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纳米SiO2暴露对人支气管上皮细胞microRNA表达水平的影响
作者:杨亚蕊 何云 龚春梅 周继昌 朱玉梅 莫俊銮

摘要:

目的  探讨纳米SiO2短期及长期暴露对人支气管上皮细胞(16HBE)microRNA(miRNA)表达水平的影响。方法  分别以5、10、15、20、25、30、40 μg/ml纳米SiO2溶液处理16HBE细胞24 h,采用CCK-8法检测细胞活性,并计算细胞增殖活性抑制率及半抑制浓度(IC50)。以10 μg/ml纳米SiO2溶液处理16HBE细胞至第10代(P10)和30代(P30),作为长期染毒组,并设立对照组(P0);以1/4 IC50、1/2 IC50、IC50浓度的纳米SiO2溶液和IC50浓度的微米SiO2溶液处理16HBE细胞24 h,作为短期染毒组,并设立无血清培养液组为对照组。用安捷伦miRNA微阵列芯片筛选P0、P10、P30组细胞差异表达的miRNA,并用逆转录荧光定量聚合酶链式反应(qRT-PCR)方法验证短期及长期染毒组与对照组miRNA的表达差异。结果  纳米SiO2溶液染剂量为5、10、15、20、25、30、40 μg/ml的16HBE细胞增殖活性抑制率分别为:(-3.33±3.80)%、(20.40±11.73)%、(39.08±5.53)%、(55.10±5.78)%、(66.42±9.60)%、(71.67±7.34)%、(81.43±5.37)%(F=129.11,P<0.001);IC50(95%CI)为18.35(15.82~20.72)μg/ml。长期染毒组中,P10、P30及P0细胞的miRNA-494-3p表达水平分别为0.45±0.08、0.28±0.07和1.00(F=60.77,P<0.001);miRNA-19a-3p表达水平分别为2.27±0.45、1.06±0.19和1.00(F=30.05,P<0.001);miRNA-148b-3p的表达水平分别为1.78±0.29、0.88±0.19和1.00(F=30.23,P<0.001)。短期染毒组中,5、10、20 μg/ml纳米SiO2溶液及20 μg/ml微米SiO2溶液染毒组的miRNA-494-3p表达水平分别为0.99±0.04、1.38±0.19、2.13±0.14、0.81±0.25(F=57.03,P<0.001);miRNA-19a-3p的表达水平分别为0.91±0.03、1.12±0.03、0.53±0.01、0.86±0.01(F=408.78,P<0.001);miRNA-148b-3p的表达水平分别为0.95±0.02、1.22±0.00、0.54±0.02、1.15±0.04(F=264.14,P<0.001)。结论  纳米SiO2短期及长期染毒可诱导16HBE细胞miRNA表达水平的变化,其中,长期和短期染毒对miRNA-494-3p的表达水平影响不同。

关键词:二氧化硅;上皮细胞;微RNAs;微阵列芯片;逆转录聚合酶链反应

Abstract:

Objective  To investigate the effect of short and long term exposure to SiO2 nanoparticles on microRNA expression level in human bronchial epithelial cells(16HBE cells).Methods  The 16HBE cells were exposed to 5, 10, 15, 20, 25, 30 and 40 μg/ml SiO2 nanoparticles for 24 h to detect the cell viability by using CCK-8 assay. The inhibition rate of proliferation activity and half inhibitory concentration (IC50) were calculated. The 16HBE cells were exposed to 10 μg/ml SiO2 nanoparticles for 10 and 30 generations, named P10 and P30, and the control P0 was set. The cells were treated with SiO2 nanoparticles at 0, 1/4 IC50, 1/2 IC50 and IC50 concentration and μm-SiO2 at IC50 concentration for 24 h, and the control serum-free culture medium was set. Agilent miRNAs microarray chip was used to screen differentially expressed miRNAs in P10, P30 and P0 groups. The expression level of miRNA was detected by reverse transcription fluorescence quantitative polymerase chain reaction (qRT-PCR).Results  The inhibition rate of proliferation activity of 5, 10, 15, 20, 25,30,40 μg/ml group were (-3.33±3.80)%, (20.40±11.73)%, (39.08±5.53)%, (55.10±5.78)%, (66.42±9.60)%, (71.67±7.34)%, (81.43±5.37)%, respectively; F=129.11, P<0.001. The IC50 (95%CI) was 18.35 (15.82-20.72) μg/ml. The expression level of miRNA-494-3p in P0, P10 and P30 were 1.00, 0.45±0.08, 0.28±0.07, respectively; F=60.77, P<0.001. miRNA-19a-3p were 1.00, 2.27±0.45, 1.06±0.19, respectively; F=30.05, P<0.001. miRNA-148b-3p were 1.00, 1.78±0.29, 0.88±0.19, respectively; F=30.23, P<0.001. Compared to control group, the expression level of miRNA-494-3p in 5, 10, 20 μg/ml SiO2 nanoparticles groups and 20 μg/ml μm-SiO2 group were 0.99±0.04, 1.38±0.19, 2.13±0.14, 0.81±0.25, respectively; F=57.03, P<0.001. miRNA-19a-3p were 0.91±0.03, 1.12±0.03, 0.53±0.01, 0.86±0.01, respectively; F=408.78, P<0.001. miRNA-148b-3p were 0.95±0.02, 1.22±0.00, 0.54±0.02, 1.15±0.04 respectively; F=264.14, P<0.001.Conclusion  Short and long term exposure to SiO2 nanoparticles can affect the expression level of miRNAs in 16HBE cells. The expressions of miRNA-494-3p after long and short period exposure are different.

Key words: Silica nanoparticle;Human bronchial epithelial cell;microRNAs;miRNAs microarray chip;qRT-PCR

发表日期:2016/3

引用本文:

图/表:

  • 10.3760/cma.j.issn.0253-9624.2016.03.011.T001:表1 纳米SiO2长期染毒组16HBE细胞miRNA表达情况

    10.3760/cma.j.issn.0253-9624.2016.03.011.T001:表1 纳米SiO2长期染毒组16HBE细胞miRNA表达情况

  • 10.3760/cma.j.issn.0253-9624.2016.03.011.F001:图1 纳米SiO2长期染毒组及对照组miRNA微阵列杂交结果

    10.3760/cma.j.issn.0253-9624.2016.03.011.F001:图1 纳米SiO2长期染毒组及对照组miRNA微阵列杂交结果

  • 10.3760/cma.j.issn.0253-9624.2016.03.011.T002:表2 纳米SiO2长期染毒组及对照组16HBE细胞miRNA相对表达水平(±s,n=3)

    10.3760/cma.j.issn.0253-9624.2016.03.011.T002:表2 纳米SiO2长期染毒组及对照组16HBE细胞miRNA相对表达水平(±s,n=3)

  • 10.3760/cma.j.issn.0253-9624.2016.03.011.T003:表3 不同剂量纳米SiO2及微米SiO2短期染毒对16HBE细胞miRNA相对表达水平的影响(±s,n=3)

    10.3760/cma.j.issn.0253-9624.2016.03.011.T003:表3 不同剂量纳米SiO2及微米SiO2短期染毒对16HBE细胞miRNA相对表达水平的影响(±s,n=3)

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