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目的 研究miR-202对多发性骨髓瘤U266细胞中B细胞活化因子(BAFF)的调节作用及其机制.方法 通过生物信息学软件预测miR-202与BAFF的潜在结合位点,构建荧光素酶报告基因载体.将人类miR-202-3P模拟物(has-miR-202-3 P-mimics)、人类miR-202-3P抑制物(has-miR-202-inhibitor)、siBAFF及各自阴性对照转染U266细胞,采用实时荧光定量PCR和Western blot法验证miR-202对BAFF的调控作用,采用细胞增殖实验和流式细胞仪检测转染后U266细胞的增殖能力和凋亡情况.结果 未转染组、has-miR-202-3 P-mimics转染组、has-miR-202-3 P-inhibitor转染组和siBAFF转染组U266细胞中BAFF mRNA的相对表达量分别为1.040±0.057、0.573±0.073、1.205±0.097和0.368±0.052.与未转染组比较,has-miR-202-3 P-mimics转染组和siBAFF转染组U266细胞中BAFF mRNA的表达明显下调(P＜0.05).Western blot法检测BAFF蛋白的表达结果与mRNA基本一致.未转染组、has-miR-202-3 P-mimics转染组、has-miR-202-3 P-inhibitor转染组和siBAFF转染组U266细胞的A450nm值分别为1.063±0.052、0.714 ±0.045、0.936±0.066和0.764±0.053.与未转染组比较,has-miR-202-3 P-mimics转染组和siBAFF转染组U266细胞的A450nm值明显降低(P＜0.05).未转染组、has-miR-202-3 P-mimics转染组、has-miR-202-3 P-inhibitor转染组和siBAFF转染组U266细胞的凋亡率分别为26.2％、49.6％、21.1％和30.7％.与未转染组比较,has-miR-202-3P-mimics转染组U266细胞的凋亡率明显增加(P＜0.05).has-miR-202DP-mimics转染组U266细胞中p-JNK蛋白的表达水平降低.结论 miR-202通过调节BAFF的表达对U266细胞发挥抑制增殖和诱导凋亡的作用.JNK/SAPK信号通路参与了miR-202对BAFF的调控作用.

Objective To explore the regulating effect of miR-202 on B cell-activating factor,and check whether the regulation influences the growth of multiple myeloma cells.Methods The potential binding sites of BAFF for miR-202 were predicted using bioinformatics software.Luciferase reporter gene analysis was used to evaluate the regulatory effect of miR-202 on BAFF.Human multiple myeloma U266 cells were transfected with has-miR-202-mimics,has-miR-202-inhibitor,siBAFF and their negative controls,respectively.After above treatments,BAFF mRNA and protein levels were detected by real-time PCR and Western blot analysis,and the proliferation and apoptosis in the multiple myeloma (MM) cells were examined by WST-1 and annexin V-FLUOS assay,respectively.Results The BAFF mRNA expression levels in the untransfected group,has-miR-202-3P-mimics transfected group,has-miR-202-3P-inhibitor transfected group and siBAFF transfected group were 1.040 ± 0.057,0.573 ± 0.073,1.205 ± 0.097 and 0.368 ± 0.052,respectively.BAFF mRNA expressions in U266 cells transfected with has-miR-202-3P-mimics and siBAFF were significantly decreased compared with that in the untransfected group (P ＜ 0.05).The BAFF protein expression level of each group was consistent with the mRNA assay result.The absorbance value in 450 nm of the untransfected group,has-miR-202-3P-mimics transfected group,hasmiR-202-3P-inhibitor transfected group and siBAFF transfected group were 1.063 ±0.052,0.714 ±0.045,0.936 ± 0.066 and 0.764 ± 0.053,respectively.In comparison with the untransfected group,the absorbance value at 450 nm of has-miR-202-3P-mimics and siBAFF transfected groups was significantly reduced (P ＜ 0.05).The cell apoptosis rates of untransfected group,has-miR-202-3P-mimics transfected group,has-miR-202-3P-inhibitor transfected group and siBAFF transfected group were 26.2％,49.6％,21.1％ and 30.7％,respectively.Therefore,the cell apoptosis rate of has-miR-202-3P-mimics transfected group was significantly increased than that of the untransfected group (P ＜ 0.05).p-JNK protein expression level was decreased in the has-miR-202-3P-mimics transfected cells.Conclusions MiR-202 can inhibit the proliferation and induce apoptosis in MM cells via regulating BAFF.JNK/SAPK signaling pathway is involved in the regulation of BAFF by miR-202.