Hepatitis B Virus Inhibits Apoptosis of Hepatoma Cells by Sponging the MicroRNA 15a/16 Cluster

Abstract

Hepatitis B virus (HBV) causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV persistence are not well understood. In this study, we found that HBV inhibited the chemotherapy drug etoposide-induced apoptosis of hepatoma cells. Further analysis revealed that HBV mRNAs possess a microRNA 15a/16 (miR-15a/16)-complementary site (HBV nucleotides [nt] 1362 to 1383) that acts as a sponge to bind and sequester endogenous miR-15a/16. Consequently, Bcl-2, known as the target of miR-15a/16, was upregulated in HBV-infected cells. The data from HBV-transgenic mice further confirmed that HBV transcripts cause the reduction of miR15a/16 and increase of Bcl-2. More importantly, we examined the levels of HBV transcripts and miR-15a/16 in HBV-infected HCC from patients and found that the amount of HBV mRNA and the level of miR-15a/16 were negatively correlated. Consistently, the level of Bcl-2 mRNA was upregulated in HBV-infected patients. In conclusion, we identified a novel HBV mRNAmiR-15a/16 -Bcl-2 regulatory pathway that is involved in inhibiting etoposide-induced apoptosis of hepatoma cells, which may contribute to facilitating chronic HBV infection and hepatoma development. There are approximately 350 million chronic hepatitis B virus (HBV) carriers worldwide, and chronic HBV infection is the major etiological factor in hepatocellular carcinoma (HCC) (1, 2). The relative risk for the development of HCC in chronic hepatitis B (CHB) patients is estimated to be 25 to 37 times higher than that in those without infection (1,3,4).HBV is an enveloped, partially double-stranded DNA virus with a genome size of 3.2 kb. The HBV genome contains four overlapping open reading frames (ORFs). The RNA transcripts are polyadenylated and capped and are named the pre-C/C or pregenomic RNA (pgRNA) and the pre-S, S, and X mRNAs. These mRNAs encode several viral proteins, including the polymerase, core, HBe, pre-S1, S2, S, and X proteins (5). HBV has been reported to play an important role in regulating apoptosis. For example, HBV core protein inhibits TRAIL-induced apoptosis of hepatocytes by blocking DR5 expression (6). HBx can bind to the C terminus of p53 and inhibit p53-mediated cellular processes, including transcriptional transactivation and apoptosis (7). But the HBx protein was also found to sensitize cells to apoptotic killing by tumor necrosis factor alpha (8) and to inhibit Fas-mediated apoptosis associated with upregulation of the SAPK/JNK pathway in Chang cells (9).MicroRNAs (miRNAs) are single-stranded noncoding RNAs which negatively regulate gene expression at the posttranscriptional level, primarily through base pairing to the 3=-untranslated region (UTR) of target mRNA (10). Growing evidence indicates that microRNAs control basic cell functions, ranging from proliferation to apoptosis, by direct targeting (11,12). For instance, miR-101 exerts a proapoptotic function by targeting Mcl-1 (13), and miR-29c inhibits ce...

Hepatitis B Virus Regulates Apoptosis and Tumorigenesis through the MicroRNA-15a-Smad7-Transforming Growth Factor Beta Pathway

“…The patients were hospitalized during June 2012 to July 2013. The clinical characteristics of enrolled subjects were listed in a previous study (12). Written informed consent was provided by all study participants.…”

“…HBV transgenic BALB/c mice were purchased from the Transgenic Engineering Lab, Infectious Disease Center (Guangzhou, China), and were described in our previous study (12).…”

“…Smad7 and Smad2 were cloned into pFLAG-CMV2 (Sigma, USA) and pCMV-Myc (Clontech, USA), respectively. pHBV1.3, carrying the full-length HBV genome, and its mutant pHBV1.3-mut, which cannot absorb miR-15a, were generated as described previously (12).…”

“…Also, HBV can promote cell proliferation and tumor formation by downregulating miR-122 (11). Previously, we found that HBV inhibits apoptosis by directly sponging miR15a and upregulating Bcl-2 expression (12). It has been reported that miR-15a can enhance prostate cancer progression and promote cell growth and survival by targeting Bcl-2, CCND1, and WNT3A (13).…”

Sponging of cellular proteins by viral RNAs

“…Indeed, there are already several examples of virally-encoded RNA sponges that do just that. These include the non-coding U-rich HSUR1 transcripts made by Herpesvirus saimiri [14] which targets cellular miR-27 for degradation, the m169 transcript of murine cytomegalovirus which also inhibits miR-27 [15], and the sponging of cellular miRNAs encoded by the 15a/16 cluster by Hepatitis B virus mRNAs [16]. Importantly, since viral transcripts are often present in very large quantities relative to natural cellular ceRNAs, these viral RNAs may very well be present in such high ratios relative to their targets that could indeed effectively sequester cellular miRNAs (thus overcoming the concerns raised by the Denzler et al study [9]).…”