Open Access

Downregulated long non-coding RNA DREH promotes cell proliferation in hepatitis B virus-associated hepatocellular carcinoma

  • Authors:
    • Dong Lv
    • Yuan Wang
    • Ying Zhang
    • Peilin Cui
    • Youqing Xu
  • View Affiliations

  • Published online on: June 21, 2017     https://doi.org/10.3892/ol.2017.6436
  • Pages: 2025-2032
  • Copyright: © Lv et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The hepatitis B virus X (HBx) protein has been characterized as an oncogene involved in epigenetic modifications during hepatocarcinogenesis; however, the underlying mechanisms are not entirely clear. Long non‑coding RNAs (lncRNAs), a type of epigenetic regulator molecules, have also been demonstrated to serve crucial roles in carcinogenesis, including hepatocellular carcinoma (HCC). In the present study, a human lncRNA DREH was identified, which inhibits cell proliferation in vitro and in vivo, and acts as a tumor suppressor in HBx‑mediated hepatocarcinogenesis. The study revealed that the expression of DREH was frequently downregulated in hepatitis B virus (HBV)‑associated HCC tissues in comparison with adjacent non‑cancerous hepatic tissues, and was inversely correlated with HBx mRNA expression in HBV‑associated HCC. In addition, the levels of DREH were inversely correlated with hepatitis B surface antigen and tumor size in HCC tissues. The forced expression of HBx in liver cell lines resulted in a significant decrease in the expression of DREH. Furthermore, suppression of DREH expression promotes the proliferation of HCC cells in vitro and in vivo. In conclusion, the present findings support the role of HBx‑downregulated lncRNA DREH in tumor suppression in HBV‑associated HCC patients. This contributes to a better understanding of epigenetic aberration of deregulated lncRNAs by HBx and the potential development of lncRNA‑based targeted approaches for the treatment of HBV‑associated HCC.

Introduction

As one of the most common cancer types in the world, hepatocellular carcinoma (HCC) has an extremely high morbidity and mortality rate, particularly in Asia and Africa (1). Overall, 50 to 55% of HCC cases are attributable to persistent hepatitis B virus (HBV) infections, which may result in end-stage liver disease, including liver cirrhosis and HCC (2). As the smallest open reading frame of the HBV genome, HBX encodes the hepatitis B virus X (HBx) protein which has been implicated in HBV-associated HCC pathogenesis, acting as a weak oncogene or a cofactor in hepatocarcinogenesis (35). However, the molecular mechanisms underlying HBx protein-mediated tumorigenesis are not entirely clear. Previous studies have demonstrated that genetic alterations alone do not account for the complexity of HBx-induced hepatocarcinogenesis, but that epigenetic changes, including DNA methylation (6), histone modifications (7) and non-coding RNA expression (6,8), are also involved in this process.

Long non-coding RNAs (lncRNAs) are a type of non-coding RNAs which are longer than 200 nucleotide transcripts and have little or no protein-coding capacity (9,10). Previous studies have demonstrated that lncRNAs are involved in diverse biological functions and pathological processes (10,11), and that altered lncRNA levels may result in aberrant gene expression through a variety of mechanisms, including transcription, post-transcriptional processing (12), chromatin modification, genomic imprinting and the regulation of protein function (13). Increasing evidence demonstrates that altered expression levels of lncRNAs contribute to a wide range of cancer types, including breast, lung, prostate and liver cancer (1417). Therefore, lncRNAs may potentially be used as diagnostic markers or therapeutic targets for cancer in the clinic.

Using lncRNA microarrays and gene sequencing technology, a large number of lncRNAs have been observed to be aberrantly expressed in HCC tissues and involved in hepatocarcinogenesis. These include highly upregulated in liver cancer (HULC), high expression in HCC (HEIH), activated by TGF-β (ATB) and HOX transcript antisense RNA (HOTAIR), which serve a role in diverse biological processes including cell proliferation, apoptosis and metastasis (1720). Several lncRNAs have been identified to be associated with the HBx protein (17,21). Huang et al (22) examined the lncRNA expression profiles in the livers of HBx transgenic and wild-type mice, and observed that certain lncRNAs are dysregulated and associated with HBx in HBx transgenic mice. These authors further investigated the biological function of the lncRNA Dreh, which may be downregulated by HBx protein, in mice. It was observed to inhibit HCC growth and metastasis, acting as a tumor suppressor in the development of HBV-HCC. The same authors also identified a human ortholog of Dreh, which was termed DREH, and observed that its expression level was frequently downregulated in HBV-associated HCC tissues. This decrement was significantly correlated with poor survival in HCC patients. However, the specific role of lncRNA DREH in HCC remains largely unknown.

In the present study, the expression levels of lncRNA DREH in 30 pairs of human HBV-positive HCC tissues and 30 pairs of HBV-negative HCC tissues and their pair-matched normal liver tissues were assessed. The results revealed that the expression level of DREH was significantly downregulated in HBV-HCC tissues compared with their adjacent non-cancerous hepatic tissues, and was inversely correlated with HBx mRNA expression in HBV-associated HCCs. Further investigation of the biological function of DREH in vivo and in vitro revealed that inhibition of DREH promotes cell proliferation in HBx-induced hepatocarcinogenesis. Together, these results suggest that DREH exerts an impact as a potential tumor repressor gene and may provide new insight into the role of HBx-associated lncRNAs in the development of HCC.

Materials and methods

Animal and patient samples

The four-week-old male BALB/c nude mice used in this study were purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). All mice were bred and maintained in a pathogen-free facility and were used in accordance with the institutional guidelines for animal care. The animal studies were approved by the Institutional Animal Care and Use Committee of the Capital Medical University, Beijing, China.

The 30 HBV-associated HCC tissues and 30 HBV-negative HCC tissues and corresponding adjacent non-cancerous liver tissues used in this study were obtained with informed consent from patients who underwent radical resection in the Peking University People's Hospital (Beijing, China). Studies using human tissues were reviewed and approved by the Committees for Ethical Review of Research Involving Human Subjects of the Capital Medical University. The clinicopathological characteristics of the 60 patients are summarized in Table I.

Table I.

Clinicopathological characteristics of 60 HCC patients.

Table I.

Clinicopathological characteristics of 60 HCC patients.

CharacteristicNumber (n=60)Percentage
Age (years)
  ≤553965.00
  >552135.00
Sex
  Male5185.00
  Female  915.00
Tumor differentiation
  I–II2135.00
  III–IV3965.00
TNM stage
  I2541.67
  II–III3558.33
Tumor size (cm)
  ≤53151.67
  >52948.33
Tumor number
  Single5286.67
  Multiple  813.33
AFP (µg/l)
  ≤201220.00
  >204880.00
Encapsulation
  Absent2948.33
  Complete3151.67
Microvascular invasion
  Absent5185.00
  Present  915.00
Macrovascular invasion
  Absent5490.00
  Present  610.00
Liver cirrhosis
  Absent1321.67
  Present4778.33
HBs antigen
  Negative3050.00
  Positive3050.00
HBe antigen
  Negative4066.67
  Positive2033.33
ALT (U/l)
  ≤402643.33
  >403456.67

[i] HCC, hepatocellular carcinoma; TNM, tumor-node-metastasis; AFP, α-fetoprotein; HBs antigen, hepatitis B surface antigen; HBe antigen, hepatitis B envelope antigen; ALT, alanine aminotransferase.

Construction of vectors

To construct HBx-expressing vectors, complementary DNA encoding HBx was PCR-amplified and sub-cloned into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All vectors were constructed according to standard methods and verified by sequencing. The polymerase chain reaction (PCR) primers used are presented in Table II.

Table II.

Sequences of primers and siRNAs used in study.

Table II.

Sequences of primers and siRNAs used in study.

Name Sequences
qPCR primers
HBxSense 5′-CCCTTCTTCATCTACCGTTCC-3′
Anti-sense 5′-CGTTGACATTGCTGCGAGT-3′
  β-actinSense 5′-TGTGTTGGCGTACAGGTCTTTG
Anti-sense 5′-GGGAAATCGTGCGTGACATTAAG
DREHSense 5′-CATTTGGCGGGACTACTTATT-3′
Anti-sense 5′-TTCAATCTGGCTTTGTTCGTT-3′
Primers for vector construction
DREH cloneSense 5′-GGGGTACCCCATGGCTGCTAGGGTGTG-3′
Anti-sense 5′-CGGGATCCCGTCAGGCAGAGGTGAAAAAG-3′
siRNA sequences
DREH siRNASense 5′-UCAUUUGGCGGGACUACUUTT-3′
Anti-sense 5′-AAGUAGUCCCGCCAAAUGATT-3′
siRNA NCSense 5′-UUCUCCGAACGUGUCACGUTT-3′
Anti-sense 5′-ACGUGACACGUUCGGAGAATT-3′
HBx siRNASense 5′-CCCACCAAAUAUUGCCCAATT-3′
Anti-sense 5′-UUGGGCAAUAUUUGGUGGGTT-3′

[i] siRNA, small interfering RNA; qPCR, quantitative polymerase chain reaction; HBx, hepatitis B virus X; NC, negative control.

Cell culture and transfection

The liver cell lines HepG2, HepG2.2.15, Hep3B, Huh-7 and SMMC-7721 were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (Gibco) and were maintained in a humidified 37°C incubator with an atmosphere of 5% CO2. The different plasmids and small interfering RNA (siRNA) sequences were transfected into cells using a Lipofectamine® 3000 kit (Invitrogen) according to the manufacturer's protocol. The siRNAs were synthesized by GenePharma (Shanghai, China). The siRNA sequences are provided in Table II.

Reverse transcription and quantitative PCR (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen). First-strand cDNA was generated using the Reverse Transcription system kit (Stratagene, La Jolla, CA, USA). Random primers (6mer; Takara Bio, Inc., Otsu, Japan) were used for RT-PCR for lncRNAs. Real-time PCR was performed using a standard SYBR-Green PCR kit protocol on a StepOne Plus system (Applied Biosystems, Thermo Fisher Scientific, Inc.). β-actin was employed as an endogenous control to normalize for the amount of total mRNA in each sample. The qPCR reactions were performed in triplicate, including no-template controls. The relative RNA expression was calculated using the comparative Cq method. The primer sequences are presented in Table II.

Cell Counting Kit-8 (CCK-8) assay

HepG2 or Huh-7 cells (2×103 cells/well) transfected with DREH siRNA or negative control were dispensed in 100-µl aliquots into 96-well plates. At the indicated time points, CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to the cells for 2 h and then the optical density was read using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All of the experiments were performed in triplicate.

Colony formation assay

For colony formation assay, cells were seeded at a density of 100 cells per well in a 12-well culture plate and cultured for 2 weeks, then cells were washed twice with phosphate-buffered saline (PBS), fixed with methanol, and the colonies were stained with 1% crystal violet and counted.

In vivo assay for tumor growth

Lentivirus-based short hairpin RNA (shRNA) constructs (GenePharma) were used to stably knock down DREH gene expression according to the manufacturer's protocol. HepG2 cells were stably transduced with DREH shRNA lentivirus. Cells transfected with DREH shRNA or control shRNA (1.0×107) were suspended in 100 µl PBS and implanted subcutaneously into the bilateral armpit of BALB/c nude mice (five in each group). The tumors were measured every three days after implantation, and the volume of each tumor was calculated as: Length × width2 × 0.4. All mice were sacrificed four weeks later.

Statistical analysis

The expression of DREH in HCC patients was compared using the paired samples t-test. The association between DREH and HBx mRNA expression was analyzed by Pearson's correlation. The correlations between DREH and clinicopathological characteristics in the 60 HCC patients were analyzed by the χ2 test or Fisher's exact probability test. Others comparisons were determined by Student's t-test. All P-values were two-sided and obtained using the SPSS 18.0 software package (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

LncRNA DREH is significantly downregulated in HBV-associated HCC tissues

To confirm the role of lncRNA DREH in HCC, DREH expression levels were first examined in 30 pairs of human HBV-associated HCC tissues and 30 pairs of HBV-negative HCC tissues and their pair-matched normal liver tissues by qPCR. The results revealed that the expression levels of DREH were significantly downregulated in HBV-HCC tissues in comparison with adjacent non-cancerous hepatic tissues from the same patient (P<0.0001, paired samples t-test); however, no significant difference was observed in the expression levels between the HBV-negative HCC tissues and the adjacent non-cancerous hepatic tissues. In addition, the expression of DREH was significantly higher in HBV-negative HCC tissues compared with HBV-positive HCC tissues (Fig. 1A).

DREH expression was further compared with clinicopathological characteristics in these 60 HCC patients, and statistical analysis revealed that lower DREH expression levels in HCC tissues were significantly positively correlated with tumor size (χ2=5.406, P=0.020, Table III) and hepatitis B surface antigen (HBsAg) (χ2=4.267, P=0.039, Table III). However, no direct correlation was identified between the expression of lncRNA DREH and other clinical characteristics, including age, sex, tumor differentiation, tumor-node-metastasis stage, tumor number, α-fetoprotein (AFP), encapsulation, microvascular invasion, macrovascular invasion, liver cirrhosis, hepatitis B envelope antigen and alanine aminotransferase (ALT) (Table III). These results indicate that DREH may be involved in HCC tumor growth and potentially associated with HBV infection.

Table III.

Correlation between lncRNA DREH expression and clinicopathological characteristics in 60 HCC patients.

Table III.

Correlation between lncRNA DREH expression and clinicopathological characteristics in 60 HCC patients.

LncRNA DREH expression

CharacteristicLow (n=30)High (n=30)P-value
Age (years) 0.787
  ≤552019
  >551011
Sex 0.278
  Male2427
  Female  6  3
Tumor differentiation 0.787
  I–II1011
  III–IV2019
TNM stage 0.190
  I1015
  II–III2015
Tumor size (cm) 0.020a
  ≤52011
  >51019
Tumor number 0.254b
  Single2428
  Multiple  6  2
AFP (µg/l) 0.333b
  ≤20  4  8
  >202622
Encapsulation 0.196
  Absent1217
  Complete1813
Microvascular invasion 1.000b
  Absent2526
  Present  5  4
Macrovascular invasion 0.671b
  Absent2628
  Present  4  2
Liver cirrhosis 0.754
  Absent  6  7
  Present2423
HBs antigen 0.039a
  Negative1119
  Positive1911
HBe antigen 0.273
  Negative1822
  Positive12  8
ALT (U/l) 0.297
  ≤401115
  >401915

a P<0.05

b Fisher's exact test; χ2 tests for all other analyses. The median expression level of lncRNA DREH was used as the cutoff. Patients with HC C were divided into an lncRNA DREH ‘low’ group (whose expression was lower than the median) and ‘high’ group (whose expression was higher than the median). LncRNA, long non-coding RNA; HCC, hepatocellular carcinoma; AFP, α-fetoprotein; HBs antigen, hepatitis B surface antigen; HBe antigen, hepatitis B envelope antigen; ALT, alanine aminotransferase.

DREH and HBx mRNA levels are inversely correlated in human HBV-associated HCC tissues

Next, it was assessed whether decreased DREH expression was correlated with the levels of HBx expression in human HBV-associated HCC tissues. The expression levels of HBx were further analyzed in the aforementioned 30 HCC tissues. A statistically significant inverse correlation was observed between DREH and HBx mRNA (n=30, r=−0.531, P=0.0033, Pearson's correlation; Fig. 1B). These data reveal the potential reciprocal regulation of DREH expression induced by HBx in human HCCs, and suggest that DREH may be involved in HCC pathogenesis as a tumor suppressor subsequent to HBx overexpression in chronic hepatitis B patients.

DREH is significantly downregulated in human HCC cell lines expressing HBx

To investigate the correlation between HBx and DREH expression, it was first determined whether DREH was differentially expressed in human HCC cells. The expression levels of DREH were assessed by RT-qPCR. The results revealed that the expression of DREH was markedly lower in HepG2.2.15 (a derivative of the human hepatoma cell line HepG2 that has been stably transformed with a head-to-tail dimer of HBV DNA) and Hep3B (a cell line containing the integrated hepatitis B viral genome) cell lines compared with HepG2, Huh-7 and SMMC-7721 cells, which do not express HBx (Fig. 2A).

Enforced HBx expression downregulates DREH in human HCC cells

In order to verify whether this downregulation was correlated with HBx expression, HepG2 and Huh-7 cells were transiently transfected with HBx expression vector pc-HBx and control vector pcDNA3.1. The levels of DREH were measured 72 h after transient transfection. The mRNA expression of HBx following infection is shown in the left panel of Fig. 2B. The results reveal that DREH was downregulated in pc-HBx-transfected cells in comparison with the pcDNA3.1 control groups (Fig. 2B).

Conversely, HBx expression was also repressed by siRNA; the knockdown efficacy of HBx siRNA is shown in the left panel of Fig. 2C. The inhibition of HBx by siRNA was observed to increase DREH expression in HepG2.2.15 and Hep3B cells which express HBx (Fig. 2C).

Inhibition of DREH promotes cell proliferation of HCC cells in vitro

The frequent downregulation of lncRNA DREH by HBx and the inverse correlation between DREH expression and tumor size in HBV-HCC patients implies that DREH may have a role in cell proliferation in HBV-associated hepatocarcinogenesis. To prove this, the effects of reduced expression of DREH on cell proliferation were investigated in two HCC cell lines. DREH expression was repressed by RNA interference, and the relative expression levels of DREH following infection of DREH siRNA or control siRNA are shown in the left panel of Fig. 3A and B. Cell-Counting Kit-8 assays demonstrated that suppression of cellular DREH enhanced the cell proliferation index compared with the control siRNA group in HepG2 and Huh-7 cells. The negative control siRNAs did not affect the cell proliferation index compared with the mock cells with no treatment (Fig. 3A and B).

Further colony formation assays also revealed that downregulation of DREH significantly enhanced the colony formation ability in HepG2 and Huh-7 cells compared with the control cells, consistent with the above results (Fig. 3C and D). Thus, these results suggest that DREH may serve a key role in HBx-induced hepatocellular proliferation.

Inhibition of DREH promotes tumor growth in vivo

To determine the effects of DREH on tumorigenesis in vivo, DREH-downregulated or control cells (HepG2 cells stably transfected with either shRNA-DREH or control shRNA) were subcutaneously injected into nude mice for xenoplantation. Mice injected with cells transfected with shRNA-DREH demonstrated significantly increased tumor growth compared with those injected with cells transfected with control shRNA (Fig. 4A and C).

As assessed by measurements of tumor volume, tumor weight and tumor weight/body weight ratio, the inhibition of DREH expression significantly promoted overall tumor growth 4 weeks after ectopic subcutaneous implantation in nude mice (Fig. 4B and D). These results further indicated that DREH was involved in the biological function of cell proliferation in HBV-associated HCC.

Discussion

HCC is a leading cause of cancer-associated mortality worldwide (23). Current guidelines recommend different therapeutic measures for the treatment of HCC patients with different stages, including surgery, chemotherapy, radiation therapy, and sorafenib and transarterial chemoembolization (24,25). Despite several recent advances and technical refinements, the long-term survival outcome of patients remains unsatisfactory (26). Therefore, it is necessary to thoroughly investigate the pathogenetic mechanism of HCC and develop new targeted treatments. The majority of recent investigations into cancer etiology have identified that epigenetics serves a critical role in cancer (27,28). Alterations in epigenetic modifications regulate all DNA-based processes, including transcription, DNA repair and replication, and are considered to be early events in tumorigenesis. There are also potential targets for therapeutic intervention using epigenetic drugs (29,30).

LncRNAs are a type of epigenetic regulator and are becoming one of the hot topics in genome research. Previous studies have revealed various functions and molecular mechanisms of these enigmatic molecules in biological processes of human health and diseases (31,32). With the development of high-throughput detection technologies including lncRNA microarray, RNA sequencing and the recent application of next-generation sequencing, thousands of lncRNAs have been observed to be aberrantly expressed and associated with various cancer types (33). The HBx protein has been reported to promote malignant transformation by epigenetic modifications and genetic regulation during hepatocarcinogenesis (34,35). HBx also alters the expression profiles of lncRNAs, and these cancer-associated lncRNAs may serve key roles in gene regulation and thus affect various aspects of cellular homeostasis (21,22).

In this study, a human lncRNA DREH was identified, which was downregulated by HBx protein. The suppression of DREH expression promotes the proliferation of HCC cells in vitro and in vivo, acting as a tumor suppressor in HBx-mediated hepatocarcinogenesis. The expression levels of DREH were examined in 30 pairs of human HBV-positive HCC tissues and 30 pairs of HBV-negative HCC tissues and their pair-matched normal liver tissues. The results revealed that the expression of DREH was frequently downregulated in HBV-associated HCC tissues compared with their adjacent non-cancerous hepatic tissues and was inversely correlated with HBx mRNA expression in HBV-associated HCCs. Clinical correlation analysis demonstrated that the levels of DREH were inversely correlated with HBsAg and tumor size in HCC tissues.

In summary, these findings suggest that lncRNA DREH exerts an impact as a potential tumor repressor gene in the development of human HBV-associated HCC. The modulation of cell proliferation by DREH may be used as a potential target for the prevention and treatment of HBV-associated HCC.

Glossary

Abbreviations

Abbreviations:

HBV

hepatitis B virus

HBx

hepatitis B virus X

HCC

hepatocellular carcinoma

lncRNA

long non-coding RNA

HBsAg

hepatitis B surface antigen

AFP

α-fetoprotein

ALT

alanine aminotransferase

RT-qPCR

reverse transcription-quantitative polymerase chain reaction

siRNA

small interfering RNA

CCK-8

Cell Counting Kit-8

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August-2017
Volume 14 Issue 2

Print ISSN: 1792-1074
Online ISSN:1792-1082

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Spandidos Publications style
Lv D, Wang Y, Zhang Y, Cui P and Xu Y: Downregulated long non-coding RNA DREH promotes cell proliferation in hepatitis B virus-associated hepatocellular carcinoma. Oncol Lett 14: 2025-2032, 2017
APA
Lv, D., Wang, Y., Zhang, Y., Cui, P., & Xu, Y. (2017). Downregulated long non-coding RNA DREH promotes cell proliferation in hepatitis B virus-associated hepatocellular carcinoma. Oncology Letters, 14, 2025-2032. https://doi.org/10.3892/ol.2017.6436
MLA
Lv, D., Wang, Y., Zhang, Y., Cui, P., Xu, Y."Downregulated long non-coding RNA DREH promotes cell proliferation in hepatitis B virus-associated hepatocellular carcinoma". Oncology Letters 14.2 (2017): 2025-2032.
Chicago
Lv, D., Wang, Y., Zhang, Y., Cui, P., Xu, Y."Downregulated long non-coding RNA DREH promotes cell proliferation in hepatitis B virus-associated hepatocellular carcinoma". Oncology Letters 14, no. 2 (2017): 2025-2032. https://doi.org/10.3892/ol.2017.6436