Oligodendrocyte lineage gene 1 plays a key role in hypoxic-ischemic brain damage and myelin repair, miRNA-9 is involved in the occurrence of many related neurological disorders. Bioin- formatics analysis demonstrated ...Oligodendrocyte lineage gene 1 plays a key role in hypoxic-ischemic brain damage and myelin repair, miRNA-9 is involved in the occurrence of many related neurological disorders. Bioin- formatics analysis demonstrated that miRNA-9 complementarily, but incompletely, bound oligodendrocyte lineage gene 1, but whether miRNA-9 regulates oligodendrocyte lineage gene 1 remains poorly understood. Whole brain slices of 3-day-old Sprague-Dawley rats were cultured and divided into four groups： control group; oxygen-glucose deprivation group （treatment with 8% O2 ＋ 92% N2 and sugar-free medium for 60 minutes）; transfection control group （after oxygen and glucose deprivation for 60 minutes, transfected with control plasmid） and miRNA-9 transfection group （after oxygen and glucose deprivation for 60 minutes, transfected with miRNA-9 plasmid）. From the third day of transfection, and with increasing culture days, oligodendrocyte lineage gene 1 expression increased in each group, peaked at 14 days, and then decreased at 21 days. Real-time quantitative PCR results, however, demonstrated that oligoden- drocyte lineage gene 1 expression was lower in the miRNA-9 transfection group than that in the transfection control group at 1, 3, 7, 14, 21 and 28 days after transfection. Results suggested that miRNA-9 possibly negatively regulated oligodendrocyte lineage gene 1 in brain tissues during hypoxic-ischemic brain damage.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
文摘目的:通过提高骨肉瘤MG-63细胞miRNA-9的表达,探讨miRNA-9对MG-63细胞增殖及凋亡的影响及可能的作用机制。方法:MG-63细胞随机分为实验组、阴性对照组和空白组。实验组转染miRNA-9(Oligo),阴性对照组转染阴性对照核苷酸序列(microRNA negative control sequence),空白组不做转染。qRT-PCR检测细胞miRNA-9、CXCR4的表达。CCK-8法检测3组细胞的增殖;流式细胞术比较3组细胞的凋亡水平。结果:与阴性对照组和空白组相比,转染组细胞中miRNA-9表达升高;miRNA-9高表达的骨肉瘤细胞增殖速率降低、细胞凋亡率增加、CXCR4 mRNA转录水平下调,差异有统计学意义。结论:CXCR4基因参与miRNA-9抑制骨肉瘤MG-63细胞增殖过程,同时促进细胞凋亡。
基金supported by the National Natural Science Foundation of China,No.81241022the Beijing Municipal Natural Science Foundation in China,No.7122045,7072023
文摘Oligodendrocyte lineage gene 1 plays a key role in hypoxic-ischemic brain damage and myelin repair, miRNA-9 is involved in the occurrence of many related neurological disorders. Bioin- formatics analysis demonstrated that miRNA-9 complementarily, but incompletely, bound oligodendrocyte lineage gene 1, but whether miRNA-9 regulates oligodendrocyte lineage gene 1 remains poorly understood. Whole brain slices of 3-day-old Sprague-Dawley rats were cultured and divided into four groups： control group; oxygen-glucose deprivation group （treatment with 8% O2 ＋ 92% N2 and sugar-free medium for 60 minutes）; transfection control group （after oxygen and glucose deprivation for 60 minutes, transfected with control plasmid） and miRNA-9 transfection group （after oxygen and glucose deprivation for 60 minutes, transfected with miRNA-9 plasmid）. From the third day of transfection, and with increasing culture days, oligodendrocyte lineage gene 1 expression increased in each group, peaked at 14 days, and then decreased at 21 days. Real-time quantitative PCR results, however, demonstrated that oligoden- drocyte lineage gene 1 expression was lower in the miRNA-9 transfection group than that in the transfection control group at 1, 3, 7, 14, 21 and 28 days after transfection. Results suggested that miRNA-9 possibly negatively regulated oligodendrocyte lineage gene 1 in brain tissues during hypoxic-ischemic brain damage.
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.
