微小RNA-184通过Janus激酶2/信号转导与转录激活因子3途径下调B细胞淋巴瘤/白血病-2样蛋白1介导骨肉瘤的化疗耐药的机制

MicroRNA-184 down-regulates B-cell lymphoma/leukemia-2 like protein 1 to mediate chemotherapy resistance in osteosarcoma through janus kinase 2/signal transducer and activators of transcription 3 signaling pathway

目的探究微小RNA(miRNA,miR)-184通过Janus激酶2(JAK2)/信号转导与转录激活因子3(STAT3)信号通路影响B细胞淋巴瘤/白血病-2样蛋白1(BCL2L1)参与骨肉瘤细胞化疗耐药性的机制。方法选用骨肉瘤细胞株MG63,采用聚合酶链反应(PCR)、蛋白质印迹法(Western blot)等方法检测其中miR-184表达水平以及JAK2/STAT3、BCL2L1蛋白表达;转染miR-184模拟物、抑制物序列,观察对MG63、顺铂培养下的MG63(DPP-MG63)的影响,另将JAK2/STAT3通路抑制剂AG490与细胞一起培养,观察细胞变化。采用独立样本t检验,多组间比较采用单因素方差分析及组内两两比较LSD-t检验进行统计学分析。结果miR-184在骨肉瘤细胞中降低(t=9.074,P<0.05),在过表达miR-184后骨肉瘤细胞的增殖、侵袭能力减弱,凋亡率升高(F=13.810、57.090、59.660,P<0.05),且其中JAK2/STAT3通路的蛋白表达、BCL2L1蛋白(0.92±0.04)均被抑制,细胞耐药性降低(F=186.200、252.900、211.600、156.200、176.500,P<0.05),而抑制miR-184则反之。同样,在经AG490培养后,细胞的增殖、侵袭能力降低,耐药性降低,BCL2L1蛋白表达(0.84±0.05)则升高(F=11.270、38.300、77.130,P<0.05)。拯救实验显示,沉默miR-184后的细胞再与AG490进行培养,细胞生物学行为、耐药性与MG63、DPP-MG63细胞比较差异均无统计学意义(t=0.612、0.059、0.050、0.612,P>0.05)。结论miR-184通过激活JAK2/STAT3信号通路,抑制BCL2L1的表达,促进骨肉瘤细胞的生长,并降低耐药性。

Objective To explore the influence of microRNA(miR)-184 on the participation of apoptosis facilitator B-cell lymphoma/leukemia-2 like protein 1(BCL2L1)in chemotherapy resistance of osteosarcoma cells through janus kinase 2(JAK2)/signal transducer and activators of transcription 3(STAT3)signaling pathway.Methods The expression of microRNA(miRNA,miR)-184,JAK2/STAT3 and BCL2L1 protein was detected in MC63 cells by polymerase chain reaction(PCR),Western blotting.MiR-184 mimic and inhibitor sequences were transfected.The effects on MG63 cells and cisplatin-treated MG63(DPP-MG63)cells were observed.AC490,an inhibitor of JAK2/STAT3 pathway,was cultured with cells.Independent sample t test was used for statistical analysis.One-way analysis of variance was used for multi-group comparison and LSD-t test for intra-group pair comparison.Results MiR-184(t=9.074)decreased in osteosarcoma cells(P<0.05).After over-expression of miR-184,the proliferation and invasion of osteosarcoma cells decreased,and the apoptosis rate increased(F=13.810,57.090,59.660,P<0.05).Moreover,JAK2/STAT3 pathway and BCL2LI protein(0.92±0.04)were inhibited,and the drug resistance of osteosarcoma cells decreased(F=186.200,252.900,211.600,156.200,176.500,P<0.05),while the inhibition of miR-184 was vice versa(P<0.05).In the same way,after being cultured with AG490,cell proliferation and invasion decreased,drug resistance decreased,and BCL2L1 protein expression(0.84±0.05)increased(F=11.270,38.300,77.130,P<0.05).The rescue experiment manifested that the cells silenced by miR-184 were cultured with AG490,and the biological behavior and drug resistance were not different from those of MG63 and DPP-MG63 cells(t=0.612,0.059,0.050,0.612,P>0.05).Conclusion MiR-184 can inhibit the BCL2L1 expression by activating JAK2/STAT3 signaling pathway,promote the growth of osteosarcoma cells and reduce drug resistance.