Abstract：Objective: To construct the recombinant lentivirus expressing vector carrying Kaposi′s sarcoma-associated herpesvirus(KSHV)-encoded miR-K7-3p, and identify its function in regulating the proliferation of endothelial cells. Methods: miR-30-stem-loop inserted with miR-K7-3p sequence and its complementary sequence was used as template for polymerase chain reaction(PCR). Sequences were then inserted into a lentiviral vector mpCDH-CMVMCSEF1tRFP to construct the recombinant lentivirus vector mpCDH-miR-K7-3p. The recombinant plasmid mpCDH-miR-K7-3p, package plasmid psPAX2 and envelope plasmid pMD2.G were co-transfected into the 293 T cells, and filtered culture media was collected. Viral titer was determined by virus gradient dilution, together with observing the expression of red fluorescent protein(RFP). The human umbilical vein endothelial cells(HUVECs) were infected with lentivirus-miR-K7-3p and lentivirus-mpCDH in the same multiplicity of infection (MOI) and efficiency of overexpression was detected by qRTPCR. At last, cell proliferation was detected by Cell Counting Kit-8（CCK-8） assays. Results: Double digestion of the plasmid and nucleic acid sequencing identified that the recombinant plasmid carrying miR-K7-3p were successfully constructed. qRT-PCR showed the elevated level of miR-K7-3p mRNA in virus infected HUVECs. Further, the CCK-8 assay and tablet clone forming experiment offered efficient evidence that miR-K7-3p can enhance cell proliferation. Conclusion: The recombinant lentivirus vector of miRK73p was successfully constructed and can effectively enhance cell proliferation.
收稿日期: 2016-11-17
通讯作者:
卢春（通讯作者），教授，博士生导师，E-mail：clu@njmu.edu.cn
作者简介: 朱凯翔（1991—），男，硕士研究生；
引用本文:
朱凯翔， 卢春. 卡波氏肉瘤病毒编码的miR-K7-3p重组慢病毒载体构建及其对血管内皮细胞增殖的影响[J]. 江苏大学学报:医学版, 2017, 27(01): 18-23.
ZHU Kai-xiang, LU Chun. Construction of overexpression plasmid carrying KSHV-encoded miR-K7-3p and identification of its role in cell proliferation. Journal of Jiangsu University(Medicine Edition), 2017, 27(01): 18-23.
[1\]Chang Y,Cesarman E,Pessin MS,et al. Identification of herpesviruslike DNA sequences in AIDSassociated Kaposi′s sarcoma［J］. Science, 1994, 266(5192): 18651869.\[2\]Mesri EA, Cesarman E,Boshoff C.Kaposi′s sarcoma and its associated herpesvirus［J］. Nat Rev Cancer, 2010, 10(10): 707-719.\[3\]Ganem D.KSHV and Kaposi′s sarcoma: the end of the beginning?［J］. Cell, 1997, 91(2): 157-160.\[4\]Bartel DP.MicroRNAs: genomics, biogenesis, mechanism, and function［J］. Cell, 2004, 116(2): 281-297.\[5\]Nachmani D, SternGinossar N, Sarid R, et al. Diverse herpesvirus microRNAs target the stressinduced immune ligand MICB to escape recognition by natural killer cells［J］. Cell Host Microbe, 2009, 5(4): 376-385.\[6\]Nicholas J.Human herpesvirus 8encoded proteins with potential roles in virusassociated neoplasia［J］. Front Biosci, 2007, 12: 265-281.\[7\]Dedicoat M,Newton R.Review of the distribution of Kaposi′s sarcomaassociated herpesvirus(KSHV) in Africa in relation to the incidence of Kaposi′s sarcoma［J］. Br J Cancer, 2003, 88(1): 1-3.\[8\]Levine AM. AIDSassociated malignant lymphoma［J］. Med Clin North Am, 1992, 76(1): 253-268.\[9\]Orenstein JM. Ultrastructure of Kaposi sarcoma［J］. Ultrastruct Pathol, 2008, 32(5): 211-220.\[10\]Wood NH,Feller L.The malignant potential of HIVassociated Kaposi sarcoma［J］. Cancer Cell Int, 2008, 8: 14.\[11\]Abend JR, Ramalingam D, KiefferKwon P, et al. Kaposi′s sarcomaassociated herpesvirus microRNAs target IRAK1 and MYD88, two components of the tolllike receptor/interleukin1R signaling cascade, to reduce inflammatorycytokine expression［J］. J Virol, 2012, 86(21): 11663-11674.\[12\]Abend JR, Uldrick T,Ziegelbauer JM. Regulation of tumor necrosis factorlike weak inducer of apoptosis receptor protein(TWEAKR) expression by Kaposi′s sarcomaassociated herpesvirus microRNA prevents TWEAKinduced apoptosis and inflammatory cytokine expression［J］. J Virol, 2010, 84(23): 12139-12151.\[13\]Dahlke C, Maul K, Christalla T, et al. A microRNA encoded by Kaposi sarcomaassociated herpesvirus promotes Bcell expansion in vivo［J］. PLoS One, 2012, 7(11): e49435.\[14\]Gottwein E, Mukherjee N, Sachse C, et al. A viral microRNA functions as an orthologue of cellular miR155［J］. Nature, 2007, 450(7172): 1096-1099.\[15\]Suffert G, Malterer G, Hausser J, et al. Kaposi′s sarcoma herpesvirus microRNAs target caspase 3 and regulate apoptosis［J］. PLoS Pathog, 2011, 7(12): e1002405.\[16\]Samols MA, Hu J, Skalsky RL, et al. Cloning and identification of a microRNA cluster within the latencyassociated region of Kaposi′s sarcomaassociated herpesvirus［J］. J Virol, 2005, 79(14): 9301-9305.\[17\]Wang JW, Wang LJ, Mao YB, et al. Control of root cap formation by MicroRNAtargeted auxin response factors in Arabidopsis［J］. Plant Cell, 2005, 17(8): 2204-2216.\[18\]Wu XJ, Pu XM, Zhao ZF, et al. The expression profiles of microRNAs in Kaposi′s sarcoma［J］.Tumour Biol, 2015, 36(1): 437-446.\[19\]Li W, Jia X, Shen C, et al. A KSHV microRNA enhances viral latency and induces angiogenesis by targeting GRK2 to activate the CXCR2/AKT pathway［J］. Oncotarget, 2016, 7(22): 32286-32305.\[20\]Hu M, Wang C, Li W, et al. A KSHV microRNA directly targets G proteincoupled receptor kinase 2 to promote the migration and invasion of endothelial cells by inducing CXCR2 and activating AKT signaling［J］. PLoS Pathog, 2015, 11(9): e1005171.\[21\]Silva JM, Li MZ, Chang K, et al. Secondgeneration shRNA libraries covering the mouse and human genomes［J］. Nat Genet, 2005, 37(11): 1281-1288.
2017, Vol. 27 
