CAJ | 학술논문

目的筛选和分析急性髓系白血病(AML)患者骨髓造血干/祖细胞(HSPC)特异性微小RNA(miRNA,miR)的表达及其水平.方法分选AML患者骨髓及造血干细胞移植供者(正常对照)外周血CD34+细胞,提取细胞总RNA,通过与miRNA芯片杂交后筛选差异表达的miRNA,随后采用实时定量PCR技术对差异表达的miR-10a和miR-220c进行定量验证,并进行DNA序列分析.结果 miRNA芯片杂交结果显示,AML患者骨髓与正常对照CD34+细胞之间,表达差异达1倍以上的miRNA有191个.差异有统计学意义的有94个,其中44个表达增高,50个表达降低.实时定量PCR结果显示,AML患者骨髓CD34+细胞miR-10a和miR-220c表达水平是正常对照的19.6%和19.0%,PCR产物克隆DNA测序以及DNA序列同源检索证实确为miR-10a和miR-220c.结论 AML患者与正常人HSPC存在多种差异表达的miRNA,miR-10a和miR-220c表达明显下调.
목적 사선화분석급성수계백혈병(AML)환자골수조혈간/조세포(HSPC)특이성미소RNA(miRNA,miR)적표체급기수평.방법 분선AML환자골수급조혈간세포이식공자(정상대조)외주혈CD34+세포,제취세포총RNA,통과여miRNA심편잡교후사선차이표체적miRNA,수후채용실시정량PCR기술대차이표체적miR-10a화miR-220c진행정량험증,병진행DNA서렬분석.결과 miRNA심편잡교결과 현시,AML환자골수여정상대조CD34+세포지간,표체차이체1배이상적miRNA유191개.차이유통계학의의적유94개,기중44개표체증고,50개표체강저.실시정량PCR결과 현시,AML환자골수CD34+세포miR-10a화miR-220c표체수평시정상대조적19.6%화19.0%,PCR산물극륭DNA측서이급DNA서렬동원검색증실학위miR-10a화miR-220c.결론 AML환자여정상인HSPC존재다충차이표체적miRNA,miR-10a화miR-220c표체명현하조.
Objective To screen and analyze CD34+ cell specific microRNAs (miRNAs) from the patients with acute myelogenous leukemia (AML) and their expression. Methods CD34+ cells were sorted from AML patients or the mobilized peripheral blood of the donors of hematopoietic stem cell transplantation (normal control subjects) and followed by the extraction of the cell total RNAs. The differentially expressed microRNAs (miRNAs,miR) were selected after hybridizing with miRNA microarray, real time polymerase chain reaction (real-time PCR) was subsequently applied to confirm the expression of the selected miRs, and PCR products were further cloned and sequenced to check their specificity. Results Of the differentially expressed miRNAs, 191 were found to be at least one-fold change in the CD34+ cells between the AML patients and the normal control subjects. Of the 191 miRNAs, the expression difference of 94 was significant(P<0.05). Among these 94 miRNAs, the expression of 44 miRNAs was increased and the other 50 miRNAs was decreased in the CD34+ cells from the bone marrow of AML patients compared with the CD34+ cells from the mobilized peripheral blood of the normal control subjects. Real time PCR verified that the expression level of miR-10a and miR-220c in the CD34+ cells from the bone marrow of AML patients was 19.6% and 19.0% of that of CD34+ cells from mobilized peripheral blood of the normal control subjects. DNA sequencing and BLAST DNA database searching results indicated that the PCR products were really miR-10a and miR-220c. Conclusion A variety of differentially expressed-miRNAs are existed between AML and normal control subjects CD34+ cells, the expression of miR-10a and miR-220c was significantly down-regulated in the CD34+ cells from the bone marrow of AML patients.