Introduction
In recent years, hepatocellular carcinoma (HCC) is one of the most common fatal malignancies worldwide (1). Whereas surgical resection is the preferred therapy, not all patients with HCC are suitable for this procedure and the post-operative recurrence rate remains high (2). Therefore, there is a requirement for the identification of alternative effective treatment options.
The type I interferons (IFNs) are a conserved family that play significant roles in antiviral, antitumoral and immunomodulatory functions (3). Binding of type I IFN to the IFN receptor leads to the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and then amplification of IFN production (4). Valuable insights into the growth-inhibitory properties of the endogenous type I IFNs have been provided from in vivo studies (5,6). Although type I IFN has long been known to have antitumoral effects, it is only relatively recently that the role of type I IFN in tumor immunosurveillance has been described (7). The majority of studies have shown that IFN is able to suppress HCC growth (8,9), as well as prevent its recurrence (10). However, sensitivity to type I IFN therapy in HCC patients tends to vary and the exact mechanisms remain unclear. The aim of the present study was to investigate the cellular factors that determine the sensitivity of hepatoma cells to type I IFN therapy.
Materials and methods
Cell lines
The human hepatoma cells Huh7 and HepG2 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin solutions in a 37°C, 5% CO2, humidified incubator. The cells were treated with 0.02% EDTA and 0.25% trypsin during passage.
MiR-122 plasmid and inhibitor
The plasmid pDC-316-EGFP-U6-miR-122 and the normal control miRNA (miR-NC) were purchased from GeneScript (Piscataway, NJ, USA). The 2′-O-methylated, anti-miR-122 oligonucleotide (AMO-122; 5′-CAAACACCAUUGUCACACUCCA-3′) and the normal control anti-miRNA (AMO-NC) were synthesized by GenePharma (Shanghai, China).
Transfection of the miR-122 plasmid and inhibitor
Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to transfect the miR-122 plasmid and inhibitor into the cells. For use in the 24-well plates, subsequent to 24 h culture in antibiotic-free medium, the Huh7 cells were separately transfected with 1.6 μg of miR-122 plasmid or miR-NC plasmid for 48 h and 60 pmol of AMO-122 or AMO-NC for 24 h, according to the manufacturer’s instructions.
Cell treatment
Human recombinant IFN-α and IFN-γ were purchased from Pestka Biomedical Laboratories (Piscataway, NJ, USA). Cells were incubated with 100 U/ml of IFN-α or IFN-β for 4 h and then harvested for RNA analysis.
RNA isolation
Total RNA was extracted with TRIzol (Invitrogen) according to the manufacturer’s instructions and 2 μl total RNA was collected to measure the concentration of total RNA using a Biophotometer (Eppendorf, Hamburg, Germany). Samples with RNA absorption values of ≥1.9 were considered for the study.
Determination of miR-122 and IFN-α/β RNA levels by quantitative real time polymerase chain reaction (qRT-PCR)
Total RNA was incubated with specific reverse primers (for miRNAs) or oligo-dT (for mRNAs). qRT-PCR was performed to detect the miRNAs and mRNAs. Fold variations between the RNA samples were calculated subsequent to normalization to U6 RNA or GAPDH mRNA. The primers of GAPDH, IFN-α, IFN-β and miR-122 were described in a previous study (11).
Statistical analyses
All values are presented as the mean ± SEM (standard error of the mean). The Student’s t-test was used to evaluate the differences between two groups. All statistical tests were two-sided and P<0.05 was considered to indicate a statistically significant difference.
Results
Differential response of the Huh7 and HepG2 cells to type I IFN stimulation
To explore the varying responses to type I IFN therapy, two common HCC cell lines, Huh7 and HepG2, were used to evaluate this phenomenon. Huh7 is miR-122-abundant while HepG2 is miR-122-deficient (12,13). First, basal type I IFN expression was detected in the two cell lines. As shown in Fig. 1, the IFN expression in the Huh7 cells was significantly higher than that in the HepG2 cells. Then the two cell lines were stimulated with exogenous IFN-α and IFN-β and the IFN-α/β mRNA expression in the cells treated with the exogenous IFNs was increased. In the IFN-α treatment, there was a 2.96- (IFN-α mRNA) and 3.13-fold (IFN-β mRNA) increase in the HepG2 cells, and a 6.42- (IFN-α mRNA) and 5.74-fold (IFN-β mRNA) increase in the Huh7 cells. In the IFN-β treatment, there was a 3.03- (IFN-α mRNA) and 3.30-fold (IFN-β mRNA) increase in the HepG2 cells, and a 9.30- (IFN-α mRNA) and 7.69-fold (IFN-β mRNA) increase in the Huh7 cells (P<0.01). The increase was more pronounced in the treated Huh7 cells (Fig. 1C and D). These results confirm that the response to IFN treatment in HCC cell lines is varied, thus implying that certain factors in cells may contribute to this inconsistency.
miR-122 expression decreases in hepatoma cells following stimulation with exogenous IFNs
miR-122 has been closely associated with HCC progression (14). We previously identified that IFN expression was reduced when miR-122 was suppressed in human oligodendrocytes (11). Therefore, in the present study, miR-122 expression in the two cell lines was evaluated subsequent to stimulation with exogenous IFNs. As shown in Fig. 2, the basal miR-122 expression in the Huh7 cells is markedly higher than in the HepG2 cells. This finding is consistent with those of other related studies (12,13). The present study also revealed that exogenous IFN treatment decreased the miR-122 expression in the cell lines. These data suggest that cellular levels of miR-122 may affect the IFN response.
Upregulation of miR-122 increases exogenous IFN-induced type I IFN expression
To confirm that miR-122 was involved in the regulation of the cellular response to IFN treatment, Huh7 cells (transfected with miR-122 plasmid and then incubated with exogenous IFN-α or IFN-β) were processed and analyzed for type I IFN expression by qRT-RCR. First, to get the best transfection efficiency, Huh7 cells were transfected at various time-points and with various doses of miR-122 plasmid or miR-NC plasmid. The results showed that the miR-122 levels in the Huh7 cells at 24 and 48 h post-transfection when using the miR-122-expressing plasmid were significantly higher than that of the control cells and that the highest expression was at 48 h (Fig. 3A). The best transfection dose for the miR-122 plasmid was identified as 1.6 μg (Fig. 3B). Therefore, considering these results, 48 h and 1.6 μg were selected for the transfection of the miR-122 plasmid in the subsequent experimental studies.
Next, the effects of miR-122 on exogenous IFN-induced type I IFN expression in the Huh7 cells were examined. qRT-PCR showed that ectopic expression of miR-122 (Fig. 4A and D) resulted in a significant increase in IFN-α/β mRNA expression following stimulation with IFN-α (Fig. 4B and C) or IFN-β (Fig. 4E and F). These data reveal that miR-122 improved the IFN response mainly by increasing type I IFN expression.
Downregulation of miR-122 decreases exogenous IFN-induced type I IFN expression
To further validate the effect of miR-122 on type I IFN expression induced by exogenous IFNs, the Huh7 cells, which were latter stimulated with 100 U/ml IFN-α or IFN-β, were transfected with AMO-122 to knock down the miR-122 expression. Verification of the best transfection efficiency for AMO-122 was also sought and the results showed that AMO-122 was able to significantly suppress the miR-122 expression in the Huh7 cells at 24 h and 60 pmol (Fig. 3C and D). Even after exogenous IFN stimulation, IFN-α/β mRNA expression remained significantly decreased when miR-122 expression was inhibited by AMO-122 (Fig. 5). These data further confirm that the expression of miR-122 affected the exogenous IFN-induced type I IFN expression and was then involved in the IFN response.
Discussion
Type I IFN has been associated with regulating the immune response to tumorigenesis and has therefore been widely used in the treatment of certain types of tumor (15,16). HCC is one of the most frequently encountered malignant tumors in clinical practice worldwide and is associated with high mortality (1). Previous studies have shown that type I IFN may be a valuable means to inhibit the proliferation of HCC (8,9) and also to prevent its recurrence following surgery (10). By binding to the IFN receptor, type I IFNs activate the JAK/STAT pathway and then amplify IFN production (4). IFN has been used as an significant agent in the treatment of HCC, however, a study has reported that IFN therapy produced a partial response in certain patients with HCC (17). The mechanism behind these differing sensitivities to IFN therapy remains unclear.
In the present study, the Huh7 and HepG2 cells had varying responses to the IFN treatment. Further investigations revealed that the expression levels of cellular miR-122 may explain this inconsistency.
First the exogenous IFN function was characterized in the HCC cell lines of the present study by comparing the HepG2 and Huh7 cells. As shown in Fig. 1, the Huh7 cells expressed a markedly higher level of IFN-α/β mRNA compared with the HepG2 cells (Fig. 1A and B). The two cell lines were then separately treated with 100 U/ml IFN-α or IFN-β. The results showed that the IFN expression was increased in the HepG2 and Huh7 cells, but that the increase was more significant in the Huh7 cells (Fig. 1C and D). These findings suggest that the Huh7 cells had a better response to IFN treatment than the HepG2 cells. Since the exogenous IFN-α/γ proteins were used as IFN stimulators in the experiments, the supernatant was a mixture of exogenous and endogenous IFNs. Therefore, the IFN protein expression was not detected and only the IFN mRNA expression was measured by qRT-PCR assay and used to represent the IFN expression level.
Endogenous type I IFN has been identified as an essential component of the defense mechanisms to restrict tumor growth (6). In vivo studies have also provided valuable insights into the tumor growth-inhibitory property of endogenous type I IFN (5,6). The dysregulation of type I IFN expression may be implicated in tumor growth, underlining the requirement to understand the host factors that contribute to IFN expression.
miRNAs are closely associated with the IFN response. Accumulated data demonstrate that miR-155 is able to enhance the immune response by an induction of IFN (18), while miR-146a (19), miR-148, miR-152 (20), miR-26a, miR-34a, miR-145 and let-7b (21) are able to effectively attenuate the IFN response. We previously identified that IFN expression was reduced when miR-122 was suppressed in human oligodendrocytes (11). Previous studies have also shown miR-122 to be significantly reduced in the HCC cell lines and the clinical HCC samples (22). A study by Coulouarn et al emphasized that miR-122 was a diagnostic and prognostic marker for HCC progression (14). Moreover, Ma et al later observed that the loss of miR-122 resulted in increased cell migration and invasion and that restoration of miR-122 levels reversed this phenotype (23). These observations emphasize the significance of miR-122 in inhibiting HCC progression. Therefore, based on all these findings, the present study sought to investigate the effect of miR-122 on the IFN response in hepatoma cells.
In the present study, cellular miR-122 levels declined with IFN treatment, but only by 10–20% (Fig. 2). This is consistent with a previous study (24). To further understand the interaction between miR-122 and IFN treatment, the effects of miR-122 overexpression and miR-122 knockdown on the exogenous IFN-induced type I IFN expression in Huh7 cells were examined. Another previous study revealed that miR-122 significantly upregulated the apoptosis of the Huh7 cells, while the HepG2 cells were not actively responsive to miR-122 due to its adaptation to the absence of miR-122 or the lack of a miR-122 target (13). The Huh7 cells were therefore used as the main model to show the alteration of type I IFN expression.
Results from the present study indicated that miR-122 overexpression significantly increased the exogenous IFN-induced IFN-α/β mRNA expression (Fig. 4), while miR-122 knockdown resulted in a decreased exogenous IFN-induced IFN-α/β mRNA expression (Fig. 5). These results further confirm that miR-122 has an significant role in exogenous IFNs-induced type I IFN expression in hepatoma cells.
In conclusion, these data suggest that cellular miR-122 levels have an effect on IFN expression and may serve as significant factors in type I IFN therapy against HCC.