CAJ | 학술논문

目的：检测microRNA－218（ miR－218）在人急性淋巴细胞白血病T淋巴细胞（ CCRF－CEM）中的表达情况和其对细胞生物学特性的影响，并观察miR－218对靶基因c－kit表达的影响，为人白血病治疗提供新方法和新策略。方法：利用实时荧光定量PCR（ quantitative real－time PCR，qRT－PCR）检测miR－218在正常人外周血T淋巴细胞和CCRF－CEM中的表达情况。在CCRF－CEM细胞中转染miR－218 mimic后48 h，qRT－PCR检测CCRF－CEM细胞中miR－218表达变化；MTT检测miR－218对CCRF－CEM细胞活力的影响。流式细胞仪分析miR－218对细胞CCRF－CEM的细胞周期和凋亡的影响。利用荧光报告酶系统检验c－kit是miR－218调控靶基因，并采用Western blot方法检测miR－218对CCRF－CEM细胞中KIT蛋白表达的影响。结果：qRT－PCR检测结果显示：与正常人外周血T淋巴细胞相比，miR－218在CCRF－CEM细胞系中的表达显著下降（ P＜0．01）。与对照组相比，在CCRF－CEM中转染miR－218 mimic 48 h后，细胞中miR－218的表达显著上升（P＜0．01）。 MTT结果显示：在CCRF－CEM细胞中过表达miR－218后，细胞活力显著下降（ P＜0．05）。流式细胞仪分析结果显示：过表达miR－218后，miR－218 mimic转染组细胞的S期细胞比例明显下降（P＜0．01）；细胞的凋亡显著增加（P＜0．01）。荧光素酶报告基因系统检测结果显示：与对照组相比，转染miR－218 mimic组的相对荧光素酶活力显著降低（ P＜0．01）。 Western blot检测结果显示：与对照组相比，在CCRF－CEM中转染miR－218 mimic 48 h后，细胞中KIT蛋白的表达显著下降（ P＜0．01）。结论：miR－218在人急性淋巴细胞白血病T淋巴细胞（ CCRF－CEM）中表达下调，miR－218可负性调节KIT蛋白的表达，并抑制CCRF－CEM细胞增殖，促进凋亡。增强miR－218表达的治疗策略有望使白血病患者受益。目的：檢測microRNA－218（ miR－218）在人急性淋巴細胞白血病T淋巴細胞（ CCRF－CEM）中的錶達情況和其對細胞生物學特性的影響，併觀察miR－218對靶基因c－kit錶達的影響，為人白血病治療提供新方法和新策略。方法：利用實時熒光定量PCR（ quantitative real－time PCR，qRT－PCR）檢測miR－218在正常人外週血T淋巴細胞和CCRF－CEM中的錶達情況。在CCRF－CEM細胞中轉染miR－218 mimic後48 h，qRT－PCR檢測CCRF－CEM細胞中miR－218錶達變化；MTT檢測miR－218對CCRF－CEM細胞活力的影響。流式細胞儀分析miR－218對細胞CCRF－CEM的細胞週期和凋亡的影響。利用熒光報告酶繫統檢驗c－kit是miR－218調控靶基因，併採用Western blot方法檢測miR－218對CCRF－CEM細胞中KIT蛋白錶達的影響。結果：qRT－PCR檢測結果顯示：與正常人外週血T淋巴細胞相比，miR－218在CCRF－CEM細胞繫中的錶達顯著下降（ P＜0．01）。與對照組相比，在CCRF－CEM中轉染miR－218 mimic 48 h後，細胞中miR－218的錶達顯著上升（P＜0．01）。 MTT結果顯示：在CCRF－CEM細胞中過錶達miR－218後，細胞活力顯著下降（ P＜0．05）。流式細胞儀分析結果顯示：過錶達miR－218後，miR－218 mimic轉染組細胞的S期細胞比例明顯下降（P＜0．01）；細胞的凋亡顯著增加（P＜0．01）。熒光素酶報告基因繫統檢測結果顯示：與對照組相比，轉染miR－218 mimic組的相對熒光素酶活力顯著降低（ P＜0．01）。 Western blot檢測結果顯示：與對照組相比，在CCRF－CEM中轉染miR－218 mimic 48 h後，細胞中KIT蛋白的錶達顯著下降（ P＜0．01）。結論：miR－218在人急性淋巴細胞白血病T淋巴細胞（ CCRF－CEM）中錶達下調，miR－218可負性調節KIT蛋白的錶達，併抑製CCRF－CEM細胞增殖，促進凋亡。增彊miR－218錶達的治療策略有望使白血病患者受益。
목적：검측microRNA－218（ miR－218）재인급성림파세포백혈병T림파세포（ CCRF－CEM）중적표체정황화기대세포생물학특성적영향，병관찰miR－218대파기인c－kit표체적영향，위인백혈병치료제공신방법화신책략。방법：이용실시형광정량PCR（ quantitative real－time PCR，qRT－PCR）검측miR－218재정상인외주혈T림파세포화CCRF－CEM중적표체정황。재CCRF－CEM세포중전염miR－218 mimic후48 h，qRT－PCR검측CCRF－CEM세포중miR－218표체변화；MTT검측miR－218대CCRF－CEM세포활력적영향。류식세포의분석miR－218대세포CCRF－CEM적세포주기화조망적영향。이용형광보고매계통검험c－kit시miR－218조공파기인，병채용Western blot방법검측miR－218대CCRF－CEM세포중KIT단백표체적영향。결과：qRT－PCR검측결과현시：여정상인외주혈T림파세포상비，miR－218재CCRF－CEM세포계중적표체현저하강（ P＜0．01）。여대조조상비，재CCRF－CEM중전염miR－218 mimic 48 h후，세포중miR－218적표체현저상승（P＜0．01）。 MTT결과현시：재CCRF－CEM세포중과표체miR－218후，세포활력현저하강（ P＜0．05）。류식세포의분석결과현시：과표체miR－218후，miR－218 mimic전염조세포적S기세포비례명현하강（P＜0．01）；세포적조망현저증가（P＜0．01）。형광소매보고기인계통검측결과현시：여대조조상비，전염miR－218 mimic조적상대형광소매활력현저강저（ P＜0．01）。 Western blot검측결과현시：여대조조상비，재CCRF－CEM중전염miR－218 mimic 48 h후，세포중KIT단백적표체현저하강（ P＜0．01）。결론：miR－218재인급성림파세포백혈병T림파세포（ CCRF－CEM）중표체하조，miR－218가부성조절KIT단백적표체，병억제CCRF－CEM세포증식，촉진조망。증강miR－218표체적치료책략유망사백혈병환자수익。
Objective:To detect the expression of microRNA-218 (miR-218) in human acute lymphocyte leukemia (ALL) T lymphocytes ( CCRF-CEM) ,explore its effects on the biological features of CCRF-CEM cells and the expression of its target gene c-kit, so as to provide new insights for leukemia treatment.Methods: Using the quantitative real-time polymerase chain reaction ( qRT-PCR) ,we detected the expression of miR-218 in the normal peripheral blood T lymphocytes and CCRF-CEM cells.Forty-eight hours after the miR-218 mimic was transfected into the CCRF-CEM cells,the expression of miR-218 in the CCRF-CEM cells was detected by qRT-PCR.The effect of miR-218 on the CCRF-CEM cell viability was detected using MTT.The effect of miR-218 on the proliferation and apoptosis of CCRF-CEM cell was analyzed using flow cytometry.c-kit gene was identified to be a target gene of miR-218 by luciferase reporter enzyme system,and the effect of miR-218 on the expression of KIT protein in cells were determined using Western blot.Results:As shown by qRT-PCR,compared with that in the normal peripheral blood T lymphocytes,the expressions of miR-218 in ALL T lymphocytes cell lines were significantly decreased ( P<0.01 ) .Compared with the control group, the expression of miR-218 increase significantly in CCRF-CEM cells transfected with miR-218 mimic for 48 hours ( P<0.01).MTT showed that the cell viability decreased significantly after the over-expression of miR-218 in the CCRF-CEM cells ( P<0.05 ) .Flow cytometry showed that the S-phase fraction significantly declined after the over-expression of miR-218 ( P<0.01 ) , and meanwhile the apoptosis of cells also significantly increased (P<0.01).Luciferase reporter gene assay showed that,compared with the control group,the relative luciferase activity significantly declined in the miR-218 mimic transfection group (P<0.01).Compared with the control group,the expression of KIT protein in the CCRF-CEM cells transfected with miR-218 mimic for 48 hours significantly decreased ( P<0.01).Conclusion:The expression of miR-218 decreases in ALL T lymphocytes cell lines.MiR-218 can negatively regulate the expression of KIT protein,inhibit the proliferation and increase the apoptosis of CCRF-CEM cells.Treatment based on the enhanced expression of miR-218 may be a promising strategy for leukemia.