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目的探讨小檗碱对皮肤黑素瘤A375细胞株增殖及细胞周期相关miRNA及其靶基因表达的影响。方法体外培养A375细胞,加入不同浓度小檗碱(0,20,40,60,80,100μmol/L),用噻唑蓝(MTT)法测定小檗碱对A375细胞生长的抑制作用;流式细胞实验检测细胞周期;实时定量RT-PCR法检测小檗碱对A375细胞miRNA-34a,miRNA-154,miRNA-26a,miRNA-124及其对应靶基因CDK4,Cyclin D1,CyclinE,CDK2表达的影响。采用SPSS13.0软件进行多因素方差分析。结果 MTT法结果显示小檗碱抑制A375细胞增殖,具有时效依赖性和量效依赖性(P<0.05)。流式细胞实验表明低浓度(20,40μmol/L)小檗碱作用后细胞周期呈S期阻滞和G2/M期阻滞(均P<0.05),高浓度(60,80μmol/L)小檗碱作用后细胞周期呈G_2/M期阻滞(P<0.05)。实时定量RT-PCR结果表明,小檗碱作用后随着浓度的增加miRNA-34a,miRNA-154,miRNA-26a,miRNA-124表达增加(均P<0.05),其对应的靶基因CDK4,CyclinD1,CyclinE,CDK2表达减弱(均P<0.05)。结论小檗碱对A375细胞的生长抑制作用呈时间和浓度依赖性;小檗碱可能通过调控细胞周期相关miRNA及其靶基因对黑素瘤A375细胞发挥抑制作用。
Objective To investigate the effects of berberine on proliferation and cell cycle related miRNAs and their target genes in cutaneous melanoma A375 cells. Methods A375 cells were cultured in vitro. Different concentrations of berberine (0, 20, 40, 60, 80 and 100 μmol / L) were added to induce the growth of A375 cells. MTT assay was used to determine the inhibitory effect of berberine on A375 cells. Flow cytometry The cell cycle was detected by real-time quantitative RT-PCR. The effect of berberine on the expression of miRNA-34a, miRNA-154, miRNA-26a, miRNA-124 and their corresponding target genes CDK4, Cyclin D1, CyclinE and CDK2 in A375 cells was detected by real- Multivariate analysis of variance was performed using SPSS 13.0 software. Results MTT assay showed that berberine inhibited the proliferation of A375 cells in a time-and dose-dependent manner (P <0.05). The results of flow cytometry showed that S-phase arrest and G2 / M arrest (P <0.05) and high concentration (60 and 80μmol / L) of cell cycle were observed after berberine treatment at low concentration The cell cycle arrest was G 2 / M after berberine treatment (P <0.05). The results of real-time quantitative RT-PCR showed that the expression of miRNA-34a, miRNA-154, miRNA-26a and miRNA-124 increased with the increase of the concentration of berberine (all P < , CyclinE, CDK2 expression decreased (all P <0.05). Conclusion Berberine inhibits the growth of A375 cells in a time and concentration-dependent manner. Berberine may inhibit the proliferation of A375 cells by regulating cell cycle related miRNAs and their target genes.