It was recently reported by the present team that 3β-hydroxysterol Δ24-reductase (DHCR24) is induced by hepatitis C virus (HCV) infection. In addition, upregulation of DHCR24 impairs p53 activity. In human hepatoma HuH-7 cells, the degree of DHCR24 expression is higher than in normal hepatic cell lines (WRL68) at the transcriptional level. The genomic promoter sequence of DHCR24 was characterized and nucleotide substitutions were observed in HuH-7 cells at nucleotide numbers −1453 (G to A), −1420 (G to T), −488 (A to C) and −200 (G to C). The mutations of these sequences from HuH-7 cell types to WRL68 cell types suppressed DHCR24 gene promoter activity. The sequences were further characterized in hepatocytes from patient tissues. Four tissues from HCV-positive patients with cirrhosis or hepatocellular carcinoma (#1, 2, 3, 5) possessed HuH-7 cell type sequences. Interestingly, one patient with liver cirrhosis (#4) possessed WRL68 cell-type sequences; this patient had been infected with HCV and was HCV negative for 17 years after interferon therapy. Next, the effect of HCV infection on these polymorphisms was examined in humanized chimeric mouse liver and HuH-7 cells. The human hepatocytes possess WRL68 cell type and did not show the nucleotide substitution after HCV infection. The HCV-replicon was removed by interferon treatment and established the cured K4 cells. These cells possess HuH-7 cell type sequences. Thus, this study showed the genomic polymorphism in DHCR24 promoter is not directly influenced by HCV infection.
Dulbecco's modified Eagle's medium
hepatitis C virus
sustained viral response
Liver cancer is one of the most prevalent forms of cancer . More than 80% of cases occur in developing countries; however, Japan also has a remarkably high incidence . Among the primary liver cancers, HCC is the most common . Its incidence is increasing: between 1975 and 2005, age-adjusted HCC rates tripled .
One crucial cause of HCC is HCV infection . DHCR24, which functions as an oxidoreductase during cholesterol biosynthesis [6, 7], is linked to HCV-associated hepatocarcinogenesis and development of HCC [8-10]. Infection of hepatocytes with HCV results in overexpression of DHCR24. This enzyme protects cells from oxidative stress and inhibits p53 activity , thus contributing to the development of HCC . These facts prompted us to investigate whether the molecular features of DHCR24 are linked to HCC development. To this end, we characterized the promoter region of DHCR24 in HCC cell lines and clinical samples.
MATERIALS AND METHODS
Cell lines and growth conditions
HuH-7 and HepG2 cells were cultured in (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS (Sigma-Aldrich). WRL68 cells were cultured in DMEM supplemented with 1 mM sodium pyruvate (Invitrogen, Carlsbad, CA, USA), 0.1 mM non-essential amino acids (Invitrogen) and 10% FCS. HuH-7 cell-based HCV replicon harboring cell lines (R6FLR-N)  were cured off HCV by interferon treatment  and designated as K4 cells.
Northern and western blotting
Northern and western blotting were performed as previously described .
Sequencing of genomic DNA and reporter plasmid construction
Genomic DNA was extracted from HuH-7 and WRL68 cells using standard methods. DNA from the promoter region of DHCR24 (∼5 kb) was amplified using PCR (sense primer: 5′-CACTCCTGCTCACCACTGAT-3′; antisense primer: 5′-GTAGTAGATATCGAAGATAAGCGAGAGCGG-3′). These fragments were individually cloned into the upstream region of the firefly luciferase gene in the pGL3-Basic vector (Promega, Madison, WI, USA) at the XhoI and NcoI sites (as we had done previously for the HepG2 cell line) . DNA sequences were determined using standard methods. Reporter plasmids that possessed chimeric promoters were constructed using restriction enzyme sites for Tth111I (position −2160) and BssHII (position −1030).
Dual luciferase reporter assay
Using Lipofectamine LTX (Invitrogen), HepG2 cells (1 × 104 cells/well in a 96-well plate) were transfected with a reporter plasmid (0.25 μg/well) together with an internal control plasmid (phRL-TK; 0.025 μg/well) encoding Renilla luciferase (Promega). Forty-eight hours after transfection, the cells were assayed with the Dual-Glo Luciferase Assay System (Promega). Luminescence was measured using a TriStar LB941 microplate reader (Berthold Technologies GmbH, Bad Wildbad, Germany).
Liver tissue samples from chimeric mice or patients infected with hepatitis C virus
Severely combined immunodeficient mice carrying human primary hepatocytes were purchased from BD BioSciences (Franklin Lakes, NJ, USA) and African American, male, 5-year-old, HCV negative mice from PhoenixBio (Hiroshima, Japan) . These “human liver chimeric” mice were inoculated or mock-inoculated with plasma collected from an HCV-positive (HCR6 strain , GenBank accession #AY045702) patient in accordance with the requirements of the Declaration of Helsinki. HCV infection in the mice thus infected was confirmed by using quantitative PCR for HCV mRNA as previously described . The protocols for the animal experiments were pre-approved by the local Ethics Committee, and the animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Informed consent for this clinical study was obtained from five patients with HCV (Table 1) at the Kumamoto University Hospital (Kumamoto, Japan), in accordance with the Helsinki Declaration prior to 2003, and the protocol was approved by the Regional Ethics Committee. LiverPool 20-donor pooled cryopreserved human hepatocytes (Celsis IVT, Baltimore, MD, USA) were purchased and used as the normal human liver tissue control. HCV RNA was detected by the COBAS TaqMan HCV test (Hoffman–La Roche, Basel, Switzerland). Liver tissue was obtained from either mice or patients and processed for DNA sequencing. Two DNA fragments (corresponding to positions −1600 to −1292 and −631 to −86) were amplified using PCR with Tth-Bss forward and reverse primers (5′-ATTTCAACATGTCATTAACA-3′ and 5′-TTCTAGCACGGTGCTTTGTG-3′) and Bss-Nco forward and reverse primers (5′-CCAGCCATAGCCTTCCATG-3′ and 5′-AATGGCGAGCCGCGCCGG-3′), respectively. The amplified fragments were directly sequenced using the same set of primers.
|Patient ID||Sex||Age (years)||Diagnosis||ALT (IU/mL)||Outcome of IFN treatment||HCV RNA detectiona|
Student's t-test was used to test the statistical significance of the results. P values of < 0.05 were considered statistically significant.
First, we measured DHCR24 expression in cell lines of noncancerous hepatocytes (WRL68) and hepatoma cells (HuH-7 and HepG2). Compared with noncancerous hepatocytes, DHCR24 expression in the two hepatoma cell lines was considerably increased with respect to both mRNA and protein (Fig. 1a, b). In addition, the different culture media used for the WRL68 and HuH-7 cells did not significantly influence the degree of expression of DHCR24 protein (Fig. 1c).
To identify the genetic characteristic(s) that govern DHCR24 upregulation, we isolated genomic DNA from these three cell lines and sequenced the DHCR24 promoter region (nucleotide positions −4976 to +113, where +1 indicates the transcription start site). For this analysis, we sequenced three molecular clones from each cell line. Alignments of WRL68 and HuH-7 sequences showed different nucleotides at four positions: (i) an A to G switch at −1453 (i.e., A in WRL68 and G in HuH-7); (ii) a T to G switch at −1420; (iii) a C to A switch at −488; and (iv) a C to G switch at −200 (Fig. 2). The two hepatoma cell lines (HuH-7 and HepG2) had no nucleotide differences within these regions.
Next, we investigated whether these small changes in the promoter sequence affect gene expression in a heterologous context. We constructed reporter plasmids that placed the firefly luciferase gene under the control of DHCR24 promoter sequences (either from HuH-7 or WRL68 cells) (Fig. 3a). We measured the promoter activity of each construct in HepG2 cells with dual-luciferase assays. The DHCR24 promoter derived from HuH-7 cells showed significantly greater activity (i.e., induced greater expression) than the WRL68 promoter (Fig. 3b). We also constructed two reporter plasmids that contained chimeric promoters. In each of these chimeras, we replaced HuH-7 fragments containing two polymorphisms with wild-type WRL68 sequences (Fig. 3a). These chimeric promoters had less activity than did intact promoters from both HuH-7 and WRL68 cells (Fig. 3b). These results indicate that the DHCR24 promoter from HuH-7 cells contributes to the strong degree of DHCR24 expression. In addition, all four nucleotide sequences of HuH-7 cell type in promoter fragments might be important for strong promoter activity.
Thereafter, we examined whether polymorphisms within the DHCR24 promoter could be detected in clinical samples. We collected samples of liver tissue from five patients infected with HCV (Table 1) and sequenced the DHCR24 promoter region (Table 2). Of the five samples tested, four (#1–3 and #5) showed all four of the polymorphisms associated with strong promoter activity (i.e., G, G, A and G nucleotides at positions −1453, −1420, −488, and −200). In contrast, promoter sequences from patient 4 (#4) had nucleotides associated with weak activity at these positions (i.e., A, T, C, and C). Intriguingly, only #4 exhibited an SVR, which is characterized by the absence of detectable HCV RNA in serum for >24 weeks following IFN treatment. The SVR status of #4 has persisted since 1995. In #5, promoter sequences were the same in cancerous and non-cancerous regions of the liver. These results suggest that the four polymorphisms within the DHCR24 promoter region may influence the susceptibility to malignancy and IFN responsiveness of hepatoma cells and thus influence the fate of patients with HCC.
|Origin of DNA sample||Nucleotide position|
|HuH-7 cells (high)a||G||G||A||G|
|WRL68 cells (low)b||A||T||C||C|
|Patient #5 (NC)||G||G||A||G|
|Patient #5 (C)||G||G||A||G|
To assess the impact of HCV infection on genomic polymorphism in DHCR24 promoter sequences, we determined the sequences in human hepatocytes that had been transplanted into severely combined immunodeficient mice that we infected or mock-infected with HCV We detected markedly high titers of HCV only in the infected mice (Table 3). Sequencing revealed that all four polymorphic nucleotide positions were of the weak activity type. Notably, we detected no nucleotide differences between HCV- and mock-infected mice in the targeted regions (Table 3). We also established cured K4 cells by treating HCV replicon cells R6FLR-N with IFN. Analysis of the genomic sequence of these cell lines showed no nucleotide differences in R6-FLR-N and K4 cells (Table 3). These results suggest that the differences in the DHCR24 promoter sequence are ingenerate rather than induced by HCV infection.
|Origin of DNA sample||HCV||Nucleotide position|
|HuH-7 cells (high)||−||G||G||A||G|
|WRL68 cells (low)||−||A||T||C||C|
|Chimeric mouse liver||−||A||T||C||C|
|HCV infected chimeric mouse liver||+a||A||T||C||C|
|HCV replicon cells (R6FLR-N)||+||G||G||A||G|
|Cured K4 cells||+||G||G||A||G|
In this study, we analyzed the promoter sequences associated with DHCR24 in hepatocytes and identified polymorphisms that regulate the degree of expression of downstream genes (Figs. 1-3). #4 had an SVR in response to IFN treatment; thus these DHCR24 promoter sequence polymorphisms are potential biomarkers for predicting patients' responsiveness to IFN treatment.
Genomic polymorphisms within the DHCR24 promoter region may influence binding of transcription factors (Supplementary Fig. S1). In fact, a T-to-G nucleotide substitution at position −1420 generates a potential binding site for the protein encoded by the caudal homeobox gene (CdxA), a homeobox transcription factor responsible for gastrointestinal tract development and epithelial differentiation . A C-to-A substitution at position −488 generates potential binding sites for nuclear factor kappa-light-chain enhancer of activated B cells and STATx , as well as a low-affinity binding site for Nkx-2 . Finally, a C-to-G substitution at position −200 potentially abolishes a p300 binding site . These changes in transcription factor binding affinities could upregulate DHCR24 expression, thereby promote carcinogenesis.
Previously, we discovered that DHCR24 is a host factor involved in HCV-associated development of HCC [8, 9]. This protein is upregulated by HCV infection , and reduced degrees of expression (via siRNA knockdown) inhibit HCV replication . These findings are consistent with the role of DHCR24 in cholesterol biosynthesis [6, 7], which is important for HCV replication . Also, because the efficiency of HCV replication might have been lower in #4 than in other patients with strongly active DHCR24 promoter, the weak DHCR24 expression in this patient (Supplementary Fig. S2) might have contributed to the efficacy of IFN treatment.
In conclusion, we have discovered polymorphisms in the promoter region of DHCR24 gene that have not been induced by HCV infection. Future study will clarify their biological significance.
The authors thank Dr Michinori Kohara, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan for his generous support, which included supplying reagents. This work was supported by grants from the Ministry of Health, Science and Welfare and the Ministry of Education, Science and Culture, Japan.
The authors have no financial relationships to disclosure.