Objective To silence annexin Ⅱ gene expression by using small interference RNA （siRNA） in prostate cancer cell line PC3. Methods For in vitro transcription, four sequences of 29-nucleotide DNA template oligonucleo...Objective To silence annexin Ⅱ gene expression by using small interference RNA （siRNA） in prostate cancer cell line PC3. Methods For in vitro transcription, four sequences of 29-nucleotide DNA template oligonucleotides were designed, and one pair of the sequences were complementary to annexin Ⅱ gene. The other pair was negative control. The 8 nucleotides at the 3＇ end of each oligonucleotide were complementary to the T7 Promoter Primer. The sense and anti-sense siRNA templates were transcribed by T7 RNA polymerase and the resulting RNA transcripts were hybridized to create dsRNA. The siRNA was transfected into prostate cancer cell PC3. For assaying the efficiency of siRNA, confocal microscopy, Northern blotting, and Western blotting were employed to examine the expression of annexin Ⅱ protein and its mRNA. ^3H thymidine was used to measure DNA synthesis. Results The siRNA sequence specific to annexin Ⅱ gene was capable of inhibiting the expression of annexin Ⅱ protein and its mRNA. And cellular DNA synthesis was significantly reduced in siRNA transfected cells.Conclusions The protocol for the synthesis of siRNA by T7 RNA polymerase is feasible. Annexin Ⅱ might be involved in DNA synthesis.展开更多
We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord trans...We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was signiifcantly enhanced in the model group. Af-ter 8 weeks, the number of horseradish peroxidase-labeled nerve ifbers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and signiifcantly higher than in the model group. The newly formed nerve ifbers and myelinated ner ve ifbers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.展开更多
Upon the discovery of RNA interference(RNAi),canonical small interfering RNA(si RNA) has been recognized to trigger sequence-specific gene silencing. Despite the benefits of si RNAs as potential new drugs,there are ob...Upon the discovery of RNA interference(RNAi),canonical small interfering RNA(si RNA) has been recognized to trigger sequence-specific gene silencing. Despite the benefits of si RNAs as potential new drugs,there are obstacles still to be overcome,including off-target effects and immune stimulation. More recently,Dicer substrate si RNA(Dsi RNA) has been introduced as an alternative to si RNA. Similarly,it also is proving to be potent and target-specific,while rendering less immune stimulation. Dsi RNA is 25–30 nucleotides in length,and is further cleaved and processed by the Dicer enzyme. As with si RNA,it is crucial to design and develop a stable,safe,and efficient system for the delivery of Dsi RNA into the cytoplasm of targeted cells. Several polymeric nanoparticle systems have been well established to load Dsi RNA for in vitro and in vivo delivery,thereby overcoming a major hurdle in the therapeutic uses of Dsi RNA. The present review focuses on a comparison of si RNA and Dsi RNA on the basis of their design,mechanism,in vitro and in vivo delivery,and therapeutics.展开更多
Cancer has become the most life-threatening disease in the world.Mutations in and aberrant expression of genes encoding proteins and mutations in noncoding RNAs,especially long noncoding RNAs(lncRNAs),have significant...Cancer has become the most life-threatening disease in the world.Mutations in and aberrant expression of genes encoding proteins and mutations in noncoding RNAs,especially long noncoding RNAs(lncRNAs),have significant effects in human cancers.LncRNAs have no protein-coding ability but function extensively in numerous physiological and pathological processes.Small nucleolar RNA host gene 3(SNHG3)is a novel lncRNA and has been reported to be differentially expressed in various tumors,such as liver cancer,gastric cancer,and glioma.However,the interaction mechanisms for the regulation between SNHG3 and tumor progression are poorly understood.In this review,we summarize the results of SNHG3 studies in humans,animal models,and cells to underline the expression and role of SNHG3 in cancer.SNHG3 expression is upregulated in most tumors and is detrimental to patient prognosis.SNHG3 expression in lung adenocarcinoma remains controversial.Concurrently,SNHG3 affects oncogenes and tumor suppressor genes through various mechanisms,including competing endogenous RNA effects.A deeper understanding of the contribution of SNHG3 in clinical applications and tumor development may provide a new target for cancer diagnosis and treatment.展开更多
Objective:The aim of this study was to analyze the expression features of hypoxia inducible factor-1α (HIF-1α) in hepatocellular carcinoma (HCC) and effects of HIF-1α silencing on HepG2 cells.Methods:HIF-1α expres...Objective:The aim of this study was to analyze the expression features of hypoxia inducible factor-1α (HIF-1α) in hepatocellular carcinoma (HCC) and effects of HIF-1α silencing on HepG2 cells.Methods:HIF-1α expression was analyzed in the self-control HCC specimens by immunohistochemistry.After HepG2 cells with miRNA transfection,the expression of HIF-1α was determined at mRNA or protein level by real-time polymerase chain reaction (PCR) or Western blotting.Vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG-2) were determined by ELISA.Alterations of cell cycles and apoptosis of HepG2 cells were measured using a flow cytometer.Results:Positive HIF-1α was brown and granule-like in the cytoplasm or nucleus.Significant difference was found between HCC (80%) and its surrounding tissues (100%,χ2=22.35,P < 0.001) and HIF-1α expression related to tumor size.At 72 h after miRNA transfection,the expression of HIF-1α in HepG2 cells was down-regulated by 87% at mRNA or 65% at protein level,with VEGF and ANG-2 decreased to 54% and 36%,respectively.After RNA interference combined with anti-cancer drug,the apoptotic rate of HepG2 cells was increasing from 22.46% ± 0.61% to 36.99% ± 0.88%,with up-regulation of G1 phase (65.68% ± 0.91%) and down-regulation of S phase (19.47 ± 1.34 %).Conclusion:Abnormal expression of HIF-1α is associated with development of HCC,and HIF-1α gene silencing can effectively inhibit HepG2 cell proliferation.展开更多
While human immunodeficiency virus 1(HIV-1) infectionis controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from th...While human immunodeficiency virus 1(HIV-1) infectionis controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from the body. Despite decades of research there is still no effective vaccine to prevent HIV-1 infection. Therefore, the possibility of an RNA interference(RNAi)-based cure has become an increasingly explored approach. Endogenous gene expression is controlled at both, transcriptional and post-transcriptional levels by noncoding RNAs, which act through diverse molecular mechanisms including RNAi. RNAi has the potential to control the turning on/off of specific genes through transcriptional gene silencing(TGS), as well as finetuning their expression through post-transcriptional gene silencing(PTGS). In this review we will describe in detail the canonical RNAi pathways for PTGS and TGS, the relationship of TGS with other silencing mechanisms and will discuss a variety of approaches developed to suppress HIV-1 via manipulation of RNAi. We will briefly compare RNAi strategies against other approaches developed to target the virus, highlighting their potential to overcome the major obstacle to finding a cure, which is the specific targeting of the HIV-1 reservoir within latently infected cells.展开更多
文摘Objective To silence annexin Ⅱ gene expression by using small interference RNA （siRNA） in prostate cancer cell line PC3. Methods For in vitro transcription, four sequences of 29-nucleotide DNA template oligonucleotides were designed, and one pair of the sequences were complementary to annexin Ⅱ gene. The other pair was negative control. The 8 nucleotides at the 3＇ end of each oligonucleotide were complementary to the T7 Promoter Primer. The sense and anti-sense siRNA templates were transcribed by T7 RNA polymerase and the resulting RNA transcripts were hybridized to create dsRNA. The siRNA was transfected into prostate cancer cell PC3. For assaying the efficiency of siRNA, confocal microscopy, Northern blotting, and Western blotting were employed to examine the expression of annexin Ⅱ protein and its mRNA. ^3H thymidine was used to measure DNA synthesis. Results The siRNA sequence specific to annexin Ⅱ gene was capable of inhibiting the expression of annexin Ⅱ protein and its mRNA. And cellular DNA synthesis was significantly reduced in siRNA transfected cells.Conclusions The protocol for the synthesis of siRNA by T7 RNA polymerase is feasible. Annexin Ⅱ might be involved in DNA synthesis.
文摘We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was signiifcantly enhanced in the model group. Af-ter 8 weeks, the number of horseradish peroxidase-labeled nerve ifbers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and signiifcantly higher than in the model group. The newly formed nerve ifbers and myelinated ner ve ifbers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.
基金financial support received from Centre of Research and Instrumentation (CRIM), Universiti Kebangsaan Malaysia
文摘Upon the discovery of RNA interference(RNAi),canonical small interfering RNA(si RNA) has been recognized to trigger sequence-specific gene silencing. Despite the benefits of si RNAs as potential new drugs,there are obstacles still to be overcome,including off-target effects and immune stimulation. More recently,Dicer substrate si RNA(Dsi RNA) has been introduced as an alternative to si RNA. Similarly,it also is proving to be potent and target-specific,while rendering less immune stimulation. Dsi RNA is 25–30 nucleotides in length,and is further cleaved and processed by the Dicer enzyme. As with si RNA,it is crucial to design and develop a stable,safe,and efficient system for the delivery of Dsi RNA into the cytoplasm of targeted cells. Several polymeric nanoparticle systems have been well established to load Dsi RNA for in vitro and in vivo delivery,thereby overcoming a major hurdle in the therapeutic uses of Dsi RNA. The present review focuses on a comparison of si RNA and Dsi RNA on the basis of their design,mechanism,in vitro and in vivo delivery,and therapeutics.
基金Supported by the National Science and Technology Major Project of China,No. 2018ZX10302206 and 2017ZX10202203
文摘Cancer has become the most life-threatening disease in the world.Mutations in and aberrant expression of genes encoding proteins and mutations in noncoding RNAs,especially long noncoding RNAs(lncRNAs),have significant effects in human cancers.LncRNAs have no protein-coding ability but function extensively in numerous physiological and pathological processes.Small nucleolar RNA host gene 3(SNHG3)is a novel lncRNA and has been reported to be differentially expressed in various tumors,such as liver cancer,gastric cancer,and glioma.However,the interaction mechanisms for the regulation between SNHG3 and tumor progression are poorly understood.In this review,we summarize the results of SNHG3 studies in humans,animal models,and cells to underline the expression and role of SNHG3 in cancer.SNHG3 expression is upregulated in most tumors and is detrimental to patient prognosis.SNHG3 expression in lung adenocarcinoma remains controversial.Concurrently,SNHG3 affects oncogenes and tumor suppressor genes through various mechanisms,including competing endogenous RNA effects.A deeper understanding of the contribution of SNHG3 in clinical applications and tumor development may provide a new target for cancer diagnosis and treatment.
基金Supported by grants from Jiang su Health Key Project(No.K201102)Nantong City Social Development Project (No. S2009027)
文摘Objective:The aim of this study was to analyze the expression features of hypoxia inducible factor-1α (HIF-1α) in hepatocellular carcinoma (HCC) and effects of HIF-1α silencing on HepG2 cells.Methods:HIF-1α expression was analyzed in the self-control HCC specimens by immunohistochemistry.After HepG2 cells with miRNA transfection,the expression of HIF-1α was determined at mRNA or protein level by real-time polymerase chain reaction (PCR) or Western blotting.Vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG-2) were determined by ELISA.Alterations of cell cycles and apoptosis of HepG2 cells were measured using a flow cytometer.Results:Positive HIF-1α was brown and granule-like in the cytoplasm or nucleus.Significant difference was found between HCC (80%) and its surrounding tissues (100%,χ2=22.35,P < 0.001) and HIF-1α expression related to tumor size.At 72 h after miRNA transfection,the expression of HIF-1α in HepG2 cells was down-regulated by 87% at mRNA or 65% at protein level,with VEGF and ANG-2 decreased to 54% and 36%,respectively.After RNA interference combined with anti-cancer drug,the apoptotic rate of HepG2 cells was increasing from 22.46% ± 0.61% to 36.99% ± 0.88%,with up-regulation of G1 phase (65.68% ± 0.91%) and down-regulation of S phase (19.47 ± 1.34 %).Conclusion:Abnormal expression of HIF-1α is associated with development of HCC,and HIF-1α gene silencing can effectively inhibit HepG2 cell proliferation.
文摘While human immunodeficiency virus 1(HIV-1) infectionis controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from the body. Despite decades of research there is still no effective vaccine to prevent HIV-1 infection. Therefore, the possibility of an RNA interference(RNAi)-based cure has become an increasingly explored approach. Endogenous gene expression is controlled at both, transcriptional and post-transcriptional levels by noncoding RNAs, which act through diverse molecular mechanisms including RNAi. RNAi has the potential to control the turning on/off of specific genes through transcriptional gene silencing(TGS), as well as finetuning their expression through post-transcriptional gene silencing(PTGS). In this review we will describe in detail the canonical RNAi pathways for PTGS and TGS, the relationship of TGS with other silencing mechanisms and will discuss a variety of approaches developed to suppress HIV-1 via manipulation of RNAi. We will briefly compare RNAi strategies against other approaches developed to target the virus, highlighting their potential to overcome the major obstacle to finding a cure, which is the specific targeting of the HIV-1 reservoir within latently infected cells.
