长链非编码RNA MTHFD2基因在胶质母细胞瘤的表达及生物学功能
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摘要
目的长链非编码RNA(lncRNA) MTHFD2基因在胶质母细胞瘤(GBM)组织与正常脑组织中的表达有明显差异,但其在肿瘤尤其是GBM的生物学功能目前尚不清楚。文中旨在研究lncRNA MTHFD2在人GBM组织和细胞系中的表达情况,并观察下调其表达对GBM细胞生物学功能的影响。方法选取2017年9月至2017年12月东部战区总医院神经外科经手术切除的9例GBM患者标本,同时取其配对的瘤旁组织作为正常对照组织。取对数生长期的U251和U-87MG细胞,接种细胞24 h后分别加入LV-MTHFD2-shRNA病毒液(U251shRNA组、U-87MGshRNA组)和空载LV-control病毒液(U251对照组、U-87MG对照组)。采用qRT-PCR检测GBM组织和细胞系中lncRNA MTHFD2的表达。CCK-8检测细胞增殖和对化疗药物替莫唑铵的耐药情况。Transwell小室评价细胞迁移能力的变化等。结果 GBM组织lncRNA MTHFD2相对表达量明显高于正常对照组织[(5.13±3.96)vs(1.27±0.58)],差异有统计学差异(P<0.05)。与U251对照组MTHFD2的相对表达量(1.02±0.08)比较,U251shRNA组(0.05±0.01)明显降低(P<0.01);U-87MGshRNA组较U-87MG对照组亦明显降低(P<0.05)。U251shRNA组穿过Transwell微孔滤膜的细胞数量与U251对照组比较明显降低[(41.4±6.99)个/视野vs (125.8±25.27)个/视野],差异有统计学意义(P<0.01);U-87MGshRNA组较U-87MG对照组亦明显降低(P<0.05)。CCK-8结果显示,在第4天,U251shRNA组A值较U251对照组明显降低,U-87MGshRNA组A值较U-87MG对照组亦明显降低(P<0.05)。细胞耐药性结果显示:U251shRNA组细胞抑制率明显高于U251对照组,U87-MGhRNA组亦明显低于U87-MG对照组(P<0.05)。结论 lncRNA MTHFD2在GBM细胞中的表达下调可降低细胞的增殖和迁移能力,增强对化疗药物替莫唑铵的敏感性,提示其可能为GBM潜在的治疗靶标。
Objective The long non-coding RNA(lncRNA)MTHFD2 gene is expressed differentially in glioblastoma(GBM)and normal brain tissues,but its biological role in tumors,and particularly in GBM,remains unclear. This study aims to in-vestigate the expression of lncRNA MTHFD2 in the GBM tissue and four GBM cell lines,and explore the effect of its down-regulated expression on the biological function of GBM cells.Methods Specimens of GBM and the paracancerous tissue(as normal control)were collected from 9 patients treated by surgical resection in our De-partment of Neurosurgery between September and December 2017 LV-MTHFD2-shRNA(U251 shRNA and U-87 MG shRNA groups)and empty LV-control solution(U251 shRNA and U-87 MG control groups)were transfected into the U251 and U-87 MG cell lines. The expressions of lncRNA MTHFD2 in the GBM tissue and the GBM cell lines were detected by qRT-PCR,the chemosensitivity and proliferation of the cells after transfection measured by CCK-8 assay,and the changes in the cell migration ability determined by Transwell assay.Results The relative expression of lncRNA MTHFD2 was significantly higher in the GBM than in the normal tissue(5.13 ± 3.96 vs 1.27 ± 0.58,P < 0.05),while that of MTHFD2 was re-markably lower in the U251 shRNA than in the U251 control group(0.05 ± 0.01 vs 1.00 ± 0.00,P < 0.01),and so was that in the U-87 MG shRNA than in the U-87 MG control(P < 0.05). The number of cells penetrating the Transwell membrane was markedly lower in the U251 shRNA group than in the U251 control(41.4 ± 6.99 vs 125.8 ± 25.27 per field of view,P < 0.01),and so was that in the U-87 MG shRNA than in the U-87 MG control(P < 0.05). CCK-8 assay showed that,at 4 days after transfection,the A value was signifi-cantly decreased in the U251 shRNA and U-87 MG shRNA groups as compared with the U251 control and U-87 MG control groups(P <0.05). Cellular drug resistance test manifested remarkably reduced fifty percent inhibitory concentrations(IC50)in the U251 shRNA and U-87 MG shRNA groups as compared with the U251 control and U-87 MG control groups(P < 0.05).Conclusion DDown-regula-tion of the expression of lncRNA MTHFD2 can inhibit the proliferation and migration of U251 and U-87 MG cells and enhance the che-mosensitivity of the cells to temozolomide,which suggests that lncRNA MTHFD2 could be a potential therapeutic target against GBM.
Objective The long non-coding RNA(lncRNA)MTHFD2 gene is expressed differentially in glioblastoma(GBM)and normal brain tissues,but its biological role in tumors,and particularly in GBM,remains unclear. This study aims to in-vestigate the expression of lncRNA MTHFD2 in the GBM tissue and four GBM cell lines,and explore the effect of its down-regulated expression on the biological function of GBM cells.Methods Specimens of GBM and the paracancerous tissue(as normal control)were collected from 9 patients treated by surgical resection in our De-partment of Neurosurgery between September and December 2017 LV-MTHFD2-shRNA(U251 shRNA and U-87 MG shRNA groups)and empty LV-control solution(U251 shRNA and U-87 MG control groups)were transfected into the U251 and U-87 MG cell lines. The expressions of lncRNA MTHFD2 in the GBM tissue and the GBM cell lines were detected by qRT-PCR,the chemosensitivity and proliferation of the cells after transfection measured by CCK-8 assay,and the changes in the cell migration ability determined by Transwell assay.Results The relative expression of lncRNA MTHFD2 was significantly higher in the GBM than in the normal tissue(5.13 ± 3.96 vs 1.27 ± 0.58,P < 0.05),while that of MTHFD2 was re-markably lower in the U251 shRNA than in the U251 control group(0.05 ± 0.01 vs 1.00 ± 0.00,P < 0.01),and so was that in the U-87 MG shRNA than in the U-87 MG control(P < 0.05). The number of cells penetrating the Transwell membrane was markedly lower in the U251 shRNA group than in the U251 control(41.4 ± 6.99 vs 125.8 ± 25.27 per field of view,P < 0.01),and so was that in the U-87 MG shRNA than in the U-87 MG control(P < 0.05). CCK-8 assay showed that,at 4 days after transfection,the A value was signifi-cantly decreased in the U251 shRNA and U-87 MG shRNA groups as compared with the U251 control and U-87 MG control groups(P <0.05). Cellular drug resistance test manifested remarkably reduced fifty percent inhibitory concentrations(IC50)in the U251 shRNA and U-87 MG shRNA groups as compared with the U251 control and U-87 MG control groups(P < 0.05).Conclusion DDown-regula-tion of the expression of lncRNA MTHFD2 can inhibit the proliferation and migration of U251 and U-87 MG cells and enhance the che-mosensitivity of the cells to temozolomide,which suggests that lncRNA MTHFD2 could be a potential therapeutic target against GBM.
引文
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[13]Chang Y,Zhang K,Hu Z,et al.Hypoxia-regulated lncRNAs in cancer[J].Gene,2016,575(1):1-8.
[14]Sun M,Kraus WL.From discovery to function:the expanding roles of longNon-Coding RNAs in physiology and disease[J].Endocr Rev,2015,36(1):25-64.
[15]Huarte M.The emerging role of lncRNAs in cancer[J].Nat Med,2015,21(11):1253-1261.
[16]Bian EB,Ma CC,He XJ,et al.Epigenetic modification ofmiR-141 regulates SKA2 by an endogenous‘sponge’HOTAIR in glioma[J].Onco Target,2016,7(21):30610-30625.
[17]Wang Y,Wang Y,Li J,et al.CRNDE,a long non-codingRNA,promotes glioma cell growth and invasion throughmTOR signaling[J].Cancer Letters,2015,367(2):122-128.
[18]Rinn JL,Chang HY.Genome regulationby long noncoding RNAs[J].Annu Rev Biochem,2012,81:145-166.
[2]杨卓,王新军,单峤,等.胶质瘤相关性癫痫患者术后痫性再发的临床特征及影响因素[J].医学研究生学报,2017,30(4):405-408.
[3]Suzuki Y,Shirai K,Oka K,et al.Higher pAkt expressionpredicts a signficant worse prognosis in gliblastomas[J].Radiat Res,2010,51(3):343-348.
[4]Wen PY,Kesari S.Malignant gliomas in adults[J].N Engl JMed,2008,359(5):492-507.
[5]Liu GX,Hu XC,Zhou GJ.Long non-coding RNA OR3A4 promotes proliferation and migration inbreast cancer[J].Biomed Pharmacother,2017,96:426-433.
[6]Ma ZH,Huang HSY,Xu YT,et al.Current advances of long non-coding RNA highly upregulated in liver cancer in human tumors[J].Onco Targets Ther,2017,10:4711-4717.
[7]Zhang JT,Ju CJ,Zhang W,et al.lncRNA SNHG20 is associated with clinical progression and enhances cell migration and invasion in osteosarcoma[J].IUBMB Life,2018,9999(9999):1-7.
[8]丛壮壮,郭仲,秦涛等.长链非编码RNA Linc00467在肺腺癌中的表达及功能[J].医学研究生学报,2017,30(8):834-838.
[9]施雪霏,宋勇.长链非编码RNAs在肿瘤中研究进展[J].医学研究生学报,2013,26(9):997-1000.
[10]Sahu A,Singhal U,Chinnaiyan AM.Long non-coding RNAs in cancer:from function to translation[J].Trends Cancer,2015,1(2):93-109.
[11]Moran VA,Perera RJ,Khalil AM.Emerging functional andmechanistic paradigms of mammalian long non-coding RNAs[J].Nucleic Acids Res,2012,40(14):6391-6400.
[12]Wilusz JE,Sunwoo H,Spector DL.Long noncoding RNAs:functionalsurprises from the RNA world[J].Genes Dev,2009,23(13):1494-504.
[13]Chang Y,Zhang K,Hu Z,et al.Hypoxia-regulated lncRNAs in cancer[J].Gene,2016,575(1):1-8.
[14]Sun M,Kraus WL.From discovery to function:the expanding roles of longNon-Coding RNAs in physiology and disease[J].Endocr Rev,2015,36(1):25-64.
[15]Huarte M.The emerging role of lncRNAs in cancer[J].Nat Med,2015,21(11):1253-1261.
[16]Bian EB,Ma CC,He XJ,et al.Epigenetic modification ofmiR-141 regulates SKA2 by an endogenous‘sponge’HOTAIR in glioma[J].Onco Target,2016,7(21):30610-30625.
[17]Wang Y,Wang Y,Li J,et al.CRNDE,a long non-codingRNA,promotes glioma cell growth and invasion throughmTOR signaling[J].Cancer Letters,2015,367(2):122-128.
[18]Rinn JL,Chang HY.Genome regulationby long noncoding RNAs[J].Annu Rev Biochem,2012,81:145-166.