1 3 Functional interplay between hepatitis B virus X protein 4 and human miR-125a in HBV infection 5 6 7 Nicola Mosca a Q1 , Filomena Castiello a , Nicola Coppola b , Maria Consiglia Trotta a , Caterina Sagnelli b , 8 Mariantonietta Pisaturo b , Evangelista Sagnelli b , Aniello Russo a,⇑ , Nicoletta Potenza a,⇑ 9 a Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, via Vivaldi 43, 81100 Caserta, Italy 10 b Department of Mental Health and Public Medicine, Section of Infectious Diseases, Second University of Naples, via L. Armanni 5, 80135 Naples, Italy 11 12 14 article info 15 Article history: 16 Received 17 April 2014 17 Available online xxxx 18 Keywords: 19 Viral hepatitis 20 miR-125a 21 Molecular mechanisms 22 Hepatocellular carcinoma 23 24 abstract 25 The hepatitis B virus (HBV) is a widespread human pathogen and chronic HBV infection is a major risk 26 factor for hepatocellular carcinoma (HCC). Some cellular microRNAs are emerging as important regula- 27 tors of virus–host interaction, indirectly or directly modulating HBV replication and pathogenesis. 28 miR-125a binds the viral transcript encoding the surface antigen and interferes with its expression, thus 29 inhibiting viral replication. Intriguingly, liver miR-125a expression has been found increased in patients 30 with high levels of hepatic HBV-DNA. The present study investigates the mechanism by which liver 31 exposure to HBV induces the expression of miR-125a. The analyses were first performed on liver biopsies 32 from HBV patients, showing that the expression of the viral transactivator X protein (HBx) paralleled the 33 increase of miR-125a expression. Then, transfection of HCC cell lines with an HBx-expressing vector 34 showed a substantial increase of miR-125a expression. Overall, the available data depict a self-inhibitory 35 feedback loop in which HBV, through HBx, increases the expression of miR-125a, that in turn interferes 36 with expression of HBV surface antigen, thus repressing viral replication. 37 Ó 2014 Published by Elsevier Inc. 38 39 40 41 1. Introduction 42 MicroRNAs (miRNAs) are small non-coding RNAs that regulate 43 gene expression at post-transcriptional level by affecting both 44 the stability and translation of complementary mRNAs [1]. miRNAs 45 play crucial role in development, cell differentiation and apoptosis 46 [2,3]. Moreover, several studies indicate that miRNA genes act as 47 oncogenes or tumor suppressors [4,5]. Finally, recent studies have 48 shown that miRNAs are important regulators of virus–host interac- 49 tions [6–8]. 50 Several reports have been dedicated to the role of miRNAs in 51 hepatitis B virus (HBV) replication and pathogenesis, since HBV 52 affects over 350 million people worldwide and is one of the leading 53 causes of liver disease, often progressing to cirrhosis and hepatocel- 54 lular carcinoma (HCC) [9–11]. Some cellular miRNAs modulate HBV 55 replication either by targeting cellular transcription factors 56 required for HBV gene expression or by a direct binding to HBV 57 transcripts. In particular, some miRNAs promote HBV replication 58 by targeting negative regulators of HBV activity (miR-372/373, 59 miR-501) [12,13] or by enhancing a positive regulator (miR-1) 60 [14]. Other miRNAs suppress HBV replication by targeting positive 61 regulators of HBV (miR-29c, miR-122 and miR-141) [15–18] or by 62 directly targeting HBV transcripts (miR-210, mir-199a-3p and 63 miR-125a-5p) [19,20]. With regard to the last regulatory mecha- 64 nisms, several reports have been dedicated to the role of human 65 miR-125a-5p (miR-125a) in HBV infection. We first demonstrated 66 that miR-125a is able to target the expression of HBV surface anti- 67 gen (HBsAg). The interaction of miR-125a with the viral sequence 68 was first demonstrated with a validation test based on cultured 69 hepatic cells and luciferase reporter genes. Then, miR-125a was 70 shown to interfere with viral translation in PLC/PRF/5, a cell line 71 that contains several fragments of HBV DNA integrated in its 72 genome and secretes HBsAg in the culture medium. In this system, 73 transfection of a miR-125a mimic induced a marked decrease of the 74 expression of HbsAg, whereas the transfection of a miR-125a 75 inhibitor enhanced the expression of HBsAg [20]. Moreover, an 76 inspection of nucleotide sequences of HBV genomes deposited at 77 NCBI revealed that the target site of miR-125a is well conserved 78 in the viral population [8], supporting a significant role in HBV–host 79 interaction. Later, an independent study has confirmed the ability http://dx.doi.org/10.1016/j.bbrc.2014.05.009 0006-291X/Ó 2014 Published by Elsevier Inc. ⇑ Corresponding authors. Fax: +39 0823 274571. E-mail addresses: nicola.mosca@unina2.it (N. Mosca), filomena.castiello@unina2. it (F. Castiello), nicola.coppola@unina2.it (N. Coppola), mconsigliatrotta@gmail.com (M.C. Trotta), caterina.sagnelli@unina2.it (C. Sagnelli), meri.pisaturo@libero.it (M. Pisaturo), evangelista.sagnelli@unina2.it (E. Sagnelli), aniello.russo@unina2.it (A. Russo), nicoletta.potenza@unina2.it (N. Potenza). Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc YBBRC 32104 No. of Pages 5, Model 5G 15 May 2014 Please cite this article in press as: N. Mosca et al., Functional interplay between hepatitis B virus X protein and human miR-125a in HBV infection, Biochem. Biophys. Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.05.009