乳腺癌生存期相关长非编码MAPT-AS1及其共表达基因筛选与验证
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摘要
【目的】筛选乳腺癌患者生存期相关差异表达长链非编码RNA(lncRNA)及其共表达基因,并验证其乳腺癌细胞中的表达情况。【方法】通过TCGA数据库筛选943例RNA-seq数据信息:(837例乳腺癌患者+106例正常对照),发现长非编码MAPT-AS1高表达,乳腺癌患者生存期更长。构建长非编码MAPT-AS1过表达和干扰质粒,将构建好的质粒转染乳腺癌细胞株T47D,并用嘌呤霉素筛选出稳定表达的T47D细胞株,通过RT-qPCR验证长非编码MAPT-AS1及其共表达基因的表达情况。【结果】荧光显微镜观察及RT-qPCR验证,成功构建长非编码MAPT-AS1过表达及干扰稳定转染乳腺癌细胞株T47D,并筛选出干扰效率最高的长非编码MAPT-AS1干扰片段shRNA3。验证其共表达基因得到MAPT、MAPT-IT1和NXNL2在转染了干扰片段shRNA3后表达量降低,与长非编码MAPT-AS1的表达趋势一致。【结论】成功构建长非编码MAPT-AS1过表达及干扰稳定转染乳腺癌细胞株T47D,验证其和共表达基因表达情况与数据库一致,为进一步研究长非编码MAPT-AS1基因在乳腺癌中的作用机制奠定基础。
【Objective】To screen survival-related differential expression of long non-coding RNA(lncRNA)and its co-expressed genes in breast cancer patients and to verify their expression in breast cancer cells.【Methods】RNA-seq data of 943 cases(837 breast cancer + 106 normal controls)by the TCGA database were screened,and found that long non-coding MAPT-AS1 highly expressed,and breast cancer patients had longer survival. The long non-coding MAPTAS1 overexpression and interference plasmid was constructed,and the constructed plasmid was transfected into breast cancer cell line T47 D,and the stably expressed T47 D cell line was screened by puromycin. The expression of long noncoding MAPT-AS1 and its co-expressed genes was verified by the methods of RT-qPCR.【Results】Fluorescence microscopy and RT-qPCR confirmed that the long non-coding MAPT-AS1 overexpression and interference-transfected breast cancer cell line T47 D were successfully constructed,and the long non-coding MATS-AS1 interference fragment shRNA3 with the highest interference efficiency was screened. The expression of MAPT,MAPT-IT1 and NXNL2 in the coexpressed gene was decreased after transfection of the shRNA3 interference fragment,which was consistent with the expression trend of the long non-coding MAPT-AS1.【Conclusion】The long non-coding MAPT-AS1 overexpression and interference plasmid transfected breast cancer cell line T47 D were successfully constructed,and the expression of the coexpressed gene was consistent with the database. The study laid the foundation for further study of the mechanism of action of long non-coding MAPT-AS1 gene in breast cancer.
【Objective】To screen survival-related differential expression of long non-coding RNA(lncRNA)and its co-expressed genes in breast cancer patients and to verify their expression in breast cancer cells.【Methods】RNA-seq data of 943 cases(837 breast cancer + 106 normal controls)by the TCGA database were screened,and found that long non-coding MAPT-AS1 highly expressed,and breast cancer patients had longer survival. The long non-coding MAPTAS1 overexpression and interference plasmid was constructed,and the constructed plasmid was transfected into breast cancer cell line T47 D,and the stably expressed T47 D cell line was screened by puromycin. The expression of long noncoding MAPT-AS1 and its co-expressed genes was verified by the methods of RT-qPCR.【Results】Fluorescence microscopy and RT-qPCR confirmed that the long non-coding MAPT-AS1 overexpression and interference-transfected breast cancer cell line T47 D were successfully constructed,and the long non-coding MATS-AS1 interference fragment shRNA3 with the highest interference efficiency was screened. The expression of MAPT,MAPT-IT1 and NXNL2 in the coexpressed gene was decreased after transfection of the shRNA3 interference fragment,which was consistent with the expression trend of the long non-coding MAPT-AS1.【Conclusion】The long non-coding MAPT-AS1 overexpression and interference plasmid transfected breast cancer cell line T47 D were successfully constructed,and the expression of the coexpressed gene was consistent with the database. The study laid the foundation for further study of the mechanism of action of long non-coding MAPT-AS1 gene in breast cancer.
引文
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[7]Feng T,Shao F,Wu Q,et al.miR-124 downregulation leads to breast cancer progression via LncRNA-MALAT1 regulation and CDK4/E2F1 signal activation[J].Oncotarget,2016,7(13):16205-16216.
[8]Coupland KG,Kim WS,Halliday GM,et al.Role of the long non-coding RNA MAPT-AS1 in regulation of microtubule associated protein Tau(MAPT)expression in Parkinson's disease[J].PloS one,2016,11(6):e0157924.
[9]Nascimento GR,Pinto IP,De Melo AV,et al.Molecular characterization of koolen De Vries syndrome in two girls with idiopathic intellectual disability from central brazil[J].Mol Syndromol,2017,8(3):155-160.
[10]Pan Y,Pan Y,Cheng Y,et al.Knockdown of Lnc RNA MAPT-AS1 inhibits proliferation and migration and sensitizes cancer cells to paclitaxel by regulating MAPT expression in ER-negative breast cancers[J].Cell Biosci,2018,8(1):7.
[11]King A,Al-Sarraj S,Troakes C,et al.Mixed tau,T DP-43 and p62 pathology in FTLD associated with a C9ORF72 repeat expansion and p.Ala239ThrMAPT(tau)variant[J].Acta Neuropathologica,2013,125(2):303-310.
[12]Wu M,Assassi S,Salazar GA,et al.Genetic susceptibility loci of idiopathic interstitial pneumonia do not represent risk for systemic sclerosis:a case control study in Caucasian patients[J].Arthr Res Ther,2016,18(1):20.
[13]Li S,Wu L,Wang Q,et al.KDM4B promotes epithelial-mesenchymal transition through up-regulation of ZEB1 in pancreatic cancer[J].Acta Biochimicaet Biophysica Sinica,2015,47(12):997-1004.
[2]Siegel RL,Miller KD,Dvm A J.Cancer statistics,2017[J].CA-Cancer J Clin,2017,67(1):7-30.
[3]Siegel RL,Miller KD,Jemal A.Cancer statistics in China,2016[J].CA-Cancer J Clin,2016,66(1):7-30.
[4]Bunch H.Gene regulation of mammalian long noncoding RNA[J].Mol Genet Genomics,2017,89(4):110-119.
[5]Wei GH,Wang X.lncRNA MEG3 inhibit proliferation and metastasis of gastric cancer via p53 signaling pathway[J].Eur Rev Med Pharmacol Sci,2017,21(17):3850-3856.
[6]Jiang YZ,Liu YR,Xu XE,et al.Transcriptome analysis of triple-negative breast cancer reveals an integrated m RNA-lncRNA signature with predictive and prognostic value[J].Cancer Res,2016,76(8):2105-2114.
[7]Feng T,Shao F,Wu Q,et al.miR-124 downregulation leads to breast cancer progression via LncRNA-MALAT1 regulation and CDK4/E2F1 signal activation[J].Oncotarget,2016,7(13):16205-16216.
[8]Coupland KG,Kim WS,Halliday GM,et al.Role of the long non-coding RNA MAPT-AS1 in regulation of microtubule associated protein Tau(MAPT)expression in Parkinson's disease[J].PloS one,2016,11(6):e0157924.
[9]Nascimento GR,Pinto IP,De Melo AV,et al.Molecular characterization of koolen De Vries syndrome in two girls with idiopathic intellectual disability from central brazil[J].Mol Syndromol,2017,8(3):155-160.
[10]Pan Y,Pan Y,Cheng Y,et al.Knockdown of Lnc RNA MAPT-AS1 inhibits proliferation and migration and sensitizes cancer cells to paclitaxel by regulating MAPT expression in ER-negative breast cancers[J].Cell Biosci,2018,8(1):7.
[11]King A,Al-Sarraj S,Troakes C,et al.Mixed tau,T DP-43 and p62 pathology in FTLD associated with a C9ORF72 repeat expansion and p.Ala239ThrMAPT(tau)variant[J].Acta Neuropathologica,2013,125(2):303-310.
[12]Wu M,Assassi S,Salazar GA,et al.Genetic susceptibility loci of idiopathic interstitial pneumonia do not represent risk for systemic sclerosis:a case control study in Caucasian patients[J].Arthr Res Ther,2016,18(1):20.
[13]Li S,Wu L,Wang Q,et al.KDM4B promotes epithelial-mesenchymal transition through up-regulation of ZEB1 in pancreatic cancer[J].Acta Biochimicaet Biophysica Sinica,2015,47(12):997-1004.