MicroRNA-27a modulates HCV infection in differentiated hepatocyte-like cells from adipose tissue-derived mesenchymal stem cells.
OBJECTIVE
Despite the discovery of hepatitis C virus (HCV) entry factor, the mechanism by which it is regulated by miRNAs remains unclear. Adipose tissue-derived human mesenchymal stem cells (AT-hMSCs) have been widely used for differentiated hepatocyte-like cells (DHCs). Here, we established an in vitro HCV infection model using DHCs from AT-hMSCs and identified miRNAs that modulate HCV infectivity.


METHODS
AT-hMSCs were differentiated into DHCs using the conditional media, and evaluated for hepatocyte characteristics using RT-PCR, immunocytochemistry, periodic acid-Schiff staining, and a urea synthesis assay. The expression of HCV candidate receptors was also verified using immunocytochemistry. The levels of candidate miRNAs targeting HCV receptors were then determined by relative quantitative RT-PCR (rqRT-PCR). Finally, DHCs were infected using HCVcc and serum from HCV-infected patients, and infectivity of the virus was measured by rqRT-PCR and transmission electron microscopy (TEM).


RESULTS
The expected changes in morphology, function and hepatic gene expression were observed during hepatic differentiation. Moreover, the expression of candidate HCV entry factors and miR-27a were altered during hepatic differentiation. The infection and replication of HCV occurred efficiently in DHCs treated with HCVcc or infected with serum from HCV-infected patients. In addition, HCV infectivity was suppressed in miR-27a-transfected DHCs, due to the inhibition of LDLR expression by miR-27a.


CONCLUSIONS
Our results demonstrate that AT-hMSCs are a good source of DHCs, which are suitable for in vitro cultivation of HCV. Furthermore, these results suggest that miR-27a modulates HCV infectivity by regulating LDLR expression.
The Catholic University Liver Research Center & WHO Collaborating Center of Viral Hepatitis, the Catholic University of Korea, Seoul, Republic of Korea.
Jung Eun
JE
Choi
The Catholic University Liver Research Center & WHO Collaborating Center of Viral Hepatitis, the Catholic University of Korea, Seoul, Republic of Korea.
Wonhee
W
Hur
The Catholic University Liver Research Center & WHO Collaborating Center of Viral Hepatitis, the Catholic University of Korea, Seoul, Republic of Korea.
Jung-Hee
JH
Kim
The Catholic University Liver Research Center & WHO Collaborating Center of Viral Hepatitis, the Catholic University of Korea, Seoul, Republic of Korea.
Tian Zhu
TZ
Li
The Catholic University Liver Research Center & WHO Collaborating Center of Viral Hepatitis, the Catholic University of Korea, Seoul, Republic of Korea.
Eun Byul
EB
Lee
The Catholic University Liver Research Center & WHO Collaborating Center of Viral Hepatitis, the Catholic University of Korea, Seoul, Republic of Korea.
Sung Won
SW
Lee
Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea.
Wonseok
W
Kang
Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea.
Eui-Cheol
EC
Shin
Department of Virology II, National Institute of Infectious Disease, Tokyo, Japan.
Takaji
T
Wakita
The Catholic University Liver Research Center & WHO Collaborating Center of Viral Hepatitis, the Catholic University of Korea, Seoul, Republic of Korea.
Seung Kew
SK
Yoon
D000273:Adipose Tissue / Q000378:metabolism*; D002454:Cell Differentiation; D016174:Hepacivirus; D006526:Hepatitis C / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism
C573412:LDLR protein, human; C531388:MIRN27 microRNA, human; D035683:MicroRNAs; D011973:Receptors, LDL