·AIM: To explore the potential mechanism of molecular hydrogen in the regulation of miRNA expression and signal-modulating activities. ·METHODS: Retinal microglia cells were activated by Lipopolysaccharides ...·AIM: To explore the potential mechanism of molecular hydrogen in the regulation of miRNA expression and signal-modulating activities. ·METHODS: Retinal microglia cells were activated by Lipopolysaccharides (LPS) and then treated with hydrogen -saturated medium or normal medium without hydrogen. qRT -PCR was used to detect the expression difference in miR-9, miR-21 and miR-199 between these two groups. Moreover, the expression of LPS -induced signaling proteins, including Myd88, IKK -β, NF -kB, and PDCD4, were detected by Western blotting. ·RESULTS: The results demonstrated a marked downregulation of miR -9 and miR -21 and up -regulation of miR-199 by hydrogen treatment; the expression of Myd88 and IKK-β was decreased after hydrogen treatment, whereas PDCD4 was increased, and there was no significant change in NF-kB expression. · CONCLUSION: The results in the present study indicate that miR -9, miR -199 and miR -21 play an important role in the anti -inflammatory regulation of LPS -activated microglia cells by molecular hydrogen, which will help to explain the protective mechanism of molecular hydrogen against inflammatory injury.·展开更多
MicroRNAs （miRNAs） play critical roles in the development and progression in various cancers. Dysfunctional miR-9 expression remains ambiguous, and no consensus on the metastatic progression of ovarian cancer has be...MicroRNAs （miRNAs） play critical roles in the development and progression in various cancers. Dysfunctional miR-9 expression remains ambiguous, and no consensus on the metastatic progression of ovarian cancer has been reached. In this study, results from the bioinformatics analysis show that the 3＇-UTR of the E- cadherin mRNA was directly regulated by miR-9. Luciferase reporter assay results confirmed that miR-9 could directly target this 3＇-UTR. miR-9 and E-cadherin expression in ovarian cancer tissue was quantified by qRT- PCR. Migration and invasion were detected by wound healing and Transwell system assay in SKOV3 and A2780. qRT-PCR and Western blot were performed to detect the epithelial-mesenchymal transition-associated mRNA and proteins. Immunofluorescence technique was used to analyze the expression and subcellular localization of E- cadherin, N-cadherin, and vimentin. The results showed that miR-9 was frequently upregulated in metastatic serous ovarian cancer tissue compared with paired primary ones. Upregulation of miR-9 could downregulate the expression of E-cadherin but upregulate the expression of mesenchymal markers （N-cadherin and vimentin）. Overexpression of miR-9 could promote the cell migration and invasion in ovarian cancer, and these processes could be effectively inhibited via miR-9 inhibitor. Thus, our study demonstrates that miR-9 may promote ovarian cancer metastasis via targeting E-cadherin and a novel potential therapeutic approach to control metastasis of ovarian cancer.展开更多
目的:探讨miR-9在阿魏酸钠(SF)改善缺氧缺血性脑损伤新生大鼠学习记忆中对海马神经元NMDAR1和NgR1的调节作用。方法:88只7日龄SD大鼠随机分为假手术组(sham)、缺氧缺血性脑损伤组(HIBD)、阿魏酸钠治疗组(HIBD+SF)、miR-9抑制组(HIBD+LNA...目的:探讨miR-9在阿魏酸钠(SF)改善缺氧缺血性脑损伤新生大鼠学习记忆中对海马神经元NMDAR1和NgR1的调节作用。方法:88只7日龄SD大鼠随机分为假手术组(sham)、缺氧缺血性脑损伤组(HIBD)、阿魏酸钠治疗组(HIBD+SF)、miR-9抑制组(HIBD+LNA-miR-9),每组各22只。假手术组仅分离左侧颈总动脉,其余组结扎左侧颈总动脉进行缺氧后制作HIBD模型。于21日龄,阿魏酸钠治疗组连续5 d腹腔注射阿魏酸钠,miR-9抑制组经左侧脑室注射一次LNA-miR-9。于30日龄行Morris水迷宫检测各组大鼠空间学习记忆能力。于26日龄,real time RT-PCR检测各组大鼠左侧海马神经元miR-9、NMDAR1mRNA和NgR1mRNA的表达,Western Blot检测NMDAR1和NgR1蛋白的表达。结果:Morris水迷宫实验显示:与假手术组比较,HIBD组逃避潜伏期延长(P<0.05)、在目标象限逗留时间缩短(P<0.05);阿魏酸钠治疗组、miR-9抑制组较HIBD组均有逃避潜伏期缩短(P<0.05)、在目标象限逗留时间延长(P<0.05)。real time RT-PCR、Western Blot检测显示:HIBD组NMDAR1和NgR1的mRNA和蛋白表达以及miR-9表达较假手术组均明显上调(P<0.05);阿魏酸钠治疗组、miR-9抑制组较HIBD组各指标均明显下调(P<0.05)。结论:阿魏酸钠可能通过下调miR-9来抑制NMDAR1和NgR1的表达从而改善缺氧缺血大鼠的学习记忆能力。展开更多
MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs.However,the functio...MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein (EGFP) gene was used as reporter gene.The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.展开更多
Background:Our previous study found that mouse embryonic neural stem cell(NSC)-derived exosomes(EXOs)regulated NSC differentiation via the miR-9/Hes1 axis.However,the effects of EXOs on brain microvascular endothelial...Background:Our previous study found that mouse embryonic neural stem cell(NSC)-derived exosomes(EXOs)regulated NSC differentiation via the miR-9/Hes1 axis.However,the effects of EXOs on brain microvascular endothelial cell(BMEC)dysfunction via the miR-9/Hes1 axis remain unknown.Therefore,the current study aimed to determine the effects of EXOs on BMEC proliferation,migration,and death via the miR-9/Hes1 axis.Methods:Immunofluorescence,quantitative real-time polymerase chain reaction,cell counting kit-8 assay,wound healing assay,calcein-acetoxymethyl/propidium iodide staining,and hematoxylin and eosin staining were used to determine the role and mechanism of EXOs on BMECs.Results:EXOs promoted BMEC proliferation and migration and reduced cell death under hypoxic conditions.The overexpression of miR-9 promoted BMEC prolifera-tion and migration and reduced cell death under hypoxic conditions.Moreover,miR-9 downregulation inhibited BMEC proliferation and migration and also promoted cell death.Hes1 silencing ameliorated the effect of amtagomiR-9 on BMEC proliferation and migration and cell death.Hyperemic structures were observed in the regions of the hippocampus and cortex in hypoxia-induced mice.Meanwhile,EXO treatment improved cerebrovascular alterations.Conclusion:NSC-derived EXOs can promote BMEC proliferation and migra-tion and reduce cell death via the miR-9/Hes1 axis under hypoxic conditions.Therefore,EXO therapeutic strategies could be considered for hypoxia-induced vascular injury.展开更多
During the development of therapeutic microRNAs(miRNAs or miRs),it is essential to define their pharmacological actions.Rather,miRNA research and therapy mainly use miRNA mimics synthesized in vitro.After experimental...During the development of therapeutic microRNAs(miRNAs or miRs),it is essential to define their pharmacological actions.Rather,miRNA research and therapy mainly use miRNA mimics synthesized in vitro.After experimental screening of unique recombinant miRNAs produced in vivo,three lead antiproliferative miRNAs against human NSCLC cells,miR-22-3p,miR-9-5p,and miR-218-5p,were revealed to target folate metabolism by bioinformatic analyses.Recombinant miR-22-3p,miR-9-5p,and miR-218-5p were shown to regulate key folate metabolic enzymes to inhibit folate metabolism and subsequently alter amino acid metabolome in NSCLC A549 and H1975 cells.Isotope tracing studies further confirmed the disruption of one-carbon transfer from serine to folate metabolites by all three miRNAs,inhibition of glucose uptake by miR-22-3p,and reduction of serine biosynthesis from glucose by miR-9-5p and-218-5p in NSCLC cells.With greater activities to interrupt NSCLC cell respiration,glycolysis,and colony formation than miR-9-5p and-218-5p,recombinant miR-22-3p was effective to reduce tumor growth in two NSCLC patient-derived xenograft mouse models without causing any toxicity.These results establish a common antifolate mechanism and differential actions on glucose uptake and metabolism for three lead anticancer miRNAs as well as antitumor efficacy for miR-22-3p nanomedicine,which shall provide insight into developing antimetabolite RNA therapies.展开更多
基金Foundation of Heilongjiang Educational Committee Project, China (No. 12511313)Heilongjiang Provincial Foundation for Youths Project, China (No.QC2011C119)Research Program of Heilongjiang Provincial Health Office, China (No. 2009-197)
文摘·AIM: To explore the potential mechanism of molecular hydrogen in the regulation of miRNA expression and signal-modulating activities. ·METHODS: Retinal microglia cells were activated by Lipopolysaccharides (LPS) and then treated with hydrogen -saturated medium or normal medium without hydrogen. qRT -PCR was used to detect the expression difference in miR-9, miR-21 and miR-199 between these two groups. Moreover, the expression of LPS -induced signaling proteins, including Myd88, IKK -β, NF -kB, and PDCD4, were detected by Western blotting. ·RESULTS: The results demonstrated a marked downregulation of miR -9 and miR -21 and up -regulation of miR-199 by hydrogen treatment; the expression of Myd88 and IKK-β was decreased after hydrogen treatment, whereas PDCD4 was increased, and there was no significant change in NF-kB expression. · CONCLUSION: The results in the present study indicate that miR -9, miR -199 and miR -21 play an important role in the anti -inflammatory regulation of LPS -activated microglia cells by molecular hydrogen, which will help to explain the protective mechanism of molecular hydrogen against inflammatory injury.·
文摘MicroRNAs （miRNAs） play critical roles in the development and progression in various cancers. Dysfunctional miR-9 expression remains ambiguous, and no consensus on the metastatic progression of ovarian cancer has been reached. In this study, results from the bioinformatics analysis show that the 3＇-UTR of the E- cadherin mRNA was directly regulated by miR-9. Luciferase reporter assay results confirmed that miR-9 could directly target this 3＇-UTR. miR-9 and E-cadherin expression in ovarian cancer tissue was quantified by qRT- PCR. Migration and invasion were detected by wound healing and Transwell system assay in SKOV3 and A2780. qRT-PCR and Western blot were performed to detect the epithelial-mesenchymal transition-associated mRNA and proteins. Immunofluorescence technique was used to analyze the expression and subcellular localization of E- cadherin, N-cadherin, and vimentin. The results showed that miR-9 was frequently upregulated in metastatic serous ovarian cancer tissue compared with paired primary ones. Upregulation of miR-9 could downregulate the expression of E-cadherin but upregulate the expression of mesenchymal markers （N-cadherin and vimentin）. Overexpression of miR-9 could promote the cell migration and invasion in ovarian cancer, and these processes could be effectively inhibited via miR-9 inhibitor. Thus, our study demonstrates that miR-9 may promote ovarian cancer metastasis via targeting E-cadherin and a novel potential therapeutic approach to control metastasis of ovarian cancer.
文摘目的:探讨miR-9在阿魏酸钠(SF)改善缺氧缺血性脑损伤新生大鼠学习记忆中对海马神经元NMDAR1和NgR1的调节作用。方法:88只7日龄SD大鼠随机分为假手术组(sham)、缺氧缺血性脑损伤组(HIBD)、阿魏酸钠治疗组(HIBD+SF)、miR-9抑制组(HIBD+LNA-miR-9),每组各22只。假手术组仅分离左侧颈总动脉,其余组结扎左侧颈总动脉进行缺氧后制作HIBD模型。于21日龄,阿魏酸钠治疗组连续5 d腹腔注射阿魏酸钠,miR-9抑制组经左侧脑室注射一次LNA-miR-9。于30日龄行Morris水迷宫检测各组大鼠空间学习记忆能力。于26日龄,real time RT-PCR检测各组大鼠左侧海马神经元miR-9、NMDAR1mRNA和NgR1mRNA的表达,Western Blot检测NMDAR1和NgR1蛋白的表达。结果:Morris水迷宫实验显示:与假手术组比较,HIBD组逃避潜伏期延长(P<0.05)、在目标象限逗留时间缩短(P<0.05);阿魏酸钠治疗组、miR-9抑制组较HIBD组均有逃避潜伏期缩短(P<0.05)、在目标象限逗留时间延长(P<0.05)。real time RT-PCR、Western Blot检测显示:HIBD组NMDAR1和NgR1的mRNA和蛋白表达以及miR-9表达较假手术组均明显上调(P<0.05);阿魏酸钠治疗组、miR-9抑制组较HIBD组各指标均明显下调(P<0.05)。结论:阿魏酸钠可能通过下调miR-9来抑制NMDAR1和NgR1的表达从而改善缺氧缺血大鼠的学习记忆能力。
基金Project supported by the National Basic Research Program (973) of China (No. 2005CB121004)the National High-Tech R & D Program (863) of China (No. 2006AA10A119)+1 种基金the Innovation Foundation for Graduate Students of Jiangsu Provincethe National Natural Science Foundation of China (No. 61001013)
文摘MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21–23 nucleotides in length,which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein (EGFP) gene was used as reporter gene.The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.
基金Program of Natural Science Foundation of Shanghai,Grant/Award Number:21ZR1453800 and 22ZR1452400Program of National Natural Science Foundation of China,Grant/Award Number:82370057+3 种基金Fundamental Research Funds for the Central Universities,Grant/Award Number:22120220562Program of Shanghai Municipal Health Commission,Grant/Award Number:20204Y0384Program of National Key Research and Development Project of China,Grant/Award Number:2023YFC2509500。
文摘Background:Our previous study found that mouse embryonic neural stem cell(NSC)-derived exosomes(EXOs)regulated NSC differentiation via the miR-9/Hes1 axis.However,the effects of EXOs on brain microvascular endothelial cell(BMEC)dysfunction via the miR-9/Hes1 axis remain unknown.Therefore,the current study aimed to determine the effects of EXOs on BMEC proliferation,migration,and death via the miR-9/Hes1 axis.Methods:Immunofluorescence,quantitative real-time polymerase chain reaction,cell counting kit-8 assay,wound healing assay,calcein-acetoxymethyl/propidium iodide staining,and hematoxylin and eosin staining were used to determine the role and mechanism of EXOs on BMECs.Results:EXOs promoted BMEC proliferation and migration and reduced cell death under hypoxic conditions.The overexpression of miR-9 promoted BMEC prolifera-tion and migration and reduced cell death under hypoxic conditions.Moreover,miR-9 downregulation inhibited BMEC proliferation and migration and also promoted cell death.Hes1 silencing ameliorated the effect of amtagomiR-9 on BMEC proliferation and migration and cell death.Hyperemic structures were observed in the regions of the hippocampus and cortex in hypoxia-induced mice.Meanwhile,EXO treatment improved cerebrovascular alterations.Conclusion:NSC-derived EXOs can promote BMEC proliferation and migra-tion and reduce cell death via the miR-9/Hes1 axis under hypoxic conditions.Therefore,EXO therapeutic strategies could be considered for hypoxia-induced vascular injury.
基金supported in part by the National Cancer Institute[R01CA225958,R01253230]National Institute of General Medical Sciences[R35GM140835]+3 种基金National Institutes of Health(USA)supported by grants from the Chinese National Key Research and Development Program(2017YFE0109900,China)supported by a scholarship from the Chinese Scholarship Council(No.201806380133,China)the Molecular Pharmacology,Flow Cytometry,and Mouse Biology Shared Resources funded by the UC Davis Comprehensive Cancer Center Support Grant awarded by the National Cancer Institute(P30CA093373),National Institutes of Health。
文摘During the development of therapeutic microRNAs(miRNAs or miRs),it is essential to define their pharmacological actions.Rather,miRNA research and therapy mainly use miRNA mimics synthesized in vitro.After experimental screening of unique recombinant miRNAs produced in vivo,three lead antiproliferative miRNAs against human NSCLC cells,miR-22-3p,miR-9-5p,and miR-218-5p,were revealed to target folate metabolism by bioinformatic analyses.Recombinant miR-22-3p,miR-9-5p,and miR-218-5p were shown to regulate key folate metabolic enzymes to inhibit folate metabolism and subsequently alter amino acid metabolome in NSCLC A549 and H1975 cells.Isotope tracing studies further confirmed the disruption of one-carbon transfer from serine to folate metabolites by all three miRNAs,inhibition of glucose uptake by miR-22-3p,and reduction of serine biosynthesis from glucose by miR-9-5p and-218-5p in NSCLC cells.With greater activities to interrupt NSCLC cell respiration,glycolysis,and colony formation than miR-9-5p and-218-5p,recombinant miR-22-3p was effective to reduce tumor growth in two NSCLC patient-derived xenograft mouse models without causing any toxicity.These results establish a common antifolate mechanism and differential actions on glucose uptake and metabolism for three lead anticancer miRNAs as well as antitumor efficacy for miR-22-3p nanomedicine,which shall provide insight into developing antimetabolite RNA therapies.
