miR-140-3p通过靶向PD-L1抑制非小细胞肺癌A549细胞的活力、迁移和侵袭
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摘要
目的:探究微小RNA-140-3p(mi R-140-3p)对程序性细胞死亡配体1(PD-L1)的靶向关系以及对非小细胞肺癌A549细胞的活力、迁移和侵袭的影响。方法:使用RT-qPCR检测HLF-1、A549和H1299不同细胞株中mi R-140-3p的表达水平,选择差异最显著的A549细胞用作后续研究对象; Target Scan软件预测和双萤光素酶报告基因实验验证mi R-140-3p和PD-L1之间的靶向关系; RT-qPCR和Western blot检测转染mi R-140-3p模拟物和抑制剂对PD-L1表达水平的影响; MTT法检测mi R-140-3p和PD-L1对A549细胞活力的影响; Transwell实验检测mi R-140-3p和PD-L1对A549细胞迁移和侵袭的影响。结果:mi R-140-3p在人肺癌A549和H1299细胞的表达中显著下调(P <0. 05); mi R-140-3p高表达能够使PD-L1表达下调,对A549细胞的活力、迁移和侵袭具有抑制作用;转染pc DNA3. 0-PD-L1能够阻断mi R-140-3p对A549细胞活力、迁移和侵袭的抑制作用。结论:mi R-140-3p可通过靶向负调控PD-L1抑制A549细胞的活力、迁移和侵袭。
AIM: To explore the target relationship between micro RNA-140-3p( mi R-140-3p) and programmed cell death ligand 1( PD-L1) and their effect on the viability,migration and invasion of non-small-cell lung cancer A549 cells. METHODS: RT-qPCR was used to detect the mi R-140-3p expression in HLF-1,A549 and H1299 cells,and then the A549 cells with the most significant difference were selected as the subsequent research object. Target Scan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between mi R-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of mi R-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells. RESULTS: mi R-140-3p was significantly down-regulated in the A549 cells and H1299 cells( P < 0. 05). Transfection with mi R-140-3p mimic decreased the expression of PD-L1 and inhibited the viability,migration and invasion of the A549 cells. Transfection with pc DNA3. 0-PD-L1 reversed the inhibitory effect of mi R-140-3p on the viability,migration and invasion of the A549 cells. CONCLUSION: mi R-140-3p inhibits the viability,migration and invasion of A549 cells by targeting PD-L1.
AIM: To explore the target relationship between micro RNA-140-3p( mi R-140-3p) and programmed cell death ligand 1( PD-L1) and their effect on the viability,migration and invasion of non-small-cell lung cancer A549 cells. METHODS: RT-qPCR was used to detect the mi R-140-3p expression in HLF-1,A549 and H1299 cells,and then the A549 cells with the most significant difference were selected as the subsequent research object. Target Scan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between mi R-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of mi R-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells. RESULTS: mi R-140-3p was significantly down-regulated in the A549 cells and H1299 cells( P < 0. 05). Transfection with mi R-140-3p mimic decreased the expression of PD-L1 and inhibited the viability,migration and invasion of the A549 cells. Transfection with pc DNA3. 0-PD-L1 reversed the inhibitory effect of mi R-140-3p on the viability,migration and invasion of the A549 cells. CONCLUSION: mi R-140-3p inhibits the viability,migration and invasion of A549 cells by targeting PD-L1.
引文
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[16]Ishida Y,Agata Y,Shibahara K,et al.Induced expression of PD-1,a novel member of the immunoglobulin gene superfamily,upon programmed cell death[J].EMBO J,1992,11(11):3887-3895.
[17]Chen L,Flies DB.Molecular mechanisms of T cell costimulation and co-inhibition[J].Nat Rev Immunol,2013,13(4):227-242.
[18]Pardoll DM.The blockade of immune checkpoints in cancer immunotherapy[J].Nat Rev Cancer,2012,12(4):252-264.
[19]Wang Y,Wang H,Yao H,et al.Regulation of PD-L1:emerging routes for targeting tumor immune evasion[J].Front Pharmacol,2018,9:536.
[20]Velcheti V,Schalper KA,Carvajal DE,et al.Programmed death ligand-1 expression in non-small cell lung cancer[J].Lab Invest,2014,94(1):107-116.
[21]Audrito V,Serra S,Stingi A,et al.PD-L1 up-regulation in melanoma increases disease aggressiveness and is mediated through mi R-17-5p[J].Oncotarget,2017,8(9):15894-15911.
[22]Spranger S,Spaapen RM,Zha Y,et al.Up-regulation of PD-L1,IDO,and Tregsin the melanoma tumor microenvironment is driven by CD8+T cells[J].Sci Transl Med,2013,5(200):200ra116.
[23]Pyo JS,Kang G,Kim JY.Prognostic role of PD-L1 in malignant solid tumors:a meta-analysis[J].Int J Biol Markers,2017,32(1):e68-e74.
[24]Thompson RH,Gillett MD,Cheville JC,et al.Costimulatory B7-H1 in renal cell carcinoma patients:indicator of tumor aggressiveness and potential therapeutic target[J].Proc Natl Acad Sci U S A,2004,101(49):17174-17179.
[25]Ambros V.The functions of animal micro RNAs[J].Nature,2004,431(7006):350-355.
[26]Dong W,Yao C,Teng X,et al.MiR-140-3p suppressed cell growth and invasion by downregulating the expression of ATP8A1 in non-small cell lung cancer[J].Tumor Biol,2016,37(3):2973-2985.
[27]Kong XM,Zhang GH,Huo YK,et al.MicroRNA-140-3p inhibits proliferation,migration and invasion of lung cancer cells by targeting ATP6AP2[J].Int J Clin Exp Pathol,2015,8(10):12845-12852.
[28]Jude JA,Dileepan M,Subramanian S,et al.mi R-140-3p regulation of TNF-α-induced CD38 expression in human airway smooth muscle cells[J].Am J Physiol Lung Cell Mol Physiol,2012,303(5):L460-L468.
[2]Siegel RL,Miller KD,Jemal A.Cancer statistics,2018[J].CA Cancer J Clin,2018,68(1):7-30.
[3]Osarogiagbon RU,Lin CC,Smeltzer MP,et al.Prevalence,prognostic implications,and survival modulators of incompletely resected non-small cell lung cancer in the U.S.National Cancer Data Base[J].J Thorac Oncol,2016,11(1):e5-e16.
[4]Lischalk JW,Woo SM,Kataria S,et al.Long-term outcomes of stereotactic body radiation therapy(SBRT)with fiducial tracking for inoperable stage I non-small cell lung cancer(NSCLC)[J].J Radiat Oncol,2016,5(4):379-387.
[5]Heist RS,Engelman JA.Snap Shot:non-small cell lung cancer[J].Cancer Cell,2012,21(3):448.e2.
[6]Benz F,Roy S,Trautwein C,et al.Circulating microRNAs as biomarkers for sepsis[J].Int J Mol Sci,2016,17(1):E78.
[7]Korpela E,Vesprini D,Liu SK.MicroRNA in radiotherapy:mi Rage or mi Rador?[J].Br J Cancer,2015,112(5):777-782.
[8]Song Y,Zuo Y,Qian XL,et al.Inhibition of micro RNA-21-5p promotes the radiation sensitivity of non-small cell lung cancer through HMSH2[J].Cell Physiol Biochem,2017,43(3):1258-1272.
[9]Kapodistrias N,Bobori C,Theocharopoulou G.MiR-140-3p downregulation in association with PDL-1 overexpression in many cancers:a review from the literature using predictive bioinformatics tools[J].Adv Exp Med Biol,2017,988:225-233.
[10]Dong W,Yao C,Teng X,et al.MiR-140-3p suppressed cell growth and invasion by downregulating the expression of ATP8A1 in non-small cell lung cancer[J].Tumor Biol,2016,37(3):2973-2985.
[11]Ma L,Xie XW,Wang HY,et al.Clinical evaluation of tumor markers for diagnosis in patients with non-small cell lung cancer in China[J].Asian Pacific J Cancer Prev,2015,16(12):4891-4894.
[12]Casaluce F,Sgambato A,Sacco PC,et al.Emerging drugs targeting PD-1 and PD-L1:reality or hope?[J].Expert Opin Emerg Drugs,2014,19(4):557-569.
[13]Detterbeck FC,Boffa DJ,Tanoue LT.The new lung cancer staging system[J].Chest,2009,136(1):260-271.
[14]Dunn GP,Old LJ,Schreiber RD.The three Es of cancer immunoediting[J].Ann Rev Immunol,2004,22(22):329-360.
[15]Keir ME,Butte MJ,Freeman GJ,et al.PD-1 and its ligands in tolerance and immunity.[J].Ann Rev Immunol,2009,26(1):677-704.
[16]Ishida Y,Agata Y,Shibahara K,et al.Induced expression of PD-1,a novel member of the immunoglobulin gene superfamily,upon programmed cell death[J].EMBO J,1992,11(11):3887-3895.
[17]Chen L,Flies DB.Molecular mechanisms of T cell costimulation and co-inhibition[J].Nat Rev Immunol,2013,13(4):227-242.
[18]Pardoll DM.The blockade of immune checkpoints in cancer immunotherapy[J].Nat Rev Cancer,2012,12(4):252-264.
[19]Wang Y,Wang H,Yao H,et al.Regulation of PD-L1:emerging routes for targeting tumor immune evasion[J].Front Pharmacol,2018,9:536.
[20]Velcheti V,Schalper KA,Carvajal DE,et al.Programmed death ligand-1 expression in non-small cell lung cancer[J].Lab Invest,2014,94(1):107-116.
[21]Audrito V,Serra S,Stingi A,et al.PD-L1 up-regulation in melanoma increases disease aggressiveness and is mediated through mi R-17-5p[J].Oncotarget,2017,8(9):15894-15911.
[22]Spranger S,Spaapen RM,Zha Y,et al.Up-regulation of PD-L1,IDO,and Tregsin the melanoma tumor microenvironment is driven by CD8+T cells[J].Sci Transl Med,2013,5(200):200ra116.
[23]Pyo JS,Kang G,Kim JY.Prognostic role of PD-L1 in malignant solid tumors:a meta-analysis[J].Int J Biol Markers,2017,32(1):e68-e74.
[24]Thompson RH,Gillett MD,Cheville JC,et al.Costimulatory B7-H1 in renal cell carcinoma patients:indicator of tumor aggressiveness and potential therapeutic target[J].Proc Natl Acad Sci U S A,2004,101(49):17174-17179.
[25]Ambros V.The functions of animal micro RNAs[J].Nature,2004,431(7006):350-355.
[26]Dong W,Yao C,Teng X,et al.MiR-140-3p suppressed cell growth and invasion by downregulating the expression of ATP8A1 in non-small cell lung cancer[J].Tumor Biol,2016,37(3):2973-2985.
[27]Kong XM,Zhang GH,Huo YK,et al.MicroRNA-140-3p inhibits proliferation,migration and invasion of lung cancer cells by targeting ATP6AP2[J].Int J Clin Exp Pathol,2015,8(10):12845-12852.
[28]Jude JA,Dileepan M,Subramanian S,et al.mi R-140-3p regulation of TNF-α-induced CD38 expression in human airway smooth muscle cells[J].Am J Physiol Lung Cell Mol Physiol,2012,303(5):L460-L468.