过表达lncRNA HOTAIR基因的风湿关节炎成纤维样滑膜细胞稳定株构建与筛选
|
摘要
目的 HOTAIR基因与类风湿关节炎(RA)血管翳的形成紧密相关。文中旨在建立并筛选过表达lncRNA HOTAIR的人类风湿关节炎成纤维样滑膜细胞(HFLS-RA)稳定株,为进一步研究lncRNA HOTAIR在RA发病中的作用奠定基础。方法采用PCR扩增lncRNA HOTAIR目的基因,连接到BamHI-HF-HF和XhoI酶切后的PMT406载体上,构建好的质粒经测序鉴定后,转染293T细胞包装慢病毒,分别用重组质粒及空载体慢病毒感染HFLS-RA,构建和筛选稳转株;采用Trizol法分别提取空白细胞、阴性转染细胞和过表达细胞的总RNA后,反转录得到cDNA进行qPCR扩增,根据检测结果进行相对表达量的计算。结果过表达细胞的lncRNA HOTAIR相对表达量(30.329±3.860)较空白细胞和阴性转染细胞(1.001±0.048、0.892±0.247)明显升高,差异有统计学意义(P<0.05);空白细胞和阴性转染细胞差异无统计学意义(P>0.05)。说明过表达lncRNA HOTAIR的HFLS-RA细胞稳定株构建成功。结论成功构建了过表达lncRNA HOTAIR的HFLS-RA细胞稳定株,为进一步探究lncRNA HOTAIR在RA发生发展中的作用提供了实验材料。
Objective The HOTAIR gene is closely related to pannus formation in rheumatoid arthritis(RA). This study aimed to construct and screen fibroblast-like synoviocytes in human RA(HFLS-RA)stably overexpressing lncRNA HOTAIR,and to pave the way for further study of the role of lncRNA HOTAIR in the pathogenesis of RA.Methods LncRNA HOTAIR was cloned and linked to the PMT406 vector digested by BamHI-HF-HF and XhoI. The constructed plasmids were sequenced,identified and then transfected into 293 T cells to pack lentivirus. The HFLS-RA cells were infected with the recombinant and empty vector lentiviruses,and purinomycin was employed to screen the lncRNA HOTAIR-overexpressed and control cell lines. The total RNA was extracted from the blank,negatively transfected and overexpressed cells by Trizol,and the cDNA obtained by reverse transcription was amplified by qPCR,followed by determination of the expression of lncRNA HOTAIR.Results The relative expression of lncRNA HOTAIR was significantly higher in the overexpression group than in the blank con-trol and negative transfection groups(30.329 ± 3.860 vs 1.001 ± 0.048 and 0.892 ± 0.247,P < 0.05),with no statistically significant difference between the latter two groups(P > 0.05).Conclusion The HFLS-RA cell line stably overexpressing lncRNA HOTAIR was successfully constructed,which has provided some experimental evidence for further investigation of the role of lncRNA HOTAIR in the pathogenesis of RA.
Objective The HOTAIR gene is closely related to pannus formation in rheumatoid arthritis(RA). This study aimed to construct and screen fibroblast-like synoviocytes in human RA(HFLS-RA)stably overexpressing lncRNA HOTAIR,and to pave the way for further study of the role of lncRNA HOTAIR in the pathogenesis of RA.Methods LncRNA HOTAIR was cloned and linked to the PMT406 vector digested by BamHI-HF-HF and XhoI. The constructed plasmids were sequenced,identified and then transfected into 293 T cells to pack lentivirus. The HFLS-RA cells were infected with the recombinant and empty vector lentiviruses,and purinomycin was employed to screen the lncRNA HOTAIR-overexpressed and control cell lines. The total RNA was extracted from the blank,negatively transfected and overexpressed cells by Trizol,and the cDNA obtained by reverse transcription was amplified by qPCR,followed by determination of the expression of lncRNA HOTAIR.Results The relative expression of lncRNA HOTAIR was significantly higher in the overexpression group than in the blank con-trol and negative transfection groups(30.329 ± 3.860 vs 1.001 ± 0.048 and 0.892 ± 0.247,P < 0.05),with no statistically significant difference between the latter two groups(P > 0.05).Conclusion The HFLS-RA cell line stably overexpressing lncRNA HOTAIR was successfully constructed,which has provided some experimental evidence for further investigation of the role of lncRNA HOTAIR in the pathogenesis of RA.
引文
[1]李兴,尹玉峰,马斌,等.157例类风湿关节炎患者合并症及并发症的临床研究[J].中华疾病控制杂志,2016,20(2):201-203.
[2]Fearon U,Canavan M,Biniecka M,et al.Hypoxia,mitochondrial dysfunction and synovial invasiveness in rheumatoid arthritis[J].Nat Rev Rheumatol,2016,12(7):385-397.
[3]Rinn JL,Kertesz M,Wang JK,et al.Functional Demarcation of Active and Silent Chromatin Domains in Human HOX Loci by Noncoding RNAs[J].NIH-PA,2007,129(7):1311-1323.
[4]张国华,徐颖,邹晨,等.长链非编码RNA DGCR5在胃癌中的表达及临床意义[J].医学研究生学报,2018,31(1):33-38.
[5]郭倚天,许斌,陈明.长链非编码RNA在前列腺癌中的作用及研究进展[J].医学研究生学报,2017,30(2):199-203.
[6]Zhang HJ,Wei QF,Wang SJ,et al.LncRNA HOTAIR alleviates rheumatoid arthritis by targeting miR-138 and inactivating NF-κB pathway[J].Int Immunopharmacol,2017,50(6):283-290.
[7]Pahlevan Kakhki M,Nikravesh A,Shirvani Farsani Z,et al.HOTAIR but notANRIL long non-coding RNA contributes to the pathogenesis of multiple sclerosis[J].Immunology,2018,153(4):479-487.
[8]Fu WM,Lu YF,Hu BG,et al.Long noncoding RNA Hotair mediated angiogenesis in nasopharyngeal carcinoma by direct and indirect signaling pathways[J].Oncotarget,2016,7(4):4712-4723.
[9]McInnes IB,Schett G.Pathogenetic insights from the treatment of rheumatoid arthritis[J].Lancet,2017,389(10086):2328-2337.
[10]中华医学会风湿病学分会.2018中国类风湿关节炎诊疗指南[J].中华内科杂志,2018,57(4):242-251.
[11]Narayan N,Owen DR,Mandhair H,et al.Translocator Protein as an Imaging Marker of Macrophage and Stromal Activation in Rheumatoid Arthritis Pannus[J].J Nucl Med,2018,59(7):1125-1132.
[12]Mikhaylova AS,Lesnyak OM.Pannus growth regulators as potential targets for biological therapy in rheumatoid arthritis[J].Mod Rheum J,2018,12(1):55-59.
[13]Trenkmann M,Brock M,Bertoncelj MF,et al.Epigenetic Repression of the Long Noncoding Rna Hotair Regulates NF-KBSignalling and the Expression of Matrix Metalloproteases in Synovial Fibroblasts[J].ARD,2013,72(Suppl 3):A201.
[14]Li J,Zhao Y,Liu N,et al.Nuclear factor of activated T cells 5maintained by Hotair suppression of mi R-568 upregulates S100calcium binding protein A4 to promote breast cancer metastasis[J].Breast Cancer Res,2018,20(5):454-467.
[15]张聘,张雷,赵建宁.LncRNA在软骨损伤与修复中作用机制的研究进展[J].医学研究生学报,2018,31(12):1325-1328.
[16]Hidetoshi S,Yutaka K.Lentiviral vector-mediated RNAi and its use for cancer research[J].Future Oncol,2007,3(6):655-664.
[17]李振宇,徐开林,潘秀英.慢病毒载体构建及结构优化[J].国外医学(分子生物学分册),2002,24(5):310-313.
[2]Fearon U,Canavan M,Biniecka M,et al.Hypoxia,mitochondrial dysfunction and synovial invasiveness in rheumatoid arthritis[J].Nat Rev Rheumatol,2016,12(7):385-397.
[3]Rinn JL,Kertesz M,Wang JK,et al.Functional Demarcation of Active and Silent Chromatin Domains in Human HOX Loci by Noncoding RNAs[J].NIH-PA,2007,129(7):1311-1323.
[4]张国华,徐颖,邹晨,等.长链非编码RNA DGCR5在胃癌中的表达及临床意义[J].医学研究生学报,2018,31(1):33-38.
[5]郭倚天,许斌,陈明.长链非编码RNA在前列腺癌中的作用及研究进展[J].医学研究生学报,2017,30(2):199-203.
[6]Zhang HJ,Wei QF,Wang SJ,et al.LncRNA HOTAIR alleviates rheumatoid arthritis by targeting miR-138 and inactivating NF-κB pathway[J].Int Immunopharmacol,2017,50(6):283-290.
[7]Pahlevan Kakhki M,Nikravesh A,Shirvani Farsani Z,et al.HOTAIR but notANRIL long non-coding RNA contributes to the pathogenesis of multiple sclerosis[J].Immunology,2018,153(4):479-487.
[8]Fu WM,Lu YF,Hu BG,et al.Long noncoding RNA Hotair mediated angiogenesis in nasopharyngeal carcinoma by direct and indirect signaling pathways[J].Oncotarget,2016,7(4):4712-4723.
[9]McInnes IB,Schett G.Pathogenetic insights from the treatment of rheumatoid arthritis[J].Lancet,2017,389(10086):2328-2337.
[10]中华医学会风湿病学分会.2018中国类风湿关节炎诊疗指南[J].中华内科杂志,2018,57(4):242-251.
[11]Narayan N,Owen DR,Mandhair H,et al.Translocator Protein as an Imaging Marker of Macrophage and Stromal Activation in Rheumatoid Arthritis Pannus[J].J Nucl Med,2018,59(7):1125-1132.
[12]Mikhaylova AS,Lesnyak OM.Pannus growth regulators as potential targets for biological therapy in rheumatoid arthritis[J].Mod Rheum J,2018,12(1):55-59.
[13]Trenkmann M,Brock M,Bertoncelj MF,et al.Epigenetic Repression of the Long Noncoding Rna Hotair Regulates NF-KBSignalling and the Expression of Matrix Metalloproteases in Synovial Fibroblasts[J].ARD,2013,72(Suppl 3):A201.
[14]Li J,Zhao Y,Liu N,et al.Nuclear factor of activated T cells 5maintained by Hotair suppression of mi R-568 upregulates S100calcium binding protein A4 to promote breast cancer metastasis[J].Breast Cancer Res,2018,20(5):454-467.
[15]张聘,张雷,赵建宁.LncRNA在软骨损伤与修复中作用机制的研究进展[J].医学研究生学报,2018,31(12):1325-1328.
[16]Hidetoshi S,Yutaka K.Lentiviral vector-mediated RNAi and its use for cancer research[J].Future Oncol,2007,3(6):655-664.
[17]李振宇,徐开林,潘秀英.慢病毒载体构建及结构优化[J].国外医学(分子生物学分册),2002,24(5):310-313.