Suppression of hepatitis B virus replication by microRNA-199a-3p and microRNA-210.

Accumulating evidence suggests that microRNAs (miRNAs) control the replication of both RNA and DNA viruses. In order to determine whether host-encoded miRNAs affect hepatitis B virus (HBV) replication, antisense oligonucleotides (ASOs) of 328 identified human miRNAs were transfected into HepG2 2.2.15 cells, respectively. ELISA and MTS assay were used to measure the expression level of HBV S protein (HBsAg), HBV e antigen (HBeAg) and cell proliferation. Compared to experimental controls, miR-199a-3p and miR-210 efficiently reduced HBsAg expression without affecting HepG2 2.2.15 cell proliferation. Quantification of HBV DNA by real-time PCR showed that both miRNAs suppressed viral replication. Bioinformatics analysis indicated a putative binding site for miR-199a-3p in the HBsAg coding region and a putative binding site for miR-210 in the HBV pre-S1 region. The direct effect of miRNAs on the target region in HBV transcripts was validated by a fluorescent reporter assay, and the suppression of HBs gene expression by both miRNAs was measured by real-time PCR and Western blot. These results suggest that up-regulation of miR-199a-3p and miR-210 in HepG2 2.2.15 cells compared to HepG2 cells may play a role in regulating HBV replication and maintenance of a suitable level of virion production in persistent infection by targeting crucial HBV genes.

[1]  G. Acs,et al.  Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Hsien-Da Huang,et al.  ViTa: prediction of host microRNAs targets on viruses , 2006, Nucleic Acids Res..

[3]  R. Miller,et al.  Hepatitis B viral DNA-RNA hybrid molecules in particles from infected liver are converted to viral DNA molecules during an endogenous DNA polymerase reaction. , 1984, Virology.

[4]  Jialing Huang,et al.  Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes , 2007, Nature Medicine.

[5]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

[6]  B. Berkhout,et al.  RISCy Business: MicroRNAs, Pathogenesis, and Viruses* , 2007, Journal of Biological Chemistry.

[7]  Yiwei Liu.  Five-finger Dextrous Robot Hand DLR/HIT Hand II , 2009 .

[8]  Stijn van Dongen,et al.  miRBase: microRNA sequences, targets and gene nomenclature , 2005, Nucleic Acids Res..

[9]  K. Livak,et al.  Real-time quantification of microRNAs by stem–loop RT–PCR , 2005, Nucleic acids research.

[10]  F. Zoulim,et al.  Altered expression profiles of microRNAs in a stable hepatitis B virus-expressing cell line. , 2009, Chinese medical journal.

[11]  Dong Kong,et al.  HBV-encoded microRNA candidate and its target , 2007, Comput. Biol. Chem..

[12]  Anton J. Enright,et al.  Identification of Virus-Encoded MicroRNAs , 2004, Science.

[13]  A. Saïb,et al.  A Cellular MicroRNA Mediates Antiviral Defense in Human Cells , 2005, Science.

[14]  J. Mendell,et al.  MicroRNAs in cell proliferation, cell death, and tumorigenesis , 2006, British Journal of Cancer.

[15]  K. Jeang,et al.  HIV-1 Tat interaction with Dicer: requirement for RNA , 2006, Retrovirology.

[16]  Olivier Voinnet,et al.  Induction and suppression of RNA silencing: insights from viral infections , 2005, Nature Reviews Genetics.

[17]  Lin He,et al.  MicroRNAs: small RNAs with a big role in gene regulation , 2004, Nature reviews genetics.

[18]  K. Jeang,et al.  HIV-1 TAR RNA Subverts RNA Interference in Transfected Cells through Sequestration of TAR RNA-binding Protein, TRBP* , 2006, Journal of Biological Chemistry.

[19]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[20]  A. Dejean,et al.  The hepatitis B virus , 1985, Nature.

[21]  P. Sarnow,et al.  Modulation of Hepatitis C Virus RNA Abundance by a Liver-Specific MicroRNA , 2005, Science.

[22]  Eric Westhof,et al.  Single Processing Center Models for Human Dicer and Bacterial RNase III , 2004, Cell.

[23]  B. Berkhout,et al.  RNAi suppressors encoded by pathogenic human viruses. , 2008, The international journal of biochemistry & cell biology.

[24]  B. Cullen,et al.  MicroRNAs expressed by herpes simplex virus 1 during latent infection regulate viral mRNAs , 2008, Nature.

[25]  K. Kosik,et al.  MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. , 2005, Cancer research.

[26]  M. Mann,et al.  miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. , 2002, Genes & development.

[27]  Y. Lévy,et al.  Suppression of HIV-1 replication by microRNA effectors , 2009, Retrovirology.