gga-miRNA-155对MDCC-MSB1细胞增殖的影响
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摘要
【目的】构建gga-miRNA-155前体基因(pre-gga-miRNA-155)真核表达载体,验证gga-miRNA-155在MDCC-MSB1细胞中的表达效果,探究其对MDCC-MSB1细胞增殖的影响。【方法】采用PCR方法从鸡肝脏基因组DNA中扩增gga-miRNA-155前体基因片段pre-gga-miRNA-155,并将其克隆入pMD-18T载体,构建克隆载体pMD-18T-pre-gga-miRNA-155。用EcoRⅠ和XhoⅠ双酶切pcDNA6.2-GW/EmGFP-miRNA真核表达载体和pMD-18T-pre-gga-miRNA-155,将pre-gga-miRNA-155酶切回收片段与pcDNA6.2-GW/EmGFP-miRNA的酶切回收片段进行连接,构建重组真核表达载体pcDNA6.2-pre-gga-miRNA-155,对其进行PCR、EcoRⅠ和XhoⅠ双酶切及DNA测序鉴定。将重组载体转染到MDCC-MSB1细胞中,应用实时荧光定量PCR (Real-time PCR)检测gga-mi-RNA-155的表达水平,利用MTT法检测细胞增殖能力的变化。【结果】PCR扩增获得了长度约154bp的鸡pre-gga-mi-RNA-155基因。成功构建了pre-gga-miRNA-155的真核表达载体pcDNA6.2-pre-gga-miRNA-155,瞬时转染MD-CC-MSB1细胞后gga-miRNA-155表达水平显著增加,且MDCC-MSB1细胞的体外增殖能力增强。【结论】成功构建了pre-gga-miRNA-155的真核表达载体,其可在MDCC-MSB1细胞中过表达gga-miRNA-155,且可促进MDCC-MSB1细胞的体外增殖。
【Objective】This study constructed the eukaryotic expression vector of gga-miRNA-155 precursor gene fragment(pre-gga-miRNA-155)and tested its expression efficiency in MDCC-MSB1 to explore the effects on proliferation of MDCC-MSB1.【Method】The precursor gene fragment of gga-miRNA-155 was amplified by PCR from the genomic DNA of chicken liver tissue,before being cloned into the pMD-18 Tvector to construct the cloning vector pMD-18 T-pre-gga-miRNA-155.The eukaryotic expression vector pcDNA6.2-GW/EmGFP-miRNA and cloning vector pMD-18 T-pre-gga-miRNA-155 were digested bythe restriction enzymes EcoRⅠand XhoⅠ,the pre-gga-miRNA-155 and pcDNA6.2-GW/EmGFP-miRNA gene fragments were recycled,and the pre-gga-miRNA-155 gene fragment was connected to the pcDNA6.2-GW/EmGFP-miRNA to construct the recombinant vector pcDNA6.2-pre-gga-miRNA-155.The accuracy of the recombinant eukaryotic expression vector was verified by PCR,EcoRⅠ and XhoⅠ double enzyme digestion,and DNA sequencing.The MDCC-MSB1 cells were transfected with the recombinant vector pcDNA6.2-gga-miRNA-155,and the expression of gga-miRNA-155 was evaluated by real-time quantitative PCR.The proliferation of MDCC-MSB1 cell was also determined by MTT.【Result】The pre-ggamiRNA-155 gene fragment with length of 154 bp was obtained by PCR.The eukaryotic expression vector of gga-miRNA-155(pcDNA6.2-pre-gga-miRNA-155)was successfully constructed.The expression level of gga-miRNA-155 was significantly increased in pcDNA6.2-pre-gga-miRNA-155 transfected MDCC-MSB1 cells.The proliferation of MDCC-MSB1 cells in vitro was also promoted.【Conclusion】The eukaryotic expression vector of pre-gga-miRNA-155 was successfully constructed.It could over-express gga-miRNA-155 in MDCC-MSB1 cell and promote the proliferation of MDCC-MSB1 cell.
【Objective】This study constructed the eukaryotic expression vector of gga-miRNA-155 precursor gene fragment(pre-gga-miRNA-155)and tested its expression efficiency in MDCC-MSB1 to explore the effects on proliferation of MDCC-MSB1.【Method】The precursor gene fragment of gga-miRNA-155 was amplified by PCR from the genomic DNA of chicken liver tissue,before being cloned into the pMD-18 Tvector to construct the cloning vector pMD-18 T-pre-gga-miRNA-155.The eukaryotic expression vector pcDNA6.2-GW/EmGFP-miRNA and cloning vector pMD-18 T-pre-gga-miRNA-155 were digested bythe restriction enzymes EcoRⅠand XhoⅠ,the pre-gga-miRNA-155 and pcDNA6.2-GW/EmGFP-miRNA gene fragments were recycled,and the pre-gga-miRNA-155 gene fragment was connected to the pcDNA6.2-GW/EmGFP-miRNA to construct the recombinant vector pcDNA6.2-pre-gga-miRNA-155.The accuracy of the recombinant eukaryotic expression vector was verified by PCR,EcoRⅠ and XhoⅠ double enzyme digestion,and DNA sequencing.The MDCC-MSB1 cells were transfected with the recombinant vector pcDNA6.2-gga-miRNA-155,and the expression of gga-miRNA-155 was evaluated by real-time quantitative PCR.The proliferation of MDCC-MSB1 cell was also determined by MTT.【Result】The pre-ggamiRNA-155 gene fragment with length of 154 bp was obtained by PCR.The eukaryotic expression vector of gga-miRNA-155(pcDNA6.2-pre-gga-miRNA-155)was successfully constructed.The expression level of gga-miRNA-155 was significantly increased in pcDNA6.2-pre-gga-miRNA-155 transfected MDCC-MSB1 cells.The proliferation of MDCC-MSB1 cells in vitro was also promoted.【Conclusion】The eukaryotic expression vector of pre-gga-miRNA-155 was successfully constructed.It could over-express gga-miRNA-155 in MDCC-MSB1 cell and promote the proliferation of MDCC-MSB1 cell.
引文
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[19]南雪,曾泉,吕洋,等.miR-155对肝癌细胞增殖的影响[J].生物技术通讯,2014,25(6):783-786.Nan X,Zeng Q,LüY,et al.Effect of miR-155on hepatocellular carcinoma cell proliferation[J].Letters in Biotechnology,2014,25(6):783-786.
[2]Iwasaki T,Tanaka K,Kawano M,et al.Tumor-suppressive microRNA-let-7ainhibits cell proliferation via targeting of E2F2in osteosarcoma cells[J].Int J Oncol,2015,46(4):1543-1550.
[3]Adams B D,Kasinski A L,Slack F J.Aberrant regulation and function of microRNAs in cancer[J].Curr Biol,2014,24(16):R762-776.
[4]连娇燕,庹必光.非编码RNA与肝细胞肝癌发生发展的研究进展[J].世界华人消化杂志,2015,23(3):396-403.Lian J Y,Tuo B G.Role of non-coding RNAs in development and progression of hepatocellular carcinoma[J].World Chinese Journal of Digestology,2015,2015;23(3):396-403.
[5]Gu J Y,Lu Z S,Ji C H,et al.Melatonin inhibits proliferation and invasion via repression of miRNA-155in glioma cells[J].Biomedicine&Pharmacotherapy,2017,93:969-975.
[6]陈倩云,范恒.miR-155调控T细胞分化与功能的研究进展[J].中国免疫学杂志,2016,32(7):1065-1069.Chen Q Y,Fan H.The research progress of T cell differentiation and function regulated by miR-155[J].Chinese Journal of Immunolog,2016,32(7):1065-1069.
[7]Jia S,Zhai H,Zhao M.MicroRNAs regulate immune system via multiple targets[J].Discov Med,2014,18(100):237-247.
[8]Kluiver J,van den Berg A,de Jong D,et al.Regulation of primicroRNA BIC transcription and processing in Burkitt lymphoma[J].Oncogene,2007,26(26):3769-3776
[9]Xu M,Zuo D M,Liu X X,et al.MiR-155contributes to Th17cells differentiation in dextran sulfate sodium(DSS)-induced colitis mice via Jarid2[J].Biochemical and Biophysical Research Communications,2017,488(1):6-14.
[10]Yin Q Y,Wang X,Roberts C,et al.Methylation status and AP1elements are involved in EBV-mediated miR-155expression in EBV positive lymphoma cells[J].Virology,2016,494(7):158-167.
[11]刘红霞,施琼,周一青,等.过表达miR-155抑制BMP9诱导间充质干细胞C3H10T1/2成骨分化[J].中国生物工程杂志,2017,37(5):9-18.Liu H X,Shi Q,Zhou Y Q,et al.Overexpression of miR-155inhibits the osteogenic differentiation of mesenchymal stem cells C3H10T1/2Induced by BMP9[J].China Biotechnology,2017,37(5):9-18.
[12]曾芙蓉,陈偲,汤立军.miR-155在常见病原微生物感染中的研究进展[J].生命科学,2014,26(7):751-755.Zeng F R,Chen C,Tang L J.The research progress of miR-155in the process of pathogenic microorganism infection[J].Chinese Bulletin of Life Sciences,2014,26(7):751-755.
[13]Yu Z H,Teng M,Sun A J,et al.Virus-encoded miR-155ortholog is an important potential regulator but not essential for the development of lymphomas induced by very virulent Marek’s disease virus[J].Virology,2014,448:55-64.
[14]Chi J Q,Teng M,Yu Z H,et al.Marek’s disease virus-encoded analog of microRNA-155activates the oncogene c-Myc by targeting LTBP1 and suppressing the TGF-beta signaling pathway[J].Virology,2015,476:72-84.
[15]Yao Y,Zhao Y,Smith L P,et al.Differential expression of microRNAs in Marek’s disease virus-transformed T-lymphoma cell lines[J].The Journal of General Virology,2009,90(7):1551-1559.
[16]Lian L,Qu L,Chen Y,et al.A systematic analysis of miRNAtranscriptome in Marek’s disease virus-induced lymphoma reveals novel and differentially expressed miRNAs[J].PloSone,2012,7(11):e51003.
[17]周明,马建明.胃癌组织miR-155的表达及其与临床预后的关系[J].中国临床医学,2016,23(6):731-734.Zhou M,Ma J M.Expression of miR-155in gastric cancer and its prognostic significance[J].Chinese Journal of Clinical Medicine,2016,23(6):731-734.
[18]蔡启茵,任广立,张卫云,等.人miR-155真核过表达载体的构建及其对HepG2.2.15细胞中HBeAg的抑制效应[J].世界华人消化杂志,2014,22(28):4217-4222.Cai Q Y,Ren G L,Zhang W Y,et al.Construction of a eukaryotic vector expressing human miR-155 and inhibitory effect of miR-155on HBeAg in HepG2.2.15cells[J].World Chinese Journal of Digestology,2014,22(28):4217-4222.
[19]南雪,曾泉,吕洋,等.miR-155对肝癌细胞增殖的影响[J].生物技术通讯,2014,25(6):783-786.Nan X,Zeng Q,LüY,et al.Effect of miR-155on hepatocellular carcinoma cell proliferation[J].Letters in Biotechnology,2014,25(6):783-786.