Abstract
To explore the effect of let-7 miRNA silencing on Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and the underling mechanism. 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. 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. 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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.