Ai Z D, Wu H Y, Chen L
Department of Basic Medicine, Medical School, Kunming University of Science and Technology, Kunming 650500, China.
Zhonghua Yi Xue Za Zhi. 2018 Jan 2;98(1):46-50. doi: 10.3760/cma.j.issn.0376-2491.2018.01.010.
microRNA targeted to chronic myeloid leukemia Bcr-Abl oncogene were screened using the deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). The proliferation inhibition effect of SAHA on chronic myeloid leukemia K562 cell was detected by MTS method, and the optimal concentration of SAHA reaction was determined. Western blot was used to detect the level of PARP protein, and making sure whether SAHA induced apoptosis of K562 cell. Effect of SAHA on Bcr-Abl Gene Transcription in K562 Cells was determined by Fluorescence Quantitative PCR. The online software Target Scan and real-time fluorescence quantitative PCR was used to screen Bcr-Abl-targeted microRNA. The viability of K562 cells and Bcr-Abl transcription levels were detected by MTS method and quantitative PCR respectively after selected microRNA were transfected into K562 cell. SAHA significantly inhibited the proliferation of K562 cells and induced apoptosis, meanwhile SAHA significantly down-regulated the transcriptional level of Bcr-Abl gene. After treatment of K562 cells with SAHA, two microRNA, miR-192 and miR-6816, which could target Bcr-Abl, were screened by Target Scan and quantitative PCR. Additionally, SAHA induced the miRNAs to up-regulate 14.5 and 5.2 times, respectively. Transfection of miR-192 and miR-6816 to K562 cells significantly inhibited K562 cell viability and down-regulated the transcriptional level of Bcr-Abl gene. Acetylation inhibitor SAHA promoted the expression of miR-192 and miR-6816 in K562 cells by acetylation regulation, miR-192 and miR-6816 further down-regulated the transcription of Bcr-Abl gene, thereby inhibiting K562 cell proliferation and induced apoptosis.
使用去乙酰化酶抑制剂辛二酰苯胺异羟肟酸（SAHA）筛选靶向慢性髓性白血病Bcr-Abl癌基因的微小RNA。采用MTS法检测SAHA对慢性髓性白血病K562细胞的增殖抑制作用，并确定SAHA反应的最佳浓度。用蛋白质免疫印迹法检测PARP蛋白水平，以确定SAHA是否诱导K562细胞凋亡。通过荧光定量PCR确定SAHA对K562细胞中Bcr-Abl基因转录的影响。利用在线软件Target Scan和实时荧光定量PCR筛选靶向Bcr-Abl的微小RNA。将筛选出的微小RNA转染到K562细胞后，分别采用MTS法和定量PCR检测K562细胞的活力和Bcr-Abl转录水平。SAHA显著抑制K562细胞的增殖并诱导凋亡，同时SAHA显著下调Bcr-Abl基因的转录水平。用SAHA处理K562细胞后，通过Target Scan和定量PCR筛选出两个可靶向Bcr-Abl的微小RNA，即miR-192和miR-6816。此外，SAHA分别诱导这两个微小RNA上调14.5倍和5.2倍。将miR-192和miR-6816转染到K562细胞中可显著抑制K562细胞活力并下调Bcr-Abl基因的转录水平。乙酰化抑制剂SAHA通过乙酰化调控促进K562细胞中miR-192和miR-6816的表达，miR-192和miR-6816进一步下调Bcr-Abl基因的转录，从而抑制K562细胞增殖并诱导凋亡。
