Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's dis...Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.展开更多
BACKGROUND Diabetic kidney disease(DKD),characterized by increased urinary microalbumin levels and decreased renal function,is the primary cause of end-stage renal di-sease.Its pathological mechanisms are complicated ...BACKGROUND Diabetic kidney disease(DKD),characterized by increased urinary microalbumin levels and decreased renal function,is the primary cause of end-stage renal di-sease.Its pathological mechanisms are complicated and multifactorial;Therefore,sensitive and specific biomarkers are needed.Urinary exosome originate from diverse renal cells in nephron segments and partially mirror the pathological changes in the kidney.The microRNAs(miRNAs)in urinary exosome are remark-ably stable and highly tissue-specific for the kidney.METHODS Type 2 diabetic mellitus(T2DM)patients were recruited from the Second Hospital of Hebei Medical University and were divided into two groups:DM,diabetic pa-tients without albuminuria[urinary albumin to creatinine ratio(UACR)<30 mg/g]and DKD,diabetic patients with albuminuria(UACR≥30 mg/g).Healthy subjects were the normal control(NC)group.Urinary exosomal miR-145-5p,miR-27a-3p,and miR-29c-3p,were detected using real-time quantitative polymerase chain reaction.The correlation between exosomal miRNAs and the clinical in-dexes was evaluated.The diagnostic values of exosomal miR-145-5p and miR-27a-3p in DKD were determined using receiver operating characteristic(ROC)analysis.Biological functions of miR-145-5p were investigated by performing RESULTS Urinary exosomal expression of miR-145-5p and miR-27a-3p was more upregulated in the DKD group than in the DM group(miR-145-5p:4.54±1.45 vs 1.95±0.93,P<0.001;miR-27a-3p:2.33±0.79 vs 1.71±0.76,P<0.05)and the NC group(miR-145-5p:4.54±1.45 vs 1.55±0.83,P<0.001;miR-27a-3p:2.33±0.79 vs 1.10±0.51,P<0.001).The exosomal miR-145-5p and miR-27a-3p positively correlated with albuminuria and serum creatinine and negatively correlated with the estimated glomerular filtration rate.miR-27a-3p was also closely related to blood glucose,gly-cosylated hemoglobin A1c,and low-density lipoprotein cholesterol.ROC analysis revealed that miR-145-5p had a better area under the curve of 0.88[95%confidence interval(CI):0.784-0.985,P<0.0001]in diagnosing DKD than miR-27a-3p with 0.71(95%CI:0.547-0.871,P=0.0239).Bioinformatics analysis revealed that the target genes of miR-145-5p were located in the actin filament,cytoskeleton,and extracellular exosome and were involved in the pathological processes of DKD,including apoptosis,inflammation,and fibrosis.CONCLUSION Urinary exosomal miR-145-5p and miR-27a-3p may serve as novel noninvasive diagnostic biomarkers or promising therapeutic targets for DKD.展开更多
目的研究肝细胞肝癌(HCC)中长链非编码RNA(LncRNA)氧化应激反应丝氨酸丰富1反义RNA1(OSER1-AS1)靶向调控微小RNA(miR)-433-3p的作用及生物学意义。方法选择唐山市第九医院手术切除的HCC组织和癌旁组织,培养HCC细胞株SMMC-7721、HepG2、M...目的研究肝细胞肝癌(HCC)中长链非编码RNA(LncRNA)氧化应激反应丝氨酸丰富1反义RNA1(OSER1-AS1)靶向调控微小RNA(miR)-433-3p的作用及生物学意义。方法选择唐山市第九医院手术切除的HCC组织和癌旁组织,培养HCC细胞株SMMC-7721、HepG2、MHCC97H及正常肝细胞株HL-7702。检测LncRNA OSER1-AS1、miR-433-3p的表达水平,比较HCC组织与癌旁组织、HCC细胞株与正常肝细胞株中LncRNA OSER1-AS1、miR-433-3p表达水平的差异。对转染MHCC97H细胞进行分组,转染阴性对照(NC)-siRNA为si-NC组,转染LncRNA OSER1-AS1-siRNA为si-OSER1-AS1组,转染LncRNA OSER1-AS1-siRNA及NC miR为si-OSER1-AS1+miR-NC组,转染LncRNA OSER1-AS1-siRNA及miR-433-3p抑制物为si-OSER1-AS1+miR-433-3p抑制物组。检测细胞增殖活力、凋亡率、增殖细胞核抗原(PCNA)及裂解型caspase-3(cleaved caspase-3)的表达水平,采用双荧光素酶报告基因实验验证LncRNA OSER1-AS1靶向miR-433-3p。结果HCC组织中LncRNA OSER1-AS1的表达水平高于癌旁组织(1.77±0.34 vs1.00±0.21)、miR-433-3p的表达水平低于癌旁组织(0.65±0.09 vs 1.00±0.28)(P<0.05)且LncRNA OSER1-AS1与miR-433-3p呈负相关(r=-0.351,P<0.05);HCC细胞株中LncRNA OSER1-AS1的表达水平高于HL-7702细胞、miR-433-3p的表达水平低于HL-7702细胞(P<0.05)且MHCC97H细胞中上述变化最显著。si-OSER1-AS1组MHCC97H细胞中LncRNA OSER1-AS1、PCNA的表达水平及增殖活力低于si-NC组(0.37±0.05 vs 1.00±0.11,0.33±0.05 vs 0.92±0.12,0.51±0.09 vs 1.03±0.12),miR-433-3p、cleaved caspase-3的表达水平及凋亡率高于si-NC组(1.88±0.25 vs 1.00±0.09,0.96±0.11 vs 0.44±0.06,9.39%±1.15%vs 3.82%±0.55%)(P<0.05);si-OSER1-AS1+miR-433-3p抑制物组MHCC97H细胞中cleaved caspase-3表达水平及凋亡率低于si-OSER1-AS1+miR-NC组(0.27±0.05 vs 0.91±0.10,6.04%±0.77%vs 11.32%±1.32%),PCNA的表达水平及增殖活力高于si-OSER1-AS1+miR-NC组(0.94±0.12 vs 0.48±0.06,0.95±0.11 vs 0.34±0.05)(P<0.05)。结论LncRNA OSER1-AS1靶向miR-433-3p调控HCC细胞的增殖和凋亡。展开更多
Aim: To reveal the exonic and 3’UTR sequences of KRAS, TP53, APC, BRAF, PIK3CA genes in sporadic colorectal tumors and to investigate the clinical relevance of 3’UTR variations in miRNA profiles. Methods: In the stu...Aim: To reveal the exonic and 3’UTR sequences of KRAS, TP53, APC, BRAF, PIK3CA genes in sporadic colorectal tumors and to investigate the clinical relevance of 3’UTR variations in miRNA profiles. Methods: In the study, the exonic and 3’UTR sequences of five genes in 12 sporadic colorectal tumors were extracted by next generation sequencing. In tumors with variation in the 3’UTR region, the changes caused by the variation in the miRNA binding profile were detected. The expression profile of these miRNAs in colorectal and other solid tumors compared to normal tissue was determined. Pathway analysis was performed to determine which signaling pathways miRNAs affect. Results: Case-10 in our study was wild type KRAS and received cetuximab treatment and developed drug resistance. In this case, it was concluded that the expression of KRAS increased and tumorigenesis progressed due to miRNAs that do not bind to this region due to variations in the 3’UTR region. Among these miRNAs, hsa-miR-124-3p was found to have decreased expression in colorectal tumors and to be associated with the ECM-receptor interaction pathway. Conclusion: Variations in the 3’UTR regions of genes critical in the process of carsinogenesis are associated with drug resistance and the process of tumorigenesis.展开更多
基金supported by the National Institute on Aging (NIA)National Institutes of Health (NIH)+3 种基金Nos.K99AG065645,R00AG065645R00AG065645-04S1 (to SK)NIH research grants,NINDS,No.R01 NS115834NINDS/NIA,No.R01 NS115834-02S1 (to LG)。
文摘Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.
基金Supported by the Nature Science Foundation of Hebei Province,No.H2023104011.
文摘BACKGROUND Diabetic kidney disease(DKD),characterized by increased urinary microalbumin levels and decreased renal function,is the primary cause of end-stage renal di-sease.Its pathological mechanisms are complicated and multifactorial;Therefore,sensitive and specific biomarkers are needed.Urinary exosome originate from diverse renal cells in nephron segments and partially mirror the pathological changes in the kidney.The microRNAs(miRNAs)in urinary exosome are remark-ably stable and highly tissue-specific for the kidney.METHODS Type 2 diabetic mellitus(T2DM)patients were recruited from the Second Hospital of Hebei Medical University and were divided into two groups:DM,diabetic pa-tients without albuminuria[urinary albumin to creatinine ratio(UACR)<30 mg/g]and DKD,diabetic patients with albuminuria(UACR≥30 mg/g).Healthy subjects were the normal control(NC)group.Urinary exosomal miR-145-5p,miR-27a-3p,and miR-29c-3p,were detected using real-time quantitative polymerase chain reaction.The correlation between exosomal miRNAs and the clinical in-dexes was evaluated.The diagnostic values of exosomal miR-145-5p and miR-27a-3p in DKD were determined using receiver operating characteristic(ROC)analysis.Biological functions of miR-145-5p were investigated by performing RESULTS Urinary exosomal expression of miR-145-5p and miR-27a-3p was more upregulated in the DKD group than in the DM group(miR-145-5p:4.54±1.45 vs 1.95±0.93,P<0.001;miR-27a-3p:2.33±0.79 vs 1.71±0.76,P<0.05)and the NC group(miR-145-5p:4.54±1.45 vs 1.55±0.83,P<0.001;miR-27a-3p:2.33±0.79 vs 1.10±0.51,P<0.001).The exosomal miR-145-5p and miR-27a-3p positively correlated with albuminuria and serum creatinine and negatively correlated with the estimated glomerular filtration rate.miR-27a-3p was also closely related to blood glucose,gly-cosylated hemoglobin A1c,and low-density lipoprotein cholesterol.ROC analysis revealed that miR-145-5p had a better area under the curve of 0.88[95%confidence interval(CI):0.784-0.985,P<0.0001]in diagnosing DKD than miR-27a-3p with 0.71(95%CI:0.547-0.871,P=0.0239).Bioinformatics analysis revealed that the target genes of miR-145-5p were located in the actin filament,cytoskeleton,and extracellular exosome and were involved in the pathological processes of DKD,including apoptosis,inflammation,and fibrosis.CONCLUSION Urinary exosomal miR-145-5p and miR-27a-3p may serve as novel noninvasive diagnostic biomarkers or promising therapeutic targets for DKD.
文摘目的研究肝细胞肝癌(HCC)中长链非编码RNA(LncRNA)氧化应激反应丝氨酸丰富1反义RNA1(OSER1-AS1)靶向调控微小RNA(miR)-433-3p的作用及生物学意义。方法选择唐山市第九医院手术切除的HCC组织和癌旁组织,培养HCC细胞株SMMC-7721、HepG2、MHCC97H及正常肝细胞株HL-7702。检测LncRNA OSER1-AS1、miR-433-3p的表达水平,比较HCC组织与癌旁组织、HCC细胞株与正常肝细胞株中LncRNA OSER1-AS1、miR-433-3p表达水平的差异。对转染MHCC97H细胞进行分组,转染阴性对照(NC)-siRNA为si-NC组,转染LncRNA OSER1-AS1-siRNA为si-OSER1-AS1组,转染LncRNA OSER1-AS1-siRNA及NC miR为si-OSER1-AS1+miR-NC组,转染LncRNA OSER1-AS1-siRNA及miR-433-3p抑制物为si-OSER1-AS1+miR-433-3p抑制物组。检测细胞增殖活力、凋亡率、增殖细胞核抗原(PCNA)及裂解型caspase-3(cleaved caspase-3)的表达水平,采用双荧光素酶报告基因实验验证LncRNA OSER1-AS1靶向miR-433-3p。结果HCC组织中LncRNA OSER1-AS1的表达水平高于癌旁组织(1.77±0.34 vs1.00±0.21)、miR-433-3p的表达水平低于癌旁组织(0.65±0.09 vs 1.00±0.28)(P<0.05)且LncRNA OSER1-AS1与miR-433-3p呈负相关(r=-0.351,P<0.05);HCC细胞株中LncRNA OSER1-AS1的表达水平高于HL-7702细胞、miR-433-3p的表达水平低于HL-7702细胞(P<0.05)且MHCC97H细胞中上述变化最显著。si-OSER1-AS1组MHCC97H细胞中LncRNA OSER1-AS1、PCNA的表达水平及增殖活力低于si-NC组(0.37±0.05 vs 1.00±0.11,0.33±0.05 vs 0.92±0.12,0.51±0.09 vs 1.03±0.12),miR-433-3p、cleaved caspase-3的表达水平及凋亡率高于si-NC组(1.88±0.25 vs 1.00±0.09,0.96±0.11 vs 0.44±0.06,9.39%±1.15%vs 3.82%±0.55%)(P<0.05);si-OSER1-AS1+miR-433-3p抑制物组MHCC97H细胞中cleaved caspase-3表达水平及凋亡率低于si-OSER1-AS1+miR-NC组(0.27±0.05 vs 0.91±0.10,6.04%±0.77%vs 11.32%±1.32%),PCNA的表达水平及增殖活力高于si-OSER1-AS1+miR-NC组(0.94±0.12 vs 0.48±0.06,0.95±0.11 vs 0.34±0.05)(P<0.05)。结论LncRNA OSER1-AS1靶向miR-433-3p调控HCC细胞的增殖和凋亡。
文摘Aim: To reveal the exonic and 3’UTR sequences of KRAS, TP53, APC, BRAF, PIK3CA genes in sporadic colorectal tumors and to investigate the clinical relevance of 3’UTR variations in miRNA profiles. Methods: In the study, the exonic and 3’UTR sequences of five genes in 12 sporadic colorectal tumors were extracted by next generation sequencing. In tumors with variation in the 3’UTR region, the changes caused by the variation in the miRNA binding profile were detected. The expression profile of these miRNAs in colorectal and other solid tumors compared to normal tissue was determined. Pathway analysis was performed to determine which signaling pathways miRNAs affect. Results: Case-10 in our study was wild type KRAS and received cetuximab treatment and developed drug resistance. In this case, it was concluded that the expression of KRAS increased and tumorigenesis progressed due to miRNAs that do not bind to this region due to variations in the 3’UTR region. Among these miRNAs, hsa-miR-124-3p was found to have decreased expression in colorectal tumors and to be associated with the ECM-receptor interaction pathway. Conclusion: Variations in the 3’UTR regions of genes critical in the process of carsinogenesis are associated with drug resistance and the process of tumorigenesis.
