一个新型小鼠成骨细胞表达miRNA的克隆及表达模式
loning and expression profile of a novel microRNA in mouse osteoblast
目的：探寻新的小鼠成骨细胞表达的microRNA，并研究其在小鼠不同组织和细胞中的表达模式。方法：提取小鼠成骨细胞小分子RNA，经Poly(A)加尾、连接5′端接头后逆转录为cDNA，进行PCR扩增，回收PCR产物后连接至pcDNA3.1 TOPO载体构建cDNA文库，进行菌落PCR，阳性克隆测序后选取19-26 nt大小的RNA进行生物信息学分析确定新的miRNAs。通过Northern blot检测新的miRNA在小鼠成骨细胞、破骨细胞、骨组织等组织的表达，并进一步检测其在BMP-2诱导的小鼠基质细胞ST2成骨分化过程中的表达模式。结果：(1) 共克隆得到162个小分子RNA序列， 68个为miRNA分子，明确2个为新克隆的miRNA，其中一个命名为miR-X，其在小鼠与人类之间具有保守性，位于小鼠第二染色体。(2) miR-X在小鼠成骨细胞、骨组织及肝脏中均有表达，其中成骨细胞中表达最高，其他组织及破骨细胞不表达。(3) 在BMP-2诱导的ST2细胞成骨分化过程中，miR-X随时间延长其表达量不断增加。结论：通过构建小鼠成骨细胞小分子RNA的cDNA文库的方法，新克隆了一个在小鼠与人类存在保守性的新miRNA--miR-X。miR-X在小鼠成骨细胞、骨组织、肝脏中表达，其中成骨细胞中表达最高，并且在小鼠成骨细胞分化过程中的表达呈时间依赖性。
Objective: To explore the novel microRNAs (miRNAs) which express in mouse osteoblasts and identify their expression profiles in different tissues and cells of mouse.Methods: Extracted small RNAs from primary mouse osteoblasts. The small RNAs were polyadenylated by using poly (A) polymerase, and ligated with a 5′ RNA adapter. Then the cDNAs were obtained by reverse transcription of tailed and ligated RNAs, and amplified by polymerase chain reaction (PCR). The purified PCR products of interest were inserted into pcDNA3.1 TOPO cloning vectors and the recombinant vectors were transformed into competent cells to construct a cDNA library of small RNAs of mouse osteoblast. At last, selected positive clones containing interested fragments from the cDNA library by colony PCR for further sequencing, and analyzed the RNAs sized from 19 to 26 nt by bioinformatic methods. Northern blot was performed with total RNA extracted respectively from primary mouse osteoblast cells, primary mouse osteoclast cells, mouse bone, liver and so on. The expression pattern of the novel miRNA in the osteoblast differentiation of mouse stromal cells ST2 was also determined by Northern blot.Results: (1) 162clones were identified by DNA sequencing and database searching. Of them, there are 68 miRNAs including 2 novel miRNAs. One of the two novel miRNAs was termed "miR-X", which was located on chromosome 2 and conserved in the human sequence. (2) miR-X was expressed in osteoblasts，bone and liver, representing a highest level in osteoblasts. But it was not detected in osteoclasts and other tissues. (3) Expression of miR-X in osteoblast differentiation of BMP-2 induced ST2 cells was increased progressively with the time. Conclusion: We cloned a novel miRNA termed "miR-X" , which was conserved in human sequence, by constructing a cDNA library of small RNAs from mouse osteoblast. miR-X was detected in mouse osteoblasts，bone and liver, representing a highest level in osteoblasts and the expression of miR-X in osteoblast differentiation of BMP-2 ST2 cells represented a time-depended pattern.
罗湘杭、郭丽娟、刘唯、李辉
分子生物学细胞生物学遗传学
microRNAcDNA文库成骨细胞克隆
microRNAcDNA librarosteoblastscloning
罗湘杭,郭丽娟,刘唯,李辉.一个新型小鼠成骨细胞表达miRNA的克隆及表达模式[EB/OL].(2012-03-09)[2025-08-02].http://www.paper.edu.cn/releasepaper/content/201203-357.点此复制
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