HIF-1α siRNA对非小细胞肺癌A549细胞TIMP1、MMP1表达的影响
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摘要
目的探讨HIF-1α基因沉默对非小细胞肺癌(NSCLC)A549细胞基质金属蛋白酶1(matrix metalloproteinase 1,MMP1)、基质金属蛋白酶抑制剂1(matrix metalloproteinase inhibitor,TIMP1)表达的影响。方法将siRNAHIF-1α质粒载体稳定转染至A549细胞中,于缺氧环境下培养。应用MMT法和Transwell试验分别检测转染细胞的增殖、迁移和侵袭能力。应用qRT-PCR和蛋白免疫印迹分别检测转染细胞内TIMP1、MMP1 mRNA和蛋白表达水平。结果与Control组相比,A549组HIF-1αmRNA和蛋白表达显著增加(均P<0.05)。与Blank组和si-NC组相比,si-HIF-1α组HIF-1αmRNA表达水平显著降低(P<0.05)。与Blank组相比,si-HIF-1α组在转染24、48和72 h后细胞增殖率均显著降低(均P<0.05)。与Blank组相比,si-HIF-1α组A549细胞在转染24 h后的迁移和侵袭数量均显著降低(P<0.05)。与Blank组相比,si-HIF-1α组细胞内MMP1表达水平显著降低,而TIMP1表达水平显著增加(均P<0.05)。结论在缺氧状态下,HIF-1αsiRNA靶向沉默HIF-1α在A549细胞内的表达,并可以通过下调MMP1表达和上调TIMP1的表达而抑制细胞的增殖、迁移和侵袭。
Objective To investigate the effect of HIF-1α gene silencing on the expression of TIMP1 and MMP1 in nonsmall cell lung cancer( NSCLC) A549 cells. Methods The siRNA-HIF-1α plasmid vector was transfected into A549 cells and cultured in hypoxic environment. The proliferation,migration and invasion of transfected cells were detected by MMT and Transwell. The expression of TIMP1,MMP1 mRNA and protein in the transfected cells were detected by realtime fluorescence quantitative and western blot. Results Compared with the control group,the expression of HIF-1αmRNA and protein in A549 group was significantly increased( P < 0. 05). Compared with the blank and si-NC groups,the expression of HIF-1α mRNA in si-HIF-1α group was significantly decreased( P < 0. 05). Compared with the blank group,the proliferation rate of si-HIF-1α group was significantly decreased at 24 h,48 h and 72 h after the transfection( P < 0. 05). Compared with blank group,the migration and invasion of A549 cells in si-HIF-1α group were significantly decreased( P < 0. 05). Compared with blank group,the expression of MMP1 in si-HIF-1α group was significantly decreased,while the expression of TIMP1 was significantly increased( P < 0. 05). Conclusion HIF-1α siRNA can target silencing HIF-1α in A549 cells under hypoxia and inhibit cell proliferation,migration and invasion by down-regulating MMP1 expression and up-regulating TIMP1 expression.
Objective To investigate the effect of HIF-1α gene silencing on the expression of TIMP1 and MMP1 in nonsmall cell lung cancer( NSCLC) A549 cells. Methods The siRNA-HIF-1α plasmid vector was transfected into A549 cells and cultured in hypoxic environment. The proliferation,migration and invasion of transfected cells were detected by MMT and Transwell. The expression of TIMP1,MMP1 mRNA and protein in the transfected cells were detected by realtime fluorescence quantitative and western blot. Results Compared with the control group,the expression of HIF-1αmRNA and protein in A549 group was significantly increased( P < 0. 05). Compared with the blank and si-NC groups,the expression of HIF-1α mRNA in si-HIF-1α group was significantly decreased( P < 0. 05). Compared with the blank group,the proliferation rate of si-HIF-1α group was significantly decreased at 24 h,48 h and 72 h after the transfection( P < 0. 05). Compared with blank group,the migration and invasion of A549 cells in si-HIF-1α group were significantly decreased( P < 0. 05). Compared with blank group,the expression of MMP1 in si-HIF-1α group was significantly decreased,while the expression of TIMP1 was significantly increased( P < 0. 05). Conclusion HIF-1α siRNA can target silencing HIF-1α in A549 cells under hypoxia and inhibit cell proliferation,migration and invasion by down-regulating MMP1 expression and up-regulating TIMP1 expression.
引文
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[14]Tong C,Hao H,Xia L,et al.Hypoxia pretreatment of bone marrowderived mesenchymal stem cells seeded in a collagen-chitosan sponge scaffold promotes skin wound healing in diabetic rats with hindlimb ischemia[J].Wound Repair&Regeneration,2016,24(1):45-56.
[15]Choi JH,Yun BL,Jung J,et al.Hypoxia Inducible Factor-1αRegulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrinα-4[J].Stem Cells Int,2016:7932185.doi:10.1155/2016/7932185.
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[3]周俭珊,叶红.HIF-1α、VEGF及HIF-1α/VEGF轴在子宫内膜癌中的表达研究进展[J].海南医学,2015,26(16):2413-2416.
[4]李李,周龙书,史文静.Vasohibin-1、VEGF及HIF-1α在子宫内膜腺癌中的表达及意义[J].河北医药,2016,38(5):662-666.
[5]刘彦琦,于红刚.缺氧诱导因子-1α和血管内皮生长因子在胃癌组织中的表达及其与肿瘤细胞增殖的关系[J].中华实验外科杂志,2015,32(9):2246-2248.
[6]杨常建,董海林,聂改红,等.HIF-1α与VEGF在非小细胞肺癌组织中的表达及临床意义[J].临床医学,2016,36(6):35-36.
[7]杨潇,范海涛,郑红淑,等.RNA干扰沉默缺氧诱导因子-1α对人膀胱尿路上皮癌T24细胞增殖和凋亡的影响[J].国际泌尿系统杂志,2016,36(1):62-65.
[8]Ji F,Wang Y,Qiu L,et al.Hypoxia inducible factor 1α-mediated LOX expression correlates with migration and invasion in epithelial ovarian cancer[J].Int J Oncol,2013,42(5):1578-1588.
[9]Schito L,Semenza GL.Hypoxia-Inducible Factors:Master Regulators of Cancer Progression[J].Trends in Cancer,2016,2(12):758-770.
[10]Marshall L,Khan A H,Buggy DJ.Can Anaesthetic and Analgesic Techniques for Cancer Surgery Affect Cancer Recurrence and Metastasis?[M].S.l.:Springer US,2015,5(2):190-202.
[11]Pisamai S,Rungsipipat A,Kalpravidh C,et al.Gene expression profiles of cell adhesion molecules,matrix metalloproteinases and their tissue inhibitors in canine oral tumors[J].Res Vet Sci,2017,113(8):94-100.
[12]Fei L,Pan LH,Lin R,et al.Differential expression of HIF-1α,AQP-1,and VEGF under acute hypoxic conditions in the non-ventilated lung of a one-lung ventilation rat model[J].Life Sci,2015,124(1):50-55.
[13]Richards R,Jenkinson MD,Haylock BJ,et al.Cell cycle progression in glioblastoma cells is unaffected by pathophysiological levels of hypoxia[J].Peerj,2016,4(1):e1755.
[14]Tong C,Hao H,Xia L,et al.Hypoxia pretreatment of bone marrowderived mesenchymal stem cells seeded in a collagen-chitosan sponge scaffold promotes skin wound healing in diabetic rats with hindlimb ischemia[J].Wound Repair&Regeneration,2016,24(1):45-56.
[15]Choi JH,Yun BL,Jung J,et al.Hypoxia Inducible Factor-1αRegulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrinα-4[J].Stem Cells Int,2016:7932185.doi:10.1155/2016/7932185.