malaysia.Conclusions:This study suggests that the S-type SSU rRNA gene of P.knowlesi isolates in Sabah,Malaysian Borneo,and Peninsular Malaysia has fewer polymorphic sites,representing the conservation of the gene.These features make the S-type SSU rRNA gene suitable for comparative studies,such as determining the evolutionary relationships and common ancestry among P.knowlesi species.展开更多
Objective:To give a brief overview of the field of epigenetics and the potential predictive power that small non-coding RNA(sncRNA)may hold in relation to improving the treatment and diagnosis of male infertility.Meth...Objective:To give a brief overview of the field of epigenetics and the potential predictive power that small non-coding RNA(sncRNA)may hold in relation to improving the treatment and diagnosis of male infertility.Methods:PRISMA-ScR was used as the scoping review guideline for this investigation.All article data here have been accessed from MEDLINE–PubMed,Science Direct,EBSCO,Scopus,Sage Journals,and Google Scholar.The terms"small non coding RNA,male,infertility,miRNA,sperm"were used in the search between 2015 and 2023.Results:The study comprised 35 publications in total.Several sncRNAs,miR-155,miR-16,miR-196,miR-525-3p,miR-891 were found to be effective in regulating the mechanism of spermatozoa processing in the infertility of men.sncRNA can be used as a biomarker of male infertility.Conclusions:sncRNAs can act as biomarkers for the diagnosis of reproductive diseases.Actually,by recognizing sncRNAs and their mechanisms,a new way to treat infertile men would be paved.The functional annotation of sncRNAs in spermatogenesis is still in its infancy but has enormous potential.This is despite the fact that many potential sncRNAs have been found to date with the use of cutting-edge technology and publicly accessible sncRNA annotation tools.展开更多
基金The Youth Foundation of Heilongjiang Province,No.QC06C061the Foundation of Education Department,Heilongjiang Province,No.11521089
文摘AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression in HepG2.2.15 cells by combination of small interfering RNAs (siRNAs). METHODS: Recombinant plasmid psiI-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay (ELISA). Intracellular viral DNA and covalently closed circular DNA (cccDNA) were quantified by real-time polymerase chain reaction (PCR). HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR (RT-PCR). RESULTS: siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantlycombination of siRNAs significantly suppressed HBV cccDNA amplification. CONCLUSION: Combination of siRNAs mediates a stronger inhibition on viral replication and antigenexpression in HepG2.2.15 cells, especially on cccDNA amplification.
基金Supported by"Fondazione Cassa di Risparmio of Trieste"the"Fondazione Benefica Kathleen Foreman Casali of Trieste"+2 种基金the"Beneficentia Stiftung"of Vaduz Liechtensteinthe Italian Minister of Instruction,UniversityResearch(MIUR),PRIN 2010-11,No.20109PLMH2(in part)
文摘Hepatocellular carcinoma(HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective,especially for the advanced forms of the disease. In the last year,short double stranded RNA molecules termed small interfering RNAs(si RNAs) and micro interfering RNAs(mi RNA),emerged as interesting molecules with potential therapeutic value for HCC. The practical use of these molecules is however limited by the identification of optimal molecular targets and especially by the lack of effective and targeted HCC delivery systems. Here we focus our discussion on the most recent advances in the identification of si RNAs/mi RNAs molecular targets and on the development of suitable si RNA/mi RNAs delivery systems.
基金Supported by the Key Science and Technology Research Project ofShaanxi Province [2003K10-G35,2004K13-G11(1)].
文摘Objective To construct an expression vector of small interfering RNA (siRNA) against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast cancer cell line MCF-7. Methods Constructed an expression vector of siRNA against survivin and transfected it into PC-2 and MCF-7 cells using lipofectamine^TM 2000. The expression of survivin was detected by semi-quanfifive RT-PCR and immunohistochemistry, and its effects on proliferation of PC-2 and MCF-7 cells were detected by MTT assay. Results The introduction of sequence-specific siRNA could efficiently suppress survivin expression at both mRNA and protein levels in the two cancer cell lines. In PC-2 cells, the expression inhibition rates were 81.25% at mRNA level and 74.24% at protein level In MCF-7 cells, the expression inhibition rates were 64.91% at mRNA level and 79. 72% at protein level The proliferation of PC-2 and MCF-7 cells was also suppressed, and24 and 48 hours after the cells were reseeded, the proliferation inhibition rates of PC-2 cells were 28. 00% and 33. 38%, and that of MCF-7 cells were 31.58% and 33.02%, respectively. Conclusions The expression vector of siRNA against survivin can block survivin expression in PC-2 and MCF-7 cells efficiently and specifically. Down regulation of survivin expression can suppress proliferation of PC-2 and MCF-7 cells. Survivin RNAi may have potential value in gene therapy of human cancers.
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
基金the grants from the Research Foundation of Science & Technology Bureau of Guangzhou(2004Z2-E0011)the Guangdong Province Natural Science Foundation(5002318)
文摘Objective: HOXB7 gene is a kind of transcription regulator over-expressed in malignant melanoma (MM) cell lines. It can specifically up-regulate the expression of angiogenic factors and tumor growth factors such as bFGF, GROa, VEGF and induce angiogenesis in melanoma, resulting in the proliferation and metastasis of tumor cells. We designed and synthesized HOXB7 specific siRNA to study its interfering effect on the expressions of HOXB7 and bFGF genes in melanoma A375 cell line and the biologic characteristics of A375 cells. Methods: Three synthesized siRNA with different sequences were separately transfected into A375 cells by lipofecter 2000. The expression of HOXB7 and bFGF mRNA in transfected cells was detected by RT-PCR 24 and 48 hours after transduction. The expression of bFGF protein in the transfected cells were detected by flowcytometry 48 hours after transfection. MTT assay was used to analyze the cell proliferation rate of siRNA transfected cells. Based on the in vitro experiment results, one effective siRNA sequence was selected for the construction of in vivo siRNA expression vector. Then, a malignant melanoma animal model was established. The siRNA expression plasmid was injected into the tumor foci and its influence on the growth and angiogenesis of tumor was observed. Results: The mRNA expressions of both HOXB7 and bFGF genes in the A375 cells reduced significantly 24 and 48 hour after transfection of siRNA. Expression level of the protein of angiogenic factor bFGF induced by HOXB7 gene in siRNA transfected cells was significantly lower than that in control cells 48 hours after transduction. Cell proliferation was also suppressed in siRNA transfected cells. Two of the three siRNA strands showed prominent interference effect. The in vivo study indicated that the tumor size and the microvessel density in the tumor both reduced after injection of HOXB7siRNA plasmid. Conclusion: Down-regulation of HOXB7 gene expression can effectively reduce the expression of angiogenic factor bFGF and the proliferation of MM cells. Besides, the growth and angiogenesis of MM tumor were also inhibited.
基金supported by a grant from Natural Science Program Foundation of Guangdong Province,China(No.04010446).
文摘OBJECTIVE To investigate whether small hairpin RNA (shRNA)targeting Bcl-2 mRNA could inhibit the growth of lymphomatransplanted subcutaneously in nude mice.METHODS Recombinant Bcl-2 shRNA expression vector withgreen fluorescence protein (GFP) gene was constructed andpreserved in our lab. We evaluated the antitumor effect of the Bcl-2shRNA in vivo which was the model of nude mice bearing Rajicells xenografts. Human Raji cells were injected subcutaneouslyinto nude mice to establish lymphoma models. When thediameters of tumor were above 0.5 cm after Raji cells injection,the mice bearing tumor were randomly divided into four groups:saline control group, negative shRNA group, plasmid vectorgroup, Bcl-2 shRNA group. The polyethylenimine (PEI) was usedto transfect shRNA into tumor. The mixed PEI and shRNA wasinjected into tumors. The growth and size of tumor were observed.Tissue was stained by H&E for its pathological morphology. Theexpression of Bcl-2 mRNA in the tumor mass was detected byreverse transcription polymerase chain reaction (RT-PCR).RESULTS A significant difference in median tumor weightwas observed in mice treated with Bcl-2 shRNA, compared withthose in the groups of negative shRNA or plasmid vector or salinesolution (P< 0.05). Pathological evaluation was completed in allexcised tumors from nude mice bearing Raji cells xenografts.The tumor tissue of the mice treated with Bcl-2 shRNA showedapoptosis, serious necrosis of the cells and inflammatory cellsinfiltration. There was no change in the morphology of cellsamong negative shRNA, plasmid vector and saline solution group.In the group of the Bcl-2 shRNA, the expression levels of Bcl-2mRNA of the tumor tissue were effectively inhibited (P < 0.05).CONCLUSION The shRNA targeting at the Bcl-2 mRNAcould inhibit the growth of human lymphoma transplantedsubcutaneously in nude mice.
文摘Hepatocellular carcinoma(HCC) is the 5th most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorablesystemic side-effects of chemotherapeutic agents and susceptibility to the degradation of small interfering RNAs(si RNAs), which can knock down a specific gene involved in the disease, have hampered their clinical application. So, it could be beneficial to develop an efficient carrier for the stabilization and specific delivery of drugs and si RNA to cells. Targeted nanoparticles have gained considerable attention as an efficient drug and gene delivery system, which is due to their capability in achieving the highest accumulation of cytotoxic agents in tumor tissue, modifiable drug pharmacokinetic- and bio-distribution, improved effectiveness of treatment, and limited sideeffects. Recent studies have shed more light on the advantages of novel drug loaded carrier systems vs free drugs. Most of the animal studies have reported improvement in treatment efficacy and survival rate using novel carrier systems. Targeted delivery may be achieved passively or actively. In passive targeting, no ligand as homing device is used, while targeting is achieved by incorporating the therapeutic agent into a macromolecule or nanoparticle that passively reaches the target organ. However, in active targeting, the therapeutic agent or carrier system is conjugated to a tissue or cell-specific receptor which is overexpressed in a special malignancy using a ligand called a homing device. This review covers a broad spectrum of targeted nanoparticles as therapeutic and nonviral si RNA delivery systems, which are developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and their characteristics and opportunities for the clinical applications of drugs and therapeutic si RNA are discussed in this article. Asialoglycoprotein receptors, low-density lipoprotein, ganglioside GM1 cell surface ligand, epidermal growth factor receptor receptors, monoclonal antibodies, retinoic acid receptors, integrin receptors targeted by Arg-Gly-Asp peptide, folate, and transferrin receptors are the most widely studied cell surface receptors which are used for the site specific delivery of drugs and si RNA-based therapeutics in HCC and discussed in detail in this article.
基金supported by the Grant No.2003AA208215 from the National High Technology Programs of Chinathe Grant No.30270311 from the National Natural Science Foundation of China.
文摘RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0~60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.
基金Supported by National Natural Science Foundation of China(81373465)
文摘Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving increased synthesis of matrix components and a failure of physiological mechanisms of matrix turnover. Activation of hepatic stellate cells(HSCs) remains a central event in fibrosis. HSCs are the main source of extracellular matrix(ECM). Transforming growth factor-beta(TGF-β), which is the fibrogenic master cytokine, can induce the activation of HSCs to produce a large amount of ECM, and is capable of inducing apoptosis of liver cells. RNA interference(RNAi) is a novel gene disruption technology. Studies have shown that small interfering RNA(si RNA) targeting TGF-β1 may inhibit the activation and proliferation of HSCs, suppress ECM synthesis and block liver fibrosis. TGF-β1 si RNA-mediated gene silencing therapy provides a new avenue for liver fibrosis. This review summarizes recent progresses in research on HSCs, TGF-β1 and TGF-β1 si RNA in liver fibrosis.
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.
基金National Nature Science Foundation of China(30570828,30471961,30170915).
文摘RNA interference(RNAi),which causes the degradation of any RNA in a sequence specific manner,is a posttranscriptional gene silencing mechanism.Targeting the invariant chain(Ii)in DCs has been used as an approach to enhance antitumor immunity.It is demonstrated in this article that transfection of H-2(K)DCs with siRNA specific for Ii gene can significantly knock down Ii.When exposed to TNF-alpha,immature DCs transfected with Ii siRNA can differentiate into mature DCs without reducing viability or IL-12p70 production.Ii siRNA-treated H-2(K)DCs exhibited an increased allostimulatory capacity in a lymphocyte proliferation assay.Furthermore,Ii siRNA-transfected H-2(K)DCs enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production,and much stronger cytotoxic activity was observed when DCs were co-transfected with Ii siRNA and an endogenous tumor antigen in vitro.Our findings indicate that silencing the Ii gene in DCs with siRNA may offer a potential approach to enhancing antitumor immunotherapy.
基金the National Natural Science Foundation of China,No.82273457Guangdong Basic and Applied Basic Research Foundation,No.2021A1515012180 and No.2023A1515012762+1 种基金Special Grant for Key Area Programs of Guangdong Department of Education,No.2021ZDZX2040and Science and Technology Special Project of Guangdong Province,No.210715216902829.
文摘Small nucleolar RNAs(snoRNAs)represent a class of non-coding RNAs that play pivotal roles in post-transcriptional RNA processing and modification,thereby contributing significantly to the maintenance of cellular functions related to protein synthesis.SnoRNAs have been discovered to possess the ability to influence cell fate and alter disease progression,holding immense potential in controlling human diseases.It is suggested that the dysregulation of snoRNAs in cancer exhibits differential expression across various cancer types,stages,metastasis,treatment response and/or prognosis in patients.On the other hand,colorectal cancer(CRC),a prevalent malignancy of the digestive system,is characterized by high incidence and mortality rates,ranking as the third most common cancer type.Recent research indicates that snoRNA dysregulation is associated with CRC,as snoRNA expression significantly differs between normal and cancerous conditions.Consequently,assessing snoRNA expression level and function holds promise for the prognosis and diagnosis of CRC.Nevertheless,current comprehension of the potential roles of snoRNAs in CRC remains limited.This review offers a comprehensive survey of the aberrant regulation of snoRNAs in CRC,providing valuable insights into the discovery of novel biomarkers,therapeutic targets,and potential tools for the diagnosis and treatment of CRC and furnishing critical cues for advancing research into CRC and the judicious selection of therapeutic targets.
基金This study was supported by the Ministry of Higher Education,Malaysia(FRGS0322-SG-1/2013)Universiti Malaysia Sabah(GUG0521-2/2020).
文摘Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype.Methods:Thirty-nine blood samples infected with P.knowlesi were collected in Sabah,Malaysian Borneo and Peninsular Malaysia.The S-type SSU rRNA gene was amplified using polymerase chain reaction,cloned into a vector,and sequenced.The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10.This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11.Results:Overall,the phylogenetic analysis showed no evidence of a geographical cluster of P.knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences.The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect.Haplotype sharing of the S-type SSU rRNA gene was observed between the P.knowlesi isolates in Sabah,Malaysian Borneo,but not between Sabah,Malaysian Borneo and Peninsular Malaysia.Conclusions:This study suggests that the S-type SSU rRNA gene of P.knowlesi isolates in Sabah,Malaysian Borneo,and Peninsular Malaysia has fewer polymorphic sites,representing the conservation of the gene.These features make the S-type SSU rRNA gene suitable for comparative studies,such as determining the evolutionary relationships and common ancestry among P.knowlesi species.
文摘Objective:To give a brief overview of the field of epigenetics and the potential predictive power that small non-coding RNA(sncRNA)may hold in relation to improving the treatment and diagnosis of male infertility.Methods:PRISMA-ScR was used as the scoping review guideline for this investigation.All article data here have been accessed from MEDLINE–PubMed,Science Direct,EBSCO,Scopus,Sage Journals,and Google Scholar.The terms"small non coding RNA,male,infertility,miRNA,sperm"were used in the search between 2015 and 2023.Results:The study comprised 35 publications in total.Several sncRNAs,miR-155,miR-16,miR-196,miR-525-3p,miR-891 were found to be effective in regulating the mechanism of spermatozoa processing in the infertility of men.sncRNA can be used as a biomarker of male infertility.Conclusions:sncRNAs can act as biomarkers for the diagnosis of reproductive diseases.Actually,by recognizing sncRNAs and their mechanisms,a new way to treat infertile men would be paved.The functional annotation of sncRNAs in spermatogenesis is still in its infancy but has enormous potential.This is despite the fact that many potential sncRNAs have been found to date with the use of cutting-edge technology and publicly accessible sncRNA annotation tools.