MicroRNA-185-5p mediates regulation of SREBP2 expression by hepatitis C virus core protein.
OBJECTIVE
To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus (HCV) core protein in HepG2 cells.


METHODS
HCV genotype 1b core protein was cloned and expressed in HepG2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2 (SREBP2) and the rate-limiting enzyme in cholesterol synthesis (HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, microRNA (miRNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular miRNA.


RESULTS
HCV core protein expression in HepG2 cells increased the total intracellular cholesterol level (4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR mRNA levels (P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism study revealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity (P = 0.004). In addition, miR-185-5p expression was tightly regulated by the HCV core protein (P = 0.041). Moreover, overexpression of miR-185-5p repressed the SREBP2 mRNA level (P = 0.022) and protein expression. In contrast, inhibition of miR-185-5p caused upregulation of SREBP2 protein expression. miR-185-5p was involved in the regulation of SREBP2 expression by HCV core protein.


CONCLUSIONS
HCV core protein disturbs the cholesterol homeostasis in HepG2 cells via the SREBP2 pathway; miR-185-5p is involved in the regulation of SREBP2 by the core protein.
Min Li, Sheng-Hu Feng, Jin-Ling Dong, Jun Cheng, Beijing Ditan Hospital, Teaching Hospital of Peking University, Beijing 100015, China.
Min
M
Li
Min Li, Sheng-Hu Feng, Jin-Ling Dong, Jun Cheng, Beijing Ditan Hospital, Teaching Hospital of Peking University, Beijing 100015, China.
Qi
Q
Wang
Min Li, Sheng-Hu Feng, Jin-Ling Dong, Jun Cheng, Beijing Ditan Hospital, Teaching Hospital of Peking University, Beijing 100015, China.
Shun-Ai
SA
Liu
Min Li, Sheng-Hu Feng, Jin-Ling Dong, Jun Cheng, Beijing Ditan Hospital, Teaching Hospital of Peking University, Beijing 100015, China.
Jin-Qian
JQ
Zhang
Min Li, Sheng-Hu Feng, Jin-Ling Dong, Jun Cheng, Beijing Ditan Hospital, Teaching Hospital of Peking University, Beijing 100015, China.
Wei
W
Ju
Min Li, Sheng-Hu Feng, Jin-Ling Dong, Jun Cheng, Beijing Ditan Hospital, Teaching Hospital of Peking University, Beijing 100015, China.
Min
M
Quan
Min Li, Sheng-Hu Feng, Jin-Ling Dong, Jun Cheng, Beijing Ditan Hospital, Teaching Hospital of Peking University, Beijing 100015, China.
Sheng-Hu
SH
Feng
Min Li, Sheng-Hu Feng, Jin-Ling Dong, Jun Cheng, Beijing Ditan Hospital, Teaching Hospital of Peking University, Beijing 100015, China.
Jin-Ling
JL
Dong
Min Li, Sheng-Hu Feng, Jin-Ling Dong, Jun Cheng, Beijing Ditan Hospital, Teaching Hospital of Peking University, Beijing 100015, China.
Ping
P
Gao
Min Li, Sheng-Hu Feng, Jin-Ling Dong, Jun Cheng, Beijing Ditan Hospital, Teaching Hospital of Peking University, Beijing 100015, China.
Jun
J
Cheng
D020413:3' Untranslated Regions; D001665:Binding Sites; D002784:Cholesterol / Q000378:metabolism*; D005786:Gene Expression Regulation; D056945:Hep G2 Cells; D006706:Homeostasis; D006801:Humans; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051782:Sterol Regulatory Element Binding Protein 2 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
D020413:3' Untranslated Regions; C552431:MIRN185 microRNA, human; D035683:MicroRNAs; C497390:SREBF2 protein, human; D051782:Sterol Regulatory Element Binding Protein 2; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus; D002784:Cholesterol; C553220:HMGCR protein, human; D006903:Hydroxymethylglutaryl CoA Reductases
Cholesterol; Hepatitis C virus core protein; Steatosis; Sterol response element binding proteins; miR-185-5p