miR-34a and its novel target, NLRC5, are associated with HPV16 persistence

Highlights

• miR-34a and NLRC5 are associated with HPV16 persistence.

• NLRC5 interacts with miR-34a.

• Upregulation of NLRC5 is concurrent with downregulation of miR-34a in human cervical samples with HPV16 persistence.

• Our data uncover a previously unknown connection between the HPV16 persistence and miR-34a which directly targets to NLRC5.

• The reverse expression pattern of NLRC5 and miR-34a might be involved in the interference of HPV16 with host defense system.

Abstract

Persistent infection with human papillomavirus (HPV), particularly type 16, is causally associated with cervical cancer and its precursors. The role of miRNAs in HPV16 persistence currently remains unclear. Preliminary analysis of miRNA profile demonstrated that HPV16 infection caused a striking downregulation of miR-34a. Through bioinformatics analysis and dual-luciferase assay with site-directed mutagenesis strategy, NLRC5, a negative regulator of NF-κB signaling, was identified to be a novel interactor of miR-34a. Transfection of miR-34a mimic strikingly downregulated NLRC5 in the HPV16-positive cervical cells, which might result in the nuclear accumulation of NF-κB p65. However, transfection of miR-34a inhibitor exhibited an opposite effect. The antagonistic expressions of NLRC5 and miR-34a were also observed in keratinocytes harboring HPV16 genome as well as in human cervical samples with persistent infection of HPV16. Our data uncover a previously unknown connection among HPV16 persistence, miR-34a and its interactor NLRC5.

Introduction

Persistent infection with any type of high-risk human papillomaviruses (hrHPV) can cause cervical cancer, which is the third most commonly diagnosed cancer (Cuschieri et al., 2004, Rodriguez et al., 2008, zur Hausen, 2009) and the fourth leading cause of cancer death in females worldwide (Hang et al., 2015, Torre et al., 2015). Although the applications of morphological examination and HPV DNA test have dramatically reduced the development of cervical cancer and associated deaths (Kitchener et al., 2006, Schiffman et al., 2011), they are still unable to distinguish the persistent from transient hrHPV infection, thus resulting in overtreatment or recurrence (Du et al., 2013, Kyrgiou et al., 2006) of the disease after conization was performed on the patients with cervical precancerous lesions. Therefore, to determine the key factors involved in the viral clearance has been critical to understand the natural history of hrHPV-related cervical disease and trace the treatment modalities.

MicroRNAs (miRNAs) are an abundant class of noncoding small RNAs about 21 nucleotides which can affect the gene expression through imperfect base pairing to the 3′ untranslated region (3′ UTR) of the target genes (Djuranovic et al., 2012, Fabian et al., 2010). Among the known miRNAs, miR-34a is a member of the miR-34 family which includes two other members, miR-34b and miR-34c (Agostini and Knight, 2014, Misso et al., 2014). These three miRNAs are all potential tumor suppressors, among which miR-34a is the best characterized one in a variety of cancer types (Bu et al., 2013, Ji et al., 2009, Yin et al., 2013). Although the role of miR-34a in tumor has been well-studied, its effect on the viral clearance still remains unclear, which promotes us to investigate the involvement of this potent tumor suppressor in hrHPV persistence. NLRC5 is a new member of the Nod-like receptor (NLR) family with high abundance in mucosal epithelial cells (Benko et al., 2010, Kuenzel et al., 2010). Similar to other classic NLR members, NLRC5 is emerging to be involved in the regulation of immune response to combat intruding microbes (Kuenzel et al., 2010, Lupfer and Kanneganti, 2013, Neerincx et al., 2010). Recent studies have demonstrated that NLRC5 negatively regulates NF-κB signaling via direct binding to the NF-κB regulators IKKα/IKKβ, thereby preventing the recruitment of IKKγ/NEMO and nuclear translocation of NF-κB (Cui et al., 2010). In addition, NLRC5 may regulate this pathway by modulating the transactivation potential of nuclear NF-κB and/or by amplifying the production of the anti-inflammatory cytokine IL-10 (Benko et al., 2010). Typically, NF-κB serves as a key point of convergence for multiple signaling pathways that are relevant to the antiviral response and subsequent activation of adaptive immune response (Hayden et al., 2006, Hiscott et al., 2006, Rabson and Lin, 2000, Vallabhapurapu and Karin, 2009). Consequently, downregulation of NLRC5 might represent a unique mechanism by which hrHPV interferes with the host defense system that may promote persistent infection. Using multiple prediction algorithms, we found that a putative miR-34a targeting site was located within the 3′-UTR of NLRC5, which was also potentially related to the hrHPV persistence.

In the present study, our data have shown, for the first time, that NLRC5 is a functional target of miR-34a, through which miR-34a acts as a positive regulator of NF-κB signaling in cervical cells harboring HPV16. In addition, we have also demonstrated a significant correlation among miR-34a, NLRC5 and HPV16 persistence in human cervical samples with transient versus persistent viral infection.