miRNA-18a和miRNA-4802通过自噬抑制淋巴瘤细胞耐药性的机制研究

Mechanism study on miRNA-18a and miRNA-4802 suppressing drug resistance of lymphoma cells via inhibiting autophagy

目的研究miRNA-18a和miRNA-4802通过自噬对淋巴瘤细胞耐药性的调控机制。方法以人Burkitt’s淋巴瘤细胞Daudi和人套细胞淋巴瘤细胞JeKo-1为研究对象,阿霉素(ADR)、长春新碱(VCR)为实验药物。以ADR、VCR处理后,采用CCK-8法考察上述2种细胞的相对活性,采用Western blot法检测凋亡标志蛋白活化型胱天蛋白酶9(cleaved caspase-9)和cleaved caspase-6的表达,考察2种细胞对ADR、VCR的耐药性。采用自噬相关蛋白LC3-Ⅱ、p62表达水平检测、自噬流实验和透射电镜观察考察2种细胞自噬活性的差异;采用荧光定量聚合酶链式反应考察2种细胞中miRNA-18a、miRNA-4802及unc-51样激酶(ULK1)、自噬相关蛋白7(ATG7)mRNA的表达差异,并检测ULK1、ATG7蛋白表达差异。再以JeKo-1细胞为研究对象,考察经2种模拟生物体内源的miRNAs(miRNA-18a mimics、miRNA-4802 mimics)处理后,细胞自噬活性和耐药性的变化情况。结果以ADR和VCR处理后,JeKo-1细胞比Daudi细胞具有更强的耐药性和自噬活性。JeKo-1细胞中miRNA-18a、miRNA-4802表达水平均显著低于Daudi细胞,ULK1、ATG7 mRNA和蛋白表达水平均显著高于Daudi细胞(P<0.001)。以miRNA-18a mimics和miRNA-4802 mimics处理JeKo-1细胞后,细胞的自噬活性和耐药性均显著降低。结论miRNA-18a和miRNA-4802可分别通过降低自噬启动基因ULK1和ATG7的表达,抑制淋巴瘤细胞的自噬活性,从而降低淋巴瘤细胞对ADR和VCR的耐药性。

OBJECTIVE To study the regulation mechanism of miRNA-18a and miRNA-4802 on drug resistance of lymphoma cells via autophagy.METHODS Using human burkitt’s lymphoma cell Daudi and human mantle cell lymphoma cell JeKo-1 as the research objects,adriamycin(ADR)and vincristine(VCR)as experimental drugs.After treatment of ADR and VCR,relative cell viability was detected with CCK-8 kit;the expression of apoptosis marker protein activated cleaved caspase-9 and cleaved caspase-6 were detected by Western blot assay.The drug resistances of the two cells to ADR and VCR were investigated.The difference of autophagy activity between the two kinds of cells by expression detection of autophagy related proteins LC3-Ⅱand p62,autophagy flow experiment and transmission electron microscope observation.Fluorescence quantitative polymerase chain reaction was used to investigate the expression differences of miRNA-18a and miRNA-4802,ULK1 and ATG7 mRNA in the two cells,and to detect the expression differences of ULK1 and ATG7 proteins.Taking JeKo-1 cells as the research object,the changes of autophagy activity and drug resistance were investigated after treatment with endogenous miRNAs(miRNA-18a mimics,miRNA-4802 mimics)of two simulated organisms.RESULTS After ADR and VCR treatment,compared with Daudi cells,JeKo-1 cells had stronger drug resistance and autophagy activity.The expression of miRNA-18a and miRNA-4802 in JeKo-1 cells were significantly lower than Daudi cells,mRNA and protein expression of ULK1 and ATG7 were significantly higher than those of Daudi cells(P<0.001).After treatment of miRNA-18a mimics and miRNA-4802 mimics,the autophagy activities and drug resistances of JeKo-1 cells were decreased significantly.CONCLUSIONS miRNA-18a and miRNA-4802 can decrease drug resistances of lymphoma cells to ADR and VCR by reducing the expression of autophagy-initiating genes ULK1 and ATG7,and inhibiting the autophagy activity of lymphoma cells.