缺氧对H9C2心肌细胞中miR-19a-3p表达的研究

The preliminary study on miR-19a-3p in rat H9C2 cardiomyocytes under hypoxia

目的检测大鼠H9C2心肌细胞缺氧后细胞水平和培养液中循环微小RNA(miRNA,miR)-19a-3p的表达,并探讨其与可能靶基因低密度脂蛋白受体相关蛋白2(LRP2)的关系。方法对H9C2心肌细胞进行缺氧(37℃,5% CO_2,1% O_2)0、4、8、12、16、20、24h处理,采用荧光实时定量聚合酶链反应(qRT-PCR)检测其在不同时间点细胞水平和培养液中循环miR-19a-3p的表达水平,使用ELISA、qRT-PCR、Western Blot检测LRP2在培养液和细胞中mRNA和蛋白表达。结果 H9C2细胞培养液中miR-19a-3p的表达在缺氧0、4、8、12、16h差异无统计学意义(P>0.05),在缺氧20h与24h时,H9C2细胞培养液中miR-19a-3p的表达与缺氧0h比较,明显高表达(P<0.05);在缺氧20h与24h时,H9C2细胞中miR-19a-3p的表达与缺氧0h比较,明显下降(P<0.05);H9C2细胞培养液中LRP2的表达在缺氧16h后表达明显升高,缺氧16h[(675.2±42.4)ng/mL,n=3]、缺氧20h[(979.4±204.2)ng/mL,n=3]和缺氧24h[(1 456.0±363.6)ng/mL,n=3]较0h[(245.0±10.84)ng/mL,n=3]明显升高(P<0.05);H9C2细胞中LRP2mRNA表达水平在缺氧16h后表达明显升高,缺氧16h[(0.000 503±0.000 100)ng/mL,n=3]、缺氧20h[(0.001 303±0.000 090)ng/mL,n=3]和缺氧24h[(0.001 617±0.000 110)ng/mL,n=3]较0h[(0.000 139 4±0.000 060 0)ng/mL,n=3]明显升高(P<0.05);20h[(0.235 000±0.003 427)ng/mL,n=3]与24h[(0.535 000±0.003 427)ng/mL,n=3]LRP2蛋白表达与0h[(0.140 10±0.018 21)ng/mL,n=3]比较,差异有统计学意义(P<0.01)。结论 miR-19a-3p可能参与大鼠H9C2心肌细胞的缺氧坏死过程,并且可能通过调控LRP2而发挥作用。

Objective To study the relationship of miRNA-19a-3p and LDL receptor related protein 2（LRP2）under hypoxia in rat H9C2 cardiomyocytes.Methods H9C2 cardiomyocytes were treated in hypoxia condition（37 ℃,5% CO2,1% O2）for 0,4,8,12,16,20,24 h.The miRNA-19a-3p level was detected by real time fluorescence quantitative PCR（qRT-PCR）in H9C2 cells and cell culture medium,and the probable target gene LRP2 level were test by ELISA,qRT-PCR and western blot in H9C2 cells and cell culture medium.Results miRNA-19a-3p expression in cell culture medium was much higher under 20 hand 24 h（P〈0.05）,miRNA-19a-3p expression in H9C2 cells was much lower under 20 hand 24 h（P〈0.05）.LRP expression in cell culture medium was much higher under 16 h[（675.20±42.40）ng/mL,n=3],20 h[（979.4±204.2）ng/mL,n=3]and 24 h[（1 456.00±363.60）ng/mL,n=3]than 0 h[（245.0±10.84）ng/mL,n=3]（P〈0.05）;the LRP2 mRNA level were much higher in 16 h[（0.000 503±0.000 100）ng/mL,n=3],20 h[（0.001 303±0.000 090）ng/mL,n=3]and 24 h[（0.001 617±0.000 110）ng/mL,n=3]（P〈0.05）;than in the 0 h[（0.000 139 4±0.000 060 0）ng/mL,n=3],the LRP2 protein level were much higher in 20 h[（0.235 000±0.003 427）,n=3]and 24 h[（0.535 000±0.003 427）ng/mL,n=3]（P〈0.05）.Conclusion miRNA-19a-3p may partipate in H9C2 cardiomyocytes necrosis through regulating LRP2.