SP-1通过miR-135b/HIF-1α轴对缺氧诱导心肌细胞损伤的影响

Low Expression of SP-1 Exerts Protective Effects on Hypoxia-Induced Cardiomyocyte Injury Through the miR-135b/HIF-1αAxis

目的探讨特异性蛋白1(SP-1)通过miR-135b/HIF-1α轴对缺氧诱导的心肌细胞损伤的作用。方法 H9c2细胞进行缺氧处理后,转染si-SP-1,miR-135b mimic,anti-miR-135b和CoCl2(HIF-1α激活剂)。采用蛋白质印迹(Western Blot,WB)法检测细胞SP-1和HIF-1α蛋白表达,实时荧光定量聚合酶链式反应(PCR)法检测miR-135b表达,CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡和活性氧簇(ROS)含量,酶联免疫吸附(ELISA)试剂盒检测肿瘤坏死因子-α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)的含量,生物信息学软件分析预测并通过荧光素酶报告基因实验验证miR-135b与HIF-1α间的靶向结合作用。结果缺氧后,H9c2细胞内SP-1蛋白表达呈时间依赖性显著升高(P <0. 05),miR-135b表达呈时间依赖性显著降低(P <0. 05)。与对照组(A组)相比,缺氧组(B组)H9c2细胞中SP-1和HIF-1α蛋白表达分别为1. 467±0. 055,1. 437±0. 082(P=0. 014,0. 017),细胞凋亡率[(14. 591±1. 863)%,P=0. 000]、细胞上清液中TNF-α[(5. 270±0. 359) pg/m L,P=0. 012]、IL-1β[(25. 060±1. 212) pg/m L,P=0. 000]、IL-6 [(82. 707±4. 550) pg/m L,P=0. 000]和ROS(1. 239±0. 075,P=0. 021)含量均显著升高(P <0. 05),miR-135b表达(0. 466±0. 071,P=0. 001)和细胞增殖能力[(67. 242±3. 490)%,P=0. 008]均显著降低(P <0. 05);与缺氧+siRNA组(C组)相比,缺氧+si-SP-1组(D组)H9c2细胞中SP-1(0. 238±0. 039,P=0. 001)和HIF-1α的蛋白表达(1. 126±0. 100,P=0. 032)、细胞凋亡率[(4. 668±0. 731)%,P=0. 000]、细胞上清液中TNF-α[(3. 901±0. 354) pg/m L,P=0. 022]、IL-1β[(17. 198±1. 216) pg/m L,P=0. 002]、IL-6 [(56. 742±4. 081) pg/m L,P=0. 017]和ROS(1. 086±0. 090,P=0. 028)含量均显著降低,miR-135b表达(0. 752±0. 094,P=0. 015)和细胞增殖能力[(83. 550±2. 933)%,P=0. 016]均显著升高。miR-135b靶向调控HIF-1α。CoCl2或anti-miR-135b会导致低表达SP-1的缺氧H9c2细胞增殖能力显著降低[(48. 470±2. 831)%比(63. 728±4. 192)%,P=0. 000,0. 021],细胞凋亡率[(41. 076±1. 117)%比(35. 935±1. 776)%,P=0. 000,0. 010]和细胞上清液中TNF-α[(6. 211±0. 408) pg/m L比(5. 250±0. 325) pg/m L,P=0. 019,0. 033]、IL-1β[(43. 417±2. 561) pg/m L比(38. 675±1. 956) pg/m L,P=0. 013,0. 021]、IL-6 [(110. 440±7. 446) pg/m L比(108. 767±6. 589) pg/m L,P=0. 026,0. 031]和ROS [(1. 351±0. 073)比(1. 327±0. 041),P=0. 073,0. 035]的含量显著升高。结论 SP-1低表达通过miR-135b/HIF-1α轴发挥对缺氧诱导的心肌细胞损伤的保护作用。

Objective To investigate the effect of specific protein-1(SP-1)on hypoxia-induced cardiomyocyte injury through miR-135b/HIF-1αaxis.Methods H9c2 cells were transfected with si-SP-1 and miR-135b mimic after hypoxia,anti-miR-135b and CoCl2(hypoxia inducible factor-1α,HIF-1αactivator),SP-1 and HIF-1αprotein expression were detected by Western Blot(WB);the detection of miR-135b expression was conducted by real-time quantitative polymerase chain reaction(PCR);cholecystokinin octapeptide-8(CCK-8)were used to detect cell proliferation;flow cytometry was used to detect apoptosis and reactive oxygen species(ROS)content;enzyme-linked immunosorbent assay(ELISA)kit was applied to detect thecontents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and IL-6;bioinformatics software analysis was applied to predict and verify the targeted binding effect between miR-135b and HIF-1αby luciferase reporter gene experiments.Results After hypoxia,the expression of SP-1 protein in H9c2 cells increased significantly in a time-dependent manner(P<0.05),and the expression of miR-135b decreased significantly in a time-dependent manner(P<0.05).Compared with the control group,the protein expression of SP-1 and HIF-1αin H9c2 cells in the hypoxia group were 1.467±0.055 and 1.437±0.082(P=0.014,P=0.017),and the apoptosis rate[(14.591±1.863)%,P=0.000],TNF-α[(5.270±0.359)pg/mL,P=0.012],IL-1β[(25.060±1.212)pg/mL,P=0.000],IL-6[(82.707±4.550)pg/mL,P=0.000],and ROS(1.239±0.075,P=0.021)content was significantly increased(P<0.05),miR-135b expression(0.466±0.071,P=0.001)and cell proliferation ability[(67.242±3.490)%,P=0.008]were significantly reduced(P<0.05).Compared with the hypoxia + siRNA group,the protein expression of SP-1( 0.238 ± 0.039,P = 0.001) and HIF-1α( 1.126 ± 0.100,P = 0.032) ,apoptoticrate [ ( 4.668 ± 0.731 )%,P = 0.000 ],TNF-α[ ( 3.901 ± 0.354 ) pg /mL,P = 0.022 ],IL-1β[ ( 17.198 ± 1.216 ) pg /mL,P = 0.002 ],IL-6 [ ( 56.742 ± 4.081 ) pg /mL,P = 0.017 ] and ROS( 1.086 ± 0.090,P = 0.028) were significantly reduced( P < 0.05),miR-135bexpression( 0.752 ± 0.094,P = 0.015) and cell proliferation capacity [ ( 83.550 ± 2.933 )%,P = 0.016 ] were significantly increased( P <0.05) .miR-135b targets HIF-1α.CoCl2 or anti-miR-135b can significantly reduce the proliferation [ ( 48.470 ± 2.831 )% vs63.728 ± 4.192 )%,P = 0.000,P = 0.021 ],and apoptosis rate [ ( 41.076 ± 1.117 )% vs 35.935 ± 1.776 )%,P = 0.000,P = 0.010 ] andTNF-α[ ( 6.211 ± 0.408 ) pg /mL vs (5.250 ± 0.325 ) pg /mL,P = 0.019,P = 0.033) ],IL-1β[ ( 43.417 ± 2.561 ) pg /mL vs ( 38.675 ±1.956 ) pg /mL,P = 0.013,P = 0.021 ],IL-6 [ ( 110.440 ± 7.446 ) pg /mL vs (108.767 ± 6.589 ) pg /mL,P = 0.026,P = 0.031 ],and ROS( 1.351 ± 0.073 ) vs (1.327 ± 0.041 ),P = 0.024,P = 0.035 ] significantly increased( P < 0.05) of hypoxia H9c2 cells with low expressionof SP-1.Conclusion The low expression of SP-1 exerts a protective effect on hypoxia-induced cardiomyocyte injurythrough the miR-135b / HIF-1α axis.