PLoS ONE. 2014 May;9(5):e91958

MicroRNA-27a Modulates HCV Infection in Differentiated Hepatocyte-like Cells from Adipose Tissue-derived Mesenchymal Stem Cells

 

Jung Eun Choi1, Wonhee Hur1, Jung-Hee Kim1, Tian Zhu Li1, Eun Byul Lee1, Sung Won Lee1,

Wonseok Kang2, Eui-Cheol Shin2, Takaji Wakita3, Seung Kew Yoon1*

1 The Catholic University Liver Research Center & WHO Collaborating Center of Viral Hepatitis, the Catholic University of Korea, Seoul, Republic of Korea, 2 Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea, 3 Department of Virology II, National Institute of Infectious Disease, Tokyo, Japan

 

Abstract

Background and Aims: 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.

 

Supplements:

Hepatitis C virus (HCV) is a major cause of chronic liver diseases because chronic HCV infection can progress to liver cirrhosis and hepatocellular carcinoma (1). Pegylated interferon in combination with ribavirin has been used as standard therapy for the treatment of chronic hepatitis C up-to-date but recently developed direct-acting agents (DAA) have shown remarkable anti-viral effect through clinical trials (2). To develop the new antiviral agents or predict the resistant mechanisms of antiviral agents, it would be very important to study regulatory mechanisms of HCV pathogenesis or host factors in realistic HCV in vitro culture system. Although various cells could be infected with HCV, HCV replication and virion production do not occur efficiently in the cells (3). Therefore, we attempted to establish an in vitro HCV infection model with new approach.

Recently, few studies have been reported that HCV in vitro infection system could be conducted using differentiated hepatocyte-like cells (DHCs) derived from induced pluripotent stem (iPS) cells (4,5) or embryonic stem (ES) cells (6). Although efforts to establish an in vitro HCV infection model, it remains unclear whether cells differentiated from mesenchymal stem cells (MSCs) could become a reservoir for viral infection. MSCs can avoid drawbacks such as teratoma formation and ethical concerns (7). In addition, human adipose-tissue derived MSCs (AT-hMSCs) are attractive source of MSCs because of high differentiation capacity as well as easily procurable large quantities through cosmetic liposuction (8-10). Thus, we used DHCs derived from AT-hMSCs to establish an in vitro HCV infection model in this study.

Figure 1

Figure 1. HCV entry: candidate HCV entry receptors and their biological functions.

Since the first candidate HCV entry receptor called CD81 was discovered in 1998 (11), many researchers identified the candidate HCV entry receptors such as scavenger receptor B type I (SR-BI) (12), low-density lipoprotein receptor (LDLR) (13), occludin (14), caludin1 (15), and epidermal growth factor receptor (EGFR) (16). In addition, the study of HCV entry mechanism has been reported continuously. Based on the previous reports, HCV was secreted as a lipoviral particle form in the body (17) and it is initially recognized by LDLR and SR-B1 in target cells (18). Subsequently, HCV enters target cells through sequentially binding CD81, occludin, and claudin1 (Figure 1). Despite many researchers found out the candidate HCV entry receptors, HCV entry receptors regulating mechanism by miRNAs remain largely unknown. Here, we further try to identify the HCV entry receptors regulating miRNAs in established an in vitro HCV infection model using DHCs derived from AT-hMSCs (Figure 2).

 

Figure 2-2

Figure 2. Experimental flow in this study.

 

To establish an in vitro HCV infection model, we first induced the differentiation of hepatocyte-like cells from AT-hMSCs using the conditional medium for 4 weeks. After that, we assessed the hepatic characteristics such as change of morphology, alteration of hepatocyte or mesenchymal cell markers expression, ability of glycogen storage, and ability of urea synthesis. Our results showed that DHCs were acquired hepatocyte characteristics whereas lose mesenchymal characteristics.

Next, we further evaluated the expression of candidate HCV entry receptors in DHCs to determine the susceptibility of DHCs to HCV infection. The expression of candidate HCV entry receptors increased in DHCs compared to AT-hMSCs. DHCs revealed more highly expression levels of HCV RNA and its proteins than AT-hMSCs after infection with HCV. Moreover, HCV particles were detected in HCV infected DHCs, but not in HCV infected AT-hMSCs. These result showed that we successfully established the in vitro HCV infection model using DHCs derived from AT-hMSCs.

Based on recent papers, miRNAs play an important role in not only the hepatic differentiation (19) but also HCV replication (20-22). However, the miRNAs associated with HCV entry were not completely identified. Hence, we focused on the miRNAs expression during hepatic differentiation to find the candidate HCV entry receptors regulating miRNAs. We finally found out six up-regulated miRNAs and two down-regulated miRNAs including miR-27a and miR-99a during hepatic differentiation. In this study we were only interested in candidate HCV entry receptors expression regulating miRNAs, we therefore excluded the six up-regulated miRNAs from further study. And, two down-regulated miRNAs were used to further experiments to determine whether they could regulate expression of the candidate HCV entry receptors.

To determine downstream target of miR-27a and miR-99a, we used miRNA prediction software (http://www.microrna.org). LDLR was identified as a direct target of miR-27a and miR-99a. Collectively prior results, the miR-27a and miR-99a expression were revealed negatively correlated with the LDLR expression during hepatic differentiation. Thus, DHCs were transfected with mimics of miR-27a and miR-99a. After transfection, miR-27a-transfected DHCs showed significantly decreasing LDLR expression whereas miR-99a-transfected DHCs had no effect on the LDLR expression. We therefore selected miR-27a to evaluate the modulation of HCV infectivity by regulation of LDLR expression. miR-27a-trasnfected DHCs were infected with HCV. After that, the expression of HCV RNA was quantified. Due to the inhibition of LDLR expression by miR-27a, HCV infectivity was suppressed in miR-27a-transfected DHCs (Figure 3).

 Figure 3

Figure 3. miR-27a regulates HCV infection by down-regulation of LDLR expression in DHCs

 

Importance of the study: Our results demonstrate that AT-hMSCs are useful source of DHCs, which are suitable for studying HCV pathogenesis and HCV entry mechanism in vitro. Furthermore, these results suggest that miR-27a-regulated LDLR expression contributes to modulate HCV infectivity.

 

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Acknowledgments

This work was supported by the Bio-Synergy Research Project (NRF-2013M3A9C4078136) of the Ministry of Science, ICT and Future Planning through the National Research Foundation.

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