Fresh frozen human HCC tissue samples and matched normal hepatocellular tissue samples were obtained from the Fourth Affiliated Hospital of Hebei Medical University. The types of all the tumors were confirmed by pathologic analysis. All the human materials were used in accordance with the policies of the Institutional Review Board of Hebei Medical University.

Seven human HCC cell lines (HepG2, Hep3B, Huh7, C3A, PLC, LO2 and SUN387) were maintained in MEMα or RPMI-1640 medium (Gibco), respectively, and supplemented with 10% fetal bovine serum (FBS), 100 IU/ml of penicillin and 100 μg/ml of streptomycin. Cells were incubated at 37°C in a humidified chamber supplemented with 5% CO₂. Transfection was performed with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol.

To confirm the direct interaction between miR-1246 and NFIB mRNA, HCC cells were simultaneously transfected with miR-1246 or miR-control and the reporter vectors in 48-well plates. The cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.2, 1% Triton X-100, 0.1% SDS) 72 h later, and the proteins were harvested. The intensities of luciferase were detected with a fluorescence spectrophotometer F-4500 (Hitachi, Tokyo, Japan).

Cell proliferation was determined using the MTT cell proliferation kit (Solarbio, Beijing, China). HCC cells (5×10³) were seeded in each 96-well plate and allowed to adhere overnight. The cells were then infected with the adenovirus vector at an MOI of 10 for the indicated times prior to being used for the MTT assay as per the manufacturer’s instructions (Bio-Rad Laboratories Inc., Irvine, CA, USA).

After transfection, cells were counted and seeded in 12-well plates in triplicate at 100 cells/well. Fresh culture medium was replaced every 3 days. The colony was counted only if it contained >50 cells, and the number of colonies was counted from day 6 after seeding. The rate of colony formation was calculated with the equation: Colony formation rate = (number of colonies/number of seeded cells) × 100%.

mRNAs or miRNAs were reverse transcribed to generate cDNA using oligo(dT) primers or stem-loop reverse transcriptase (RT) primers (18), respectively. Then, U6 snRNA (for miRNA) or GAPDH (for mRNA) was considered as the endogenous control. Target genes and controls were treated under the same condition and analyzed by real-time RT-PCR using SYBR Premix Ex Taq™ (Takara, Dalian, China) according to the manufacturer’s protocol. The primers used in the present study are listed in Table I.

Protein extracts were prepared with RIPA lysis buffer in the presence of proteinase inhibitors. Western blot analysis was performed using antibodies against NFIB (1:600; Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:400; Beijing Biosynthesis Biotechnology Co., Beijing, China). The blots were then developed using the enhanced chemiluminescence (ECL) reagent (Amersham Biosciences, USA).

The anti-NFIB antibody (1:500; Abcam) was used for IHC using standard methods.

At 48 h after transfection, the HepG2 cells were detached from the plates by trypsin incubation, rinsed with phosphate-buffered saline (PBS) and fixed in 70% (v/v) ethanol. The cells were then rehydrated in PBS and incubated with RNase (100 μg/ml) and propidium iodide (PI) (60 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA). Cells were analyzed using the FACSCalibur system (BD Biosciences, San Jose, CA, USA).

Statistical analysis utilized the two-tailed Student’s t-test. Statistical significance was set as p<0.05.

We enforced the expression of p53 in HepG2 or Hep-3B cells and found that they both had induced expression of miR-1246 (Fig. 1A and B). In contrast, the knockdown of p53 abrogated the induction of miR-1246 expression in the two cell lines (Fig. 1A and B). In addition, we detected the expression of miR-1246 and p53 in 26 HCC tumor tissues, and found that miR-1246 was consistent with the p53 expression (Fig. 1C). Next, we detected the miR-1246 expression level in 6 HCC cell lines (two of them expressed wild-type p53, one of them expressed null p53, and three of them expressed mutant p53). The finding also suggested that the expression of miR-1246 was consistent with p53 (Fig. 1D).

To further explore the role of miR-1246 in HCC, we transfected either miR-1246 mimics or miR-1246 antisense oligonucleotides (miR-1246 ASO) into HCC cells. As shown in Fig. 1E and F, miR-1246 expression was increased ~8.0-fold in the Hep-3B cells when the miR-1246 mimics were induced, while, miR-1246 ASO resulted in ~74% reduction in miR-1246 levels in the HepG2 cells. Cell viability of HCC cells transfected with miR-1246 or miR-1246 ASO was evaluated by the MTT assay. miR-1246 reduced cell viability at 36 or 48 h after transfection, whereas miR-1246 ASO increased HCC cell viability (Fig. 1G and I). In parallel, we analyzed colony formation and cellular proliferation to assess the effect of miR-1246 on the proliferative capacity of HCC cells. Compared with the control group, transfection with miR-1246 was ~50% less than that of Hep3B cells transfected with the miR-control (Fig. 1H). Conversely, the colony formation rate of the HepG2 cells after transfection with miR-1246 ASO was increased 2.3-fold when compared with the control group (Fig. 1J). These results indicate that miR-1246 inhibits the cell proliferation of HCC cells.

miR-1246 has been demonstrated to play an essential role in the regulation of growth and apoptosis of cells (17–21). To determine the mechanism of miR-1246-mediated cell proliferation and apoptosis regulation in HCC cells, we next identified target genes that could be responsible for the effect of miR-1246. The oncogene NFIB, which was predicted to have two miR-1246 binding sites in its 3′-untranslated region (3′UTR) (Fig. 2A), was chosen for further study. To confirm whether miR-1246 could bind to this predicted region and suppress the protein expression of NFIB, we constructed a luciferase reporter vector in which the 3′UTR fragment of NFIB, including the putative binding site, was inserted downstream of the luciferase coding region. HepG2 cells were transfected with the reporter vector together with either miR-1246 ASO or miR-1246. As shown in Fig. 2B, the intensity of luciferase was higher in the miR-1246-blocked group as compared to the control group, whereas ectopic expression of miR-1246 decreased the intensity of luciferase intensity compared with the control group. In addition, we constructed two other luciferase reporter vectors containing mutations in the miR-1246 binding sites (Fig. 2A). Neither blocking miR-1246 with ASO nor overexpressing miR-1246 had any effect on the intensity of fluorescence from the vector with the mutated miR-1246 binding region (Fig. 2B). These results showed that miR-1246 binds directly to the 3′UTR of NFIB.

To determine whether miR-1246 negatively regulates NFIB expression at the mRNA or protein level, we assessed endogenous NFIB expression in HepG2 or Hep3B cells with altered miR-1246 expression. Both cell lines were transfected with miR-1246 or miR-1246 ASO to overexpress or block miR-1246, respectively. Notably, miR-1246 showed an inability to alter NFIB expression at the mRNA level in both cell groups transfected with miR-1246 or miR-1246 ASO (Fig. 2C). However, miR-1246 downregulated NFIB protein expression in the LO2, HepG2 and C3A cells, while miR-1246 ASO induced increased NFIB protein expression in the Hep-3B, Huh7 and PLC cells (Fig. 2D). These results suggest that miR-1246 regulates endogenous NFIB expression by targeting mRNAs and triggering translation repression.

Sequence-specific small interfering RNAs (siRNAs) can effectively suppress gene expression. An siRNA targeting NFIB (siR-NFIB) was used for the present study. Western blot assay showed that the level of NFIB expression was reduced by ~90% in the HepG2 cells that were transfected with siR-NFIB, as compared to HepG2 cells transfected with a control siRNA (Fig. 3A). Inhibition of NFIB expression inhibited cell growth and increased HepG2 cell apoptosis as compared to the control group (Fig. 3B–D), which was consistent with the effects of miR-1246 overexpression.

To further explore the miR-1246-induced HCC cell proliferation inhibition mediated by NFIB, we transfected HepG2 cells with the miR-control, miR-1246 or miR-1246/NFIB expression plasmid (pcDNA3/NFIB), respectively. Western blotting confirmed that the NFIB expression vector was effective (Fig. 3E). Based on the results, ectopic expression of NFIB reversed the effects of miR-1246 on HCC cell phenotypes (Fig. 3F–J). These results revealed that NFIB was a key mediator in the miR-1246-induced cell growth inhibition in HCC.

To explore the relevance of NFIB in relation to miR-1246 in human HCC cells, the expression of NFIB was evaluated in HCC cells by western blotting using the anti-NFIB antibody. The expression of NFIB was increased in all mutant or null p53-containing HCC cell lines with p53 wild-type cells, which was inversely correlated with miR-1246 (Fig. 4A and B). In addition, IHC staining showed predominantly cytoplasmic localization of NFIB, and the expression of NFIB was markedly increased in all malignant tumor cells from the examined archival HCC samples relative to the adjacent non-tumor tissues (Fig. 4C and D). In total, we found that the expression of miR-1246 could be induced by wild-type p53 in the HCC cell lines. Overexpression of miR-1246 inhibited the proliferation of HCC cells by targeting NFIB. In contrast, the expression of miR-1246 was reduced in the mutant or null p53 HCC cells, and downregulation of miR-1246 resulted in upregulation of NFIB, subsequently promoting HCC cell proliferation (Fig. 4E).