Zhou Ying, He Dongmei, Zeng Jinrong, Bao Shijie, Lai Jing, Weng Yujun, Chen Shengting
Departmemt of Hematology, The First Affiliated Hospital, Jinan University Guangzhou, China.
School of Medicine, Institute of Hematology, Jinan University Guangzhou, China.
Front Pharmacol. 2017 Mar 17;8:127. doi: 10.3389/fphar.2017.00127. eCollection 2017.
The effects of microRNA-20a (miR-20a) antisense oligonucleotides (ASODNs) on the proliferation and apoptosis of K562 cells were investigated, and the effects of these ASODNs in combination with imatinib on K562 cells were preliminarily observed. miR-20a ASODNs and scrambled oligonucleotides (SODNs) were chemically synthesized, and the later was used as the control. miR-20a ASODNs were transfected into K562 cells using Lipofectamine 2000 transfection reagent, and the expression of miR-20a was detected using real-time quantitative RT-PCR (qRT-PCR). The CCK8 assay was performed to detect the inhibition of the cell growth rate. The cells were stained by Hoechst 33258 to detect apoptotic cell morphology. Annexin V/PI double staining was used to detect the cell apoptosis rate using flow cytometry. The protein expression levels of E2F1, P21, and Bim in the K562 cell line were detected using western blotting. The qRT-PCR results showed that the expression level of miR-20a in K562 cells transfected with miR-20a ASODNs was lower than those in the normal control, SODN and blank transfection groups ( < 0.05). miR-20a ASODNs significantly inhibited the growth of K562 cells as compared to the controls ( < 0.05). The Hoechst staining results showed morphological changes, suggesting apoptosis. The cell apoptosis rates in the ASODN group was (13.9 ± 1.5)%, which was significantly higher than that in the normal control group (1.84 ± 0.21)%, blank transfection group (3.21 ± 0.32)%, and SODN group (3.72 ± 0.44)% ( < 0.05). The protein expression of E2F1 and P21 in K562 cells transfected with miR-20a ASODNs were higher, while the level of Bim protein was significantly lower than that in the control groups. When miR-20a ASODNs were combined with imatinib, the growth of K562 cells was significantly inhibited as compared to the ASODN treatment alone, imatinib alone, and SODN+imatinib groups ( < 0.05). miR-20a ASODNs could induce apoptosis and inhibit the proliferation of K562 cells. In addition, imatinib combined with miR-20a ASODNs can increase the inhibitory effect on K562 cell proliferation.
研究了微小RNA-20a（miR-20a）反义寡核苷酸（ASODNs）对K562细胞增殖和凋亡的影响，并初步观察了这些ASODNs与伊马替尼联合应用对K562细胞的影响。化学合成了miR-20a ASODNs和乱序寡核苷酸（SODNs），后者用作对照。使用Lipofectamine 2000转染试剂将miR-20a ASODNs转染至K562细胞中，并通过实时定量逆转录聚合酶链反应（qRT-PCR）检测miR-20a的表达。进行CCK8测定以检测细胞生长速率的抑制情况。用Hoechst 33258对细胞进行染色以检测凋亡细胞形态。采用Annexin V/PI双染法通过流式细胞术检测细胞凋亡率。使用蛋白质免疫印迹法检测K562细胞系中E2F1、P21和Bim的蛋白质表达水平。qRT-PCR结果显示，转染miR-20a ASODNs的K562细胞中miR-20a的表达水平低于正常对照组、SODN组和空白转染组（P<0.05）。与对照组相比，miR-20a ASODNs显著抑制了K562细胞的生长（P<0.05）。Hoechst染色结果显示出形态学变化，提示细胞凋亡。ASODN组的细胞凋亡率为（13.9±1.5）%，显著高于正常对照组（1.84±0.21）%、空白转染组（3.21±0.32）%和SODN组（3.72±0.44）%（P<0.05）。转染miR-20a ASODNs的K562细胞中E2F1和P21的蛋白质表达较高，而Bim蛋白水平显著低于对照组。当miR-20a ASODNs与伊马替尼联合应用时，与单独ASODN处理组、单独伊马替尼组和SODN+伊马替尼组相比，K562细胞的生长受到显著抑制（P<0.05）。miR-20a ASODNs可诱导K562细胞凋亡并抑制其增殖。此外，伊马替尼与miR-20a ASODNs联合应用可增强对K562细胞增殖的抑制作用。
