SYBR^®-Green Polymerase Chain Reaction (PCR) Master mix, TRIzol reagent and Lipofectamine™ 2000 were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). ReverTra Ace quantitative PCR (qPCR) RT kit and KOD DNA polymerase were obtained from Toyobo (Shanghai, China). 2X Taq DNA polymerase, TIANgel Midi Purification kit and TIANprep Mini Plasmid kit were obtained from Tiangen Biotech Co., Ltd. (Beijing, China). DNA markers, pEASY-T₁ vector and Escherichia coli DH5α were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). Restriction enzymes and T4 DNA ligase were from Takara Bio, Inc. (Shiga, Japan). RPMI-1640 and fetal calf serum (FCS) were obtained from Gibco-BRL (Carlsbad, CA, USA). Polyclonal rabbit anti-ZHX1 antibody was from Abcam (Cambridge, MA, USA) and the monoclonal mouse anti-β-actin antibody was from Sigma-Aldrich (St. Louis, MO, USA). Cell counting kit-8 (CCK-8) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). The SMMC-7721 liver cancer cell line was obtained from the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Science (Shanghai, China). PCR primers were synthesized by the Shanghai Biosune Biotechnology Co., Ltd., (Shanghai, China).

This study was approved by the Ethics Committee of the Provincial Hospital affiliated to Shandong University for Clinical Investigation. The study included 12 patients with HCC who underwent surgery for radical resection. Patients were excluded if they had received any other therapy prior to the surgery. Specimens of cancer liver tissue were obtained from the patients during surgery, when written consent had been obtained. Specimens of adjacent cirrhotic liver tissues were obtained as controls during the same time period. Tissues were stored at −70ºC for qPCR. For each patient, the diagnosis was confirmed by a paraffin section.

The full length of the human ZHX1 (hZHX1) gene coding sequence (CDS) is 2622 bp (GenBank accession no. NM_001017926.2), which is difficult to obtain from one PCR amplification. To acquire full-length hZHX1 complementary DNA (cDNA), two pairs of primers were designed as follows: P1F 5′-CGCggatccATG GCAAGCAGGCGAAAAT-3′, which contained a BamHI site and P1R, 5′-GGTGGGAATGCTATTAACAGGGATTAAG-3′ for the N-terminal fragment of ZHX1 (ZHX1-N); P2F, 5′-CTTCCTGGATTGGCACAGGTGAT-3′ and P2R, 5′-GCt ctagaCAAAGATTGGGCAAATTCACAG-3′, which contained an XbaI site for the C-terminal fragment of ZHX1 (ZHX1-C). Lowercase letters indicate restrictions sites. ZHX1-N and ZHX1-C overlapped and contained an endogenous EcoRI site (Fig. 1A).

cDNA was synthesized from total RNA, which was isolated from frozen paraneoplastic liver tissues. Using the cDNA as a template, ZHX1-N and ZHX1-C gene fragments were amplified with P1F/P1R and P2F/P2R, respectively. PCR mixtures were prepared in a total volume of 50 μl, including 5.0 μl of 10X buffer, 5.0 μl dNTP mixture (2 mM), 3.0 μl MgSO₄ (25 mM), 1.5 μl of each paired primer (10 μM), 2.0 μl template cDNA, 1.0 μl KOD DNA polymerase (1.0 U/μl) and double distilled water. Parameters for PCR were as follows: 5 min pre-denaturation at 94ºC; 35 cycles of denaturation at 94ºC for 45 sec, annealing at 58ºC for 45 sec and extension at 72ºC for 1 min (ZHX1-N) or 2 min (ZHX1-C), followed by a final 10-min extension at 72ºC. To obtain poly(A) tails, the PCR products were mixed with an equal volume of 2X Taq DNA polymerase in a water bath at 72ºC for 30 min. The PCR products were subjected to agarose gel electrophoresis and purified using the TIANgel Midi Purification kit, according to the manufacturer’s instructions.

ZHX1-N and ZHX1-C were respectively cloned into the pEASY-T1 vector by TA strategy resulting in pEASY-T1-ZHX1-N and pEASY-T1-ZHX1-C. DNA sequencing was used to analyze the two ZHX1 gene fragments.

To obtain the recombinant expression vector containing the full length ZHX1 gene, BamHI/EcoRI fragments of pEASY-T1-ZHX1-N were subcloned into pcDNA3 to produce pcDNA3-ZHX1-N. ZHX1-C was obtained by EcoRI and XbaI digestion with pEASY-T1-ZHX1-C and subcloned into EcoRI/XbaI of pcDNA3-ZHX1-N, yielding the recombinant eukaryotic expression plasmid, pcDNA3-ZHX1.

The construction strategy of recombinant eukaryotic expression plasmid pcDNA3-ZHX1 is presented in a schematic representation (Fig. 1B).

SMMC-7721 cells were cultured in RPMI-1640 medium supplemented with 10% FCS, in an incubator at 37ºC, with 5% CO₂ and saturated humidity. The day prior to transfection, 2.5×10⁵ cells were inoculated in a 24-well plate. When cells reached 70–80% confluency, transfections were performed with pcDNA3 and pcDNA3-ZHX1, respectively, following the Lipofectamine 2000 manufacturer’s instructions. Cells were collected 48 h following transfection.

RT-PCR was used to analyze the ZHX1 mRNA expression in SMMC-7721 cells. Briefly, total RNA was extracted from transfected SMMC-7721 cells using TRIzol reagent, according to the manufacturer’s instructions. cDNA was subsequently synthesized from the total RNA with the ReverTra Ace qPCR RT kit (Toyobo). Primers, designed using the software Primer Premier v5.0 (Premier Biosoft, Palo Alto, CA, USA), were as follows: Sense: 5′-GAAATCAAACCAGACCGTGAAGA-3′ and antisense: 5′-ATGCAGCATTGTAGGTGGGAA-3′ for ZHX1; and sense: 5′-AGTTGCGTTACACCCTTTC-3′ and antisense: 5′-CCTTCACCGTTCCAGTTT-3′ for β-actin. PCR amplifications were performed for 26 (β-actin ) or 30 (ZHX1) cycles of denaturation at 94ºC for 35 sec, annealing at 58ºC for 35 sec and extension at 72ºC for 35 sec, followed by a final 10-min extension at 72ºC. PCR products were separated on 1.5% agarose gel by electrophoresis and visualized by ethidium bromide staining.

To quantify the ZHX1 mRNA expression in HCC patients, qPCR was performed. Total RNA of liver tissue samples from 12 HCC patients was prepared using TRIzol reagent and converted to cDNA with the ReverTra Ace qPCR RT kit. qPCR was performed in a mixture of 20 μl containing10 μl SYBR-Green PCR Master mix, 1.5–3 μl cDNA and 0.4 μl of each paired primer (10 μM). qPCR was performed on the LightCycler 2.0 Instrument (Roche Diagnostics GmbH, Mannheim, Germany). Parameters were as follows: A cycle of 95ºC for 30 sec and 40 consecutive cycles of 5 sec at 95ºC, 5 sec at 58ºC (for ZHX1 and β-actin) and 30 sec at 72ºC. The expression levels of ZHX1 gene in HCC patients were normalized to that of β-actin.

Transfected SMMC-7721 cells were collected and lysed in cell lysis buffer. The protein concentration was measured with the bicinchoninic acid assay. Following boiling for 10 min, cell lyses with an equal quantity of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. ZHX1 was immunoblotted with the rabbit anti-ZHX1 antibody as follows: Membranes were blocked with phosphate-buffered saline buffer containing 3% non-fat milk overnight, followed by incubation with polyclonal rabbit anti-ZHX1 antibody (1:1500) at room temperature for 1 h and subsequently incubated with the secondary horseradish peroxidase-Immunoglobulin G antibody (1:10,000) at room temperature for 1 h. After three washes, the immunoreactive proteins were visualized by enhanced chemiluminescence.

To evaluate the effect of ZHX1 on SMMC-7721 cell proliferation, a CCK-8 assay was employed. SMMC-7721 cells were seeded into 96-well plates with 6,000 cells/well and transfected with pcDNA3-ZHX1 (experimental group) or pcDNA3 (control group). The cell number was quantified daily by CCK-8 according to the manufacturer’s instructions. Cell absorbance was read using a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) with a 450 nm filter and expressed as optical density values.

GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used for data analysis. Two-way analysis of variance was employed to analyze statistical differences between different groups of SMMC-7721 cells at various time points. Wilcoxon matched pairs test was applied to evaluate the differences between cancerous and adjacent cirrhotic liver tissues from patients. P<0.05 or P<0.001 was considered to indicate a statistically significant difference.

Two specific PCR amplifications were performed, one with P1F/P1R and another with P2F/P2R. The PCR products were subsequently analyzed by agarose gel electrophoresis. A band between 750 and 1 kb (ZHX1-N) and another at ~2 kb (ZHX1-C) were separated (Fig. 2A) and were consistent with the expected molecular mass. ZHX-N and ZHX-C were respectively cloned into pEASY-T1 vectors, which were identified by restriction enzymes and DNA sequencing. The identified ZHX1-N and ZHX1-C fragments were subcloned into a pcDNA3 vector to produce pcDNA3-ZHX1. To confirm the construction of pcDNA3-ZHX1, HindIII and BamHI/XbaI restriction enzymes and agarose gel electrophoresis was used. Two DNA bands with 6.2 and 1.9 kb were obtained with HindIII digestion and two DNA bands of 5.4 and 2.7 kb were obtained with BamHI and XbaI digestion (Fig. 2B). DNA sequencing further confirmed the successful construction of pcDNA3-ZHX1 (data not shown).

Forty-eight hours after transfection, SMMC-7721 cells were collected for RT-PCR and western blot analysis. As shown in Fig. 3A and B, SMMC-7721 cells transfected with pcDAN3-ZHX1 showed significantly increased levels of ZHX1 mRNA and protein expression, while cells without transfection or transfected with empty plasmid pcDNA3 showed low-level expression of endogenous ZHX1, indicating that a functional eukaryotic expression plasmid pcDNA3-ZHX1 was obtained.

To investigate the involvement of ZHX1 in HCC, a CCK-8 assay was employed to evaluate cell proliferation. As shown in Fig. 4, SMMC-7721 cells transfected with pcDNA3-ZHX1 exhibited a decreased proliferation rate compared with cells transfected with pcDNA3, indicating that the overexpression of ZHX1 may inhibit the proliferation of SMMC-7721 cells.

qPCR was used to evaluate the differential expression of ZHX1 in adjacent cirrhotic and cancer liver tissue from the same HCC patients. Following normalizing against the house-keeping gene, 7/12 cases showed a 2–5-fold reduction of ZHX1 mRNA compared with the adjacent cirrhotic liver tissues (Fig. 5). A significant difference was confirmed by Wilcoxon matched pairs test. These results demonstrated that reduced ZHX1 expression was widespread among cancer tissues from HCC patients and indicated that ZHX1 may be responsible for hepatocarcinogenesis.