siRNA介导的PD-L1沉默可增强人CD8~+T淋巴细胞的体外杀伤作用
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摘要
目的设计并筛选多条可高效特异性降低肿瘤细胞表面沉默程序性死亡受体-配体1(PD-L1)表达的小干扰RNA(siRNA)序列,并研究其增强人CD8~+T淋巴细胞对人胶质瘤细胞(U87 MG)免疫杀伤作用。方法根据siRNA设计法则,并通过siDirect软件在线设计针对PD-L1基因的几种不同的siRNA序列,并运用BLAST进行同源性分析,最终确定候选序列;通过RT-qPCR、Western blot及流式细胞术实验,选出高效抑制PD-L1 mRNA及蛋白表达的siRNA序列;通过流式细胞术及CCK8检测PD-L1siRNA沉默U87 MG细胞的PD-L1后,人CD8~+T细胞对U87 MG的细胞凋亡及增殖的影响。结果设计了10条针对人PD-L1基因具有不同核苷酸序列的siRNA。采用RT-qPCR、Western blot及流式细胞术在mRNA及蛋白水平上检测siRNA的沉默效率,与对照组相比,siPD-L1-1、siPD-L1-2、siPD-L1-3、siPD-L1-4、siPD-L1-5及siPD-L1-8均显著降低U87 MG中PD-L1基因的表达(P<0.05),其中siPD-L1-3的沉默效率最高。与对照组相比,流式细胞术及CCK8结果显示,siPD-L1-3及siPD-L1-8均可显著增强人CD8~+T细胞对U87 MG细胞的杀伤作用,且该杀伤作用可显著抑制U87 MG细胞增殖(P<0.05)。结论本文设计并筛选了能有效沉默PD-L1基因表达的siPD-L1-1、siPD-L1-2、siPD-L1-3、siPD-L1-4、siPD-L1-5及siPD-L1-8的6条序列,其中siPD-L1-3和siPD-L1-8可高效增强T淋巴细胞对U87 MG细胞免疫杀伤作用,且siPD-L1-3的作用最为显著。本文设计的PD-L1siRNA分子可以用于设计和制备预防、治疗多种癌症的核酸药物。
Objective To investigate the effect of small interfering RNA(siRNA)-mediated silencing of programmed cell deathligand 1(PD-L1) in human glioma cells on the cytotoxicity of human CD8~+T lymphocytes against the modified tumor cells.Methods A siRNA sequence targeting PD-L1 gene was designed and transfected into human glioma U87 MG cells via lipofectamine 2000, and the gene silencing effect was validated using RT-qPCR, Western blotting, and flow cytometry. The transfected cells were co-cultured with human CD8~+T lymphocytes, and the apoptosis of the tumor cells was analyzed with flow cytometry. Results The siRNA sequence showed strong PD-L1 gene-silencing effect at both mRNA and protein levels in U87 MG cells. Compared with the control cells, the transfected U87 MG cells showed significantly increased vulnerability to the cytotoxicity of human CD8~+T cells and an obvious reduction of proliferative activity in the co-culture(P<0.05). Conclusion Transfection of human glioma U87 MG cells with the specific siRNA targeting PD-L1 obviously enhances the toxicity of human T lymphocytes in the co-culture.
Objective To investigate the effect of small interfering RNA(siRNA)-mediated silencing of programmed cell deathligand 1(PD-L1) in human glioma cells on the cytotoxicity of human CD8~+T lymphocytes against the modified tumor cells.Methods A siRNA sequence targeting PD-L1 gene was designed and transfected into human glioma U87 MG cells via lipofectamine 2000, and the gene silencing effect was validated using RT-qPCR, Western blotting, and flow cytometry. The transfected cells were co-cultured with human CD8~+T lymphocytes, and the apoptosis of the tumor cells was analyzed with flow cytometry. Results The siRNA sequence showed strong PD-L1 gene-silencing effect at both mRNA and protein levels in U87 MG cells. Compared with the control cells, the transfected U87 MG cells showed significantly increased vulnerability to the cytotoxicity of human CD8~+T cells and an obvious reduction of proliferative activity in the co-culture(P<0.05). Conclusion Transfection of human glioma U87 MG cells with the specific siRNA targeting PD-L1 obviously enhances the toxicity of human T lymphocytes in the co-culture.
引文
[1]Ning YM,Suzman D,Maher VE,et al.FDA approval summary:atezolizumab for the treatment of patients with progressive advanced urothelial carcinoma after Platinum-Containing chemotherapy[J].Oncologist,2017,22(6):743-8.
[2]Kim ES.Avelumab:first global approval[J].Drugs,2017,77(8):929-37.
[3]Syed YY.Durvalumab:first global approval[J].Drugs,2017,77(12):1369-76.
[4]Kythreotou A,Siddique A,Mauri FA,et al.PD-L1[J].J Clin Pathol,2018,71(3):189-94.
[5]Wang XH,Bao ZQ,Zhang XJ,et al.Effectiveness and safety of PD-1/PD-L1 inhibitors in the treatment of solid tumors:a systematic review and meta-analysis[J].Oncotarget,2017,8(35):59901-14.
[6]Hobo W,Hutten TJ,Schaap NP.Immune checkpoint molecules in acute myeloid leukaemia:managing the double-edged sword[J].Br J Haematol,2018,181(1):38-53.
[7]Kourtidis A,Anastasiadis PZ.Close encounters of the RNAi kind:the silencing Life of the adherens junctions[J].Curr Opin Cell Biol,2018,54(4):30-6.
[8]Haussecker D.Current issues of RNAi therapeutics delivery and development[J].J Control Release,2014,195(2):49-54.
[9]Swaika A,Hammond WA,Joseph RW.Current state of anti-PD-L1and anti-PD-1 agents in cancer therapy[J].Mol Immunol,2015,67(2Pt A):4-17.
[10]Kwak G,Kirn D,Nam GH,et al.Programmed cell death protein ligand-1 silencing with Polyethylenimine-Dermatan sulfate complex for dual inhibition of melanoma growth[J].ACS Nano,2017,11(10):10135-46.
[11]Luo X,Peng X,Hou JY,et al.Folic acid-functionalized polyethylenimine superparamagnetic iron oxide nanoparticles as theranostic agents for magnetic resonance imaging and PD-L1 si RNA delivery for gastric cancer[J].Int J Nanomedicine,2017,12(1):5331-43.
[12]Teo PY,Yang C,Whilding LM,et al.Ovarian Cancer immunotherapy using PD-L1 si RNA targeted delivery from folic Acid-Functionalized polyethylenimine:strategies to enhance T cell killing[J].Adv Healthc Mater,2015,4(8):1180-9.
[13]Wang D,Wang T,Liu J,et al.Acid-Activatable versatile micelleplexes for PD-L1 Blockade-Enhanced cancer photodynamic immunotherapy[J].Nano Lett,2016,16(9):5503-13.
[14]Wittrup A,Lieberman J.Knocking down disease:a progress report on si RNA therapeutics[J].Nat Rev Genet,2015,16(9):543-52.
[15]Ozcan G,Ozpolat B,Coleman RL,et al.Preclinical and clinical development of si RNA-based therapeutics[J].Adv Drug Deliv Rev,2015,87(7):108-19.
[16]Ui-Tei K,Naito Y,Nishi K,et al.Thermodynamic stability and Watson-Crick base pairing in the seed duplex are major determinants of the efficiency of the si RNA-based off-target effect[J].Nucleic Acids Res,2008,36(22):7100-9.
[17]Ui-Tei K,Naito Y,Takahashi F,et al.Guidelines for the selection of highly effective si RNA sequences for mammalian and chick RNA interference[J].Nucleic Acids Res,2004,32(3):936-48.
[18]Reynolds A,Leake D,Boese Q,et al.Rational si RNA design for RNA interference[J].Nat Biotechnol,2004,22(3):326-30.
[19]Sun C,Mezzadra R,Schumacher TN.Regulation and function of the PD-L1 checkpoint[J].Immunity,2018,48(3):434-52.
[20]Goodman A,Patel SP,Kurzrock R.PD-1-PD-L1 immune-checkpoint blockade in B-cell lymphomas[J].Nat Rev Clin Oncol,2017,14(4):203-20.
[21]Gravelle P,Burroni B,Pericart S,et al.Mechanisms of PD-1/PD-L1expression and prognostic relevance in non-Hodgkin lymphoma:a summary of immunohistochemical studies[J].Oncotarget,2017,8(27):44960-75.
[22]Chen J,Jiang CC,Jin L,et al.Regulation of PD-L1:a novel role of pro-survival signalling in cancer[J].Ann Oncol,2016,27(3):409-16.
[23]Dowdy SF.Overcoming cellular barriers for RNA therapeutics[J].Nat Biotechnol,2017,35(3):222-9.
[24]Chiu YL,Rana TM.si RNA function in RNAi:A chemical modification analysis[J].RNA,2003,9(9):1034-48.
[25]Selvam C,Mutisya D,Prakash S,et al.Therapeutic potential of chemically modified si RNA:Recent trends[J].Chem Biol Drug Des,2017,90(5):665-78.
[26]Lee SH,Kang YY,Jang HE,et al.Current preclinical small interfering RNA(si RNA)-based conjugate systems for RNA therapeutics[J].Adv Drug Deliv Rev,2016,104(8):78-92.
[27]Ohaegbulam KC,Assal A,Lazar-Molnar E,et al.Human Cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway[J].Trends Mol Med,2015,21(1):24-33.
[28]Saldi T,Cortazar MA,Sheridan RM,et al.Coupling of RNA polymerase II transcription elongation with Pre-m RNA splicing[J].J Mol Biol,2016,428(12,SI):2623-35.
[29]Martinez T,Wright N,Lopez-Fraga M,et al.Silencing human genetic diseases with oligonucleotide-based therapies[J].Hum Genet,2013,132(5):481-93.
[30]Preusser M,Berghoff AS,Wick W,et al.Clinical neuropathology mini-review 6-2015:PD-L1:emerging biomarker in glioblastoma[J]?Clin Neuropathol,2015,34(6):313-21.
[2]Kim ES.Avelumab:first global approval[J].Drugs,2017,77(8):929-37.
[3]Syed YY.Durvalumab:first global approval[J].Drugs,2017,77(12):1369-76.
[4]Kythreotou A,Siddique A,Mauri FA,et al.PD-L1[J].J Clin Pathol,2018,71(3):189-94.
[5]Wang XH,Bao ZQ,Zhang XJ,et al.Effectiveness and safety of PD-1/PD-L1 inhibitors in the treatment of solid tumors:a systematic review and meta-analysis[J].Oncotarget,2017,8(35):59901-14.
[6]Hobo W,Hutten TJ,Schaap NP.Immune checkpoint molecules in acute myeloid leukaemia:managing the double-edged sword[J].Br J Haematol,2018,181(1):38-53.
[7]Kourtidis A,Anastasiadis PZ.Close encounters of the RNAi kind:the silencing Life of the adherens junctions[J].Curr Opin Cell Biol,2018,54(4):30-6.
[8]Haussecker D.Current issues of RNAi therapeutics delivery and development[J].J Control Release,2014,195(2):49-54.
[9]Swaika A,Hammond WA,Joseph RW.Current state of anti-PD-L1and anti-PD-1 agents in cancer therapy[J].Mol Immunol,2015,67(2Pt A):4-17.
[10]Kwak G,Kirn D,Nam GH,et al.Programmed cell death protein ligand-1 silencing with Polyethylenimine-Dermatan sulfate complex for dual inhibition of melanoma growth[J].ACS Nano,2017,11(10):10135-46.
[11]Luo X,Peng X,Hou JY,et al.Folic acid-functionalized polyethylenimine superparamagnetic iron oxide nanoparticles as theranostic agents for magnetic resonance imaging and PD-L1 si RNA delivery for gastric cancer[J].Int J Nanomedicine,2017,12(1):5331-43.
[12]Teo PY,Yang C,Whilding LM,et al.Ovarian Cancer immunotherapy using PD-L1 si RNA targeted delivery from folic Acid-Functionalized polyethylenimine:strategies to enhance T cell killing[J].Adv Healthc Mater,2015,4(8):1180-9.
[13]Wang D,Wang T,Liu J,et al.Acid-Activatable versatile micelleplexes for PD-L1 Blockade-Enhanced cancer photodynamic immunotherapy[J].Nano Lett,2016,16(9):5503-13.
[14]Wittrup A,Lieberman J.Knocking down disease:a progress report on si RNA therapeutics[J].Nat Rev Genet,2015,16(9):543-52.
[15]Ozcan G,Ozpolat B,Coleman RL,et al.Preclinical and clinical development of si RNA-based therapeutics[J].Adv Drug Deliv Rev,2015,87(7):108-19.
[16]Ui-Tei K,Naito Y,Nishi K,et al.Thermodynamic stability and Watson-Crick base pairing in the seed duplex are major determinants of the efficiency of the si RNA-based off-target effect[J].Nucleic Acids Res,2008,36(22):7100-9.
[17]Ui-Tei K,Naito Y,Takahashi F,et al.Guidelines for the selection of highly effective si RNA sequences for mammalian and chick RNA interference[J].Nucleic Acids Res,2004,32(3):936-48.
[18]Reynolds A,Leake D,Boese Q,et al.Rational si RNA design for RNA interference[J].Nat Biotechnol,2004,22(3):326-30.
[19]Sun C,Mezzadra R,Schumacher TN.Regulation and function of the PD-L1 checkpoint[J].Immunity,2018,48(3):434-52.
[20]Goodman A,Patel SP,Kurzrock R.PD-1-PD-L1 immune-checkpoint blockade in B-cell lymphomas[J].Nat Rev Clin Oncol,2017,14(4):203-20.
[21]Gravelle P,Burroni B,Pericart S,et al.Mechanisms of PD-1/PD-L1expression and prognostic relevance in non-Hodgkin lymphoma:a summary of immunohistochemical studies[J].Oncotarget,2017,8(27):44960-75.
[22]Chen J,Jiang CC,Jin L,et al.Regulation of PD-L1:a novel role of pro-survival signalling in cancer[J].Ann Oncol,2016,27(3):409-16.
[23]Dowdy SF.Overcoming cellular barriers for RNA therapeutics[J].Nat Biotechnol,2017,35(3):222-9.
[24]Chiu YL,Rana TM.si RNA function in RNAi:A chemical modification analysis[J].RNA,2003,9(9):1034-48.
[25]Selvam C,Mutisya D,Prakash S,et al.Therapeutic potential of chemically modified si RNA:Recent trends[J].Chem Biol Drug Des,2017,90(5):665-78.
[26]Lee SH,Kang YY,Jang HE,et al.Current preclinical small interfering RNA(si RNA)-based conjugate systems for RNA therapeutics[J].Adv Drug Deliv Rev,2016,104(8):78-92.
[27]Ohaegbulam KC,Assal A,Lazar-Molnar E,et al.Human Cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway[J].Trends Mol Med,2015,21(1):24-33.
[28]Saldi T,Cortazar MA,Sheridan RM,et al.Coupling of RNA polymerase II transcription elongation with Pre-m RNA splicing[J].J Mol Biol,2016,428(12,SI):2623-35.
[29]Martinez T,Wright N,Lopez-Fraga M,et al.Silencing human genetic diseases with oligonucleotide-based therapies[J].Hum Genet,2013,132(5):481-93.
[30]Preusser M,Berghoff AS,Wick W,et al.Clinical neuropathology mini-review 6-2015:PD-L1:emerging biomarker in glioblastoma[J]?Clin Neuropathol,2015,34(6):313-21.