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
10.3760/cma.j.issn.0253-3766.2016.07.002
- VernacularTitle:下调let-7微小RNA对卡波西肉瘤相关疱疹病毒裂解复制和丝裂原激活蛋白激酶激酶激酶激酶4及其下游因子的影响
- Author:
Jinxia ZHANG
1
;
Xiaohua TAN
;
Zhen YUAN
;
Yanhu LI
;
Yan QI
;
Xi NAN
;
Mingjian QI
;
He GAO
;
Fuzhi LIAN
;
Lei YANG
Author Information
1. 832000,石河子大学医学院基础医学系
- Keywords:
Subject words] Herpesvirus 8,human;
Mitogen-activated protein kinase kinases;
Let-7 miRNA;
Kaposi sarcoma
- From:
Chinese Journal of Oncology
2016;38(7):485-491
- CountryChina
- Language:Chinese
-
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.
