title: [Let-7 miRNA silencing promotes Kaposi's sarcoma-associated herpesvirus lytic replication via activating mitogen-activated protein kinase kinase kinase kinase 4 and its downstream factors].
abstract: OBJECTIVE
  To explore the effect of let-7 miRNA silencing on Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and the underling mechanism.
  
  
  METHODS
  The pEGFP-C2-let-7 sponge vector was transfected into BCBL-1 and 293T cells with Lipofectamine 2000 to silence the expression of let-7 miRNAs. Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of let-7 miRNAs, the transcriptional levels of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), cyclooxygenase-2 (COX-2) and matrix metalloproteinase 13 (MMP-13), and the DNA copy numbers of KSHV open reading frame 50 (ORF50) and open reading frame 72 (ORF72). Western blot was used to detect the total and phosphorylated protein levels of MAP4K4, COX-2, extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK.
  
  
  RESULTS
  The expression of let-7 miRNAs was dramatically decreased in the let-7 sponge transfected BCBL-1 and 293T cells compared with that in the vector-transfected cells (P<0.05 for all). The gene copy number and mRNA transcriptional level of KSHV ORF50 were significantly increased in the let-7 sponge transfected BCBL-1 cells compared with that in the vector-transfected cells (1.00±0.10 vs. 2.33±0.18 and 1.08±0.48 vs 3.22±0.27, respectively,P<0.001 for both). The gene copy number and mRNA transcriptional level of KSHV ORF72 were also significantly increased in let-7 sponge transfected BCBL-1 cells compared with those in the vector-transfected cells(1.07±0.49 vs 1.67±0.45 and 1.01±0.19 vs 1.54±0.11, respectively,P<0.05 for both). Furthermore, the mRNA transcriptional levels of MAP4K4, COX-2 and MMP-13 were significantly increased in the let-7 sponge transfected BCBL-1 cells compared with those in the vector-transfected cells (1.00±0.05 vs 5.73±0.96, 1.00±0.05 vs 2.68±0.19, 1.00±0.02 vs 2.69±0.25, respectively,P<0.001 for all). Let-7 miRNAs silencing also increased the protein expression levels of MAP4K4, COX-2 and phospho-ERK1/2, while the phospho-JNK and phospho-p38 were not changed in the BCBL-1 and 293T cells.
  
  
  CONCLUSIONS
  Let-7 silencing may activate the replication of KSHV, possibly through up-regulating MAP4K4 and its downstream molecules COX-2, MMP-13, and phosphorylation of ERK1/2, finally results in the progression of Kaposi sarcoma.
authors.affiliation: School of Medicine, Shihezi University, Shihezi 832000, China.
authors.forename: J X
authors.initials: JX
authors.lastname: Zhang
authors.affiliation: Department of Preventive Medicine, School of Clinical Medicine, Hangzhou Normal University, Hangzhou 310018, China.
authors.forename: X H
authors.initials: XH
authors.lastname: Tan
authors.affiliation: Department of Preventive Medicine, School of Clinical Medicine, Hangzhou Normal University, Hangzhou 310018, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Yuan
authors.affiliation: School of Medicine, Shihezi University, Shihezi 832000, China.
authors.forename: Y H
authors.initials: YH
authors.lastname: Li
authors.affiliation: Department of Preventive Medicine, School of Clinical Medicine, Hangzhou Normal University, Hangzhou 310018, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Qi
authors.affiliation: School of Medicine, Shihezi University, Shihezi 832000, China.
authors.forename: X
authors.initials: X
authors.lastname: Nan
authors.affiliation: School of Medicine, Shihezi University, Shihezi 832000, China.
authors.forename: M J
authors.initials: MJ
authors.lastname: Qi
authors.affiliation: School of Medicine, Shihezi University, Shihezi 832000, China.
authors.forename: H
authors.initials: H
authors.lastname: Gao
authors.affiliation: Department of Preventive Medicine, School of Clinical Medicine, Hangzhou Normal University, Hangzhou 310018, China.
authors.forename: F Z
authors.initials: FZ
authors.lastname: Lian
authors.affiliation: Department of Preventive Medicine, School of Clinical Medicine, Hangzhou Normal University, Hangzhou 310018, China.
authors.forename: L
authors.initials: L
authors.lastname: Yang
mesh_terms: D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D018450:Disease Progression; D018628:Gene Dosage; D005822:Genetic Vectors; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D053509:Matrix Metalloproteinase 13 / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012514:Sarcoma, Kaposi / Q000821:virology*; D014162:Transfection; D014779:Virus Replication; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D035683:MicroRNAs; C487931:mirnlet7 microRNA, human; D051546:Cyclooxygenase 2; C117090:MAP4K4 protein, human; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13