Sixty cases of HCC tissue excised in the surgery of First Hospital of Jilin University from 2009 to 2010 were selected for the experiment which had all been pathologically confirmed to be HCC and the patients had not received any treatment prior to the surgery. At the same time, 20 cases of normal liver tissues from liver trauma and next to the hepatic hemangioma were selected for comparative study. All the specimens have been discussed and approved by the ethics committee of The First Hospital of Jilin University.

Human liver cancer HepG2 cells and LO2 cells were purchased from ATCC (ATCC, USA), BEL7402 cells were a gift from Laboratory of Molecular Biology of Chinese Academy of Sciences (Shanghai, China). Lipofectamine 2000 was purchased from Invitrogen, USA, siRNA was synthesized by GenePharma (Shanghai, China), and RT-PCR kit [Takara RNA PCR kit (AMV) version 3.0] was purchased from Takara Biotechnology Co., Ltd. (Japan). PI was purchased from Sigma (USA), MOR phosphorylated MKK7, MKK7, phosphorylated JNK and JNK antibody were all purchased from Cell Signaling Technology (USA).

Cells after cell passage was inoculated in the DMEM culture medium (Gibco-BRL, NY, USA) containing 10% fetal calf serum (Hyclone Laboratories, UT, USA), 100 U/ml penicillin and 100 U/ml streptomycin. Then it was cultured in the incubator containing 5% CO₂ and 95% oxygen at 37°C.

Cells of various experimental groups in logarithmic growth phase were taken and 6 duplicate wells were set for each group with a negative control group. All the cells were put into 5% CO₂ incubator for further culture prior to color reaction. Each well was added with 20 μl of MTT (5 mg/ml) and cultured in CO₂ incubator for 4 h before the culture solution was disposed of. DMSO (200 μl) was added to each well for room temperature oscillation for 10 min and the OD values were measured with microplate reader at a wavelength of 490 nm.

Paraffin specimen section was conventionally dewaxed to water and dehydrated with gradient alcohol, for antigen retrieval, incubated for 10 min in 3% H₂O₂ solution at room temperature. Drowise 5 μl of 10% goat serum was added and then sealed for 10 min at room temperature. Rabbit anti-human (50 μl) MOR monoclonal antibody (1:50) was added and placed at 4°C overnight. Biotin labeled goat anti-human antibody (50 μl) was added and incubated for 30 min at room temperature. DAB stain with haematin was applied for 2 min with hydrochloric alcohol differentiation. Dehydration was performed with gradient alcohol. Evaluation was performed by microscopy and images were taken.

The cells were inoculated in the 6-well culture plate at the density of 5×10⁵/ml. Cells were cultured in medium free of antibiotics to >60% confluence. The culture medium free of serum and antibiotics was replaced. Purified cells were transfected for 24 h with siRNAs to μ-opioid receptor (MOR siRNAs; Santa Cruz Biotechnology, CA) at a concentration of 20 nM. Transfected cells were placed into the CO₂ incubator to cultured for 4 h, then the culture medium with serum was used to continue the culture. Non-specific Control siRNA was used as the control group. siRNA interfering efficiency was tested with RT-PCR and western blotting.

Instructions of the RNAiso^™ Plus (Takara, Japan) for total RNA extraction were followed, employing the RT-PCR reagent (Takara) for RT-PCR reaction with the steps as shown in the instructions. MOP forward primer: 5′-TCTGGCTCCAAAGAAAAGGA-3′, reverse primer: 5′-CAATGCAGAAGTGCCAAGAA-3′. MKK7 forward primer: 5′-GCCAGACTGGGAAGAAATCTG-3′, reverse primer: 5′-GGCGGACACACACTCATAAA ACAGA-3′. β-actin forward primer: 5′-CTGGGACGACATGGAGA AAA-3′, reverse primer: 5′-AAGGAAGGCTGGAAGAG TGC-3′. PCR reaction system 50 μl at the following reaction conditions: 94°C 2 min, 94°C degeneration 30 sec, 58°C anneal 30 sec, 72°C extension 30 sec, total 31 cycles. The PCR product was electrophoresed with 1.0% agarose gel, then scanned and analyzed with gel imaging system.

Trypsin was digested to collect the cells, and the cells were washed twice with pre-cooled PBS, and single cell suspension were prepared. Annexin V and PI staining fluid were added, respectively, in accordance with the steps of the instructions for apoptosis reagent kit (Annexin V-FITC kit, Biosea Biotechnology Co., Beijing, China) for 15 min at room temperature before the analysis with flow cytometry for apoptosis.

Trypsin (0.25%) was digested to collect the cells and washed with PBS 2 times, then fixed at 4°C with 70% cold ethanol overnight. The ethanol was discarded the following day, and 2 more washes with PBS followed. PI comprehensive dye medium (1 ml) (with 10 μg of RNase, and 5 μl of Triton X-100) was added, then stored in a dark for 30 min at 4°C before analysis with flow cytometry (Becton-Dickinson, USA).

Total protein was separated with SDS-PAGE. Proteins were placed to PVDF membranes, with the semidry method, and seal with 5% skim milk powder at 4°C overnight. The membrane was washed with TBST and the first antibody added at 37°C for hybridization for 1 h. The second antibody was then applied at 37°C for hybridization for 1 h, before the use of TBST and color reaction for 5 min with autoradiography. Quantity One for optical density value analysis and measurement was used. The results are shown as specimen optical density value/β-actin optical density.

Ten male nude mice, aged 6–8 weeks and weighing ~20 g were used. The anumals were cared for by the Animal Experimental Center of Jilin University (Changchun, China). The animal experimental plan was approved by the medical ethics committee of Jilin University. The mice were randomly divided into two groups of 5 mice each. The animals were vaccinated with the cells of control group and MOR siRNA group, respectively. Each mouse was injected with 1×10⁵ cells. The growth of the nude mice were observed every day, and after four weeks the mice were sacrificed. The subcutaneous transplanted tumor tissue was cut, under aseptic condition, for index analysis.

SPSS 17.0 statistical software was used for the statistical analysis. The values are shown as the mean ± SD. The statistical analysis was performed using the Student’s t-test, and the differences between the groups were considered to be statistically significant at p<0.05.

RT-PCR and western blotting were employed by us to analyze the expression of MOR mRNA and protein in human hepatoma carcinoma cells. It was found that there was high expression of MOR mRNA in HepG2 cells and BEL7402 cells (Fig. 1A). The expression of MOR protein in HepG2 cells and BEL7402 cells was similar to that of mRNA (Fig. 1B). Moreover, it was found by the immunohistochemical analysis that MOR expression in human hepatoma carcinoma tissues was obviously higher than that in normal human liver tissues (Fig. 1C).

We used MOR siRNA and negative control oligonucleotide to transfect HepG2 cells and observed the MOR mRNA and protein expression of transfections under various conditions. We found that 45 nM of MOR siRNA transfection for 24 h could significantly reduce the expression level of MOR mRNA, and the expression level of MOR protein declined accordingly. This efficiency can endure at least for 72 h (Fig. 2A and B). This indicates that MOR siRNA transfection could effectively silence the MOR gene and affect the expression of MOR expression in turn.

In order to verify the impact of downregulating MOR on human hepatoma carcinoma cell growth, Naloxone of various concentrations (0, 0.1, 1.0, 10, 100 μM) and MOR siRNA of various concentrations (0, 25, 35, 45, 55 nM) were administered to treat human hepatoma carcinoma HepG2 cells for 24, 48 and 72 h before MTT analysis on cell viability (Fig. 3A and B). As indicated in the results, with the increase of the concentrations of Naloxone and MOR siRNA, A490 value of HepG2 cells reduced. Such a result means that downregulating MOR can inhibit the growth of human hepatoma carcinoma cells.

In order to explore whether downregulating MOR results in apoptosis of human hepatoma carcinoma cells, we used Hoechst 33342 stain and flow cytometry to observe apoptosis of human hepatoma carcinoma cells. The results indicated that after the administration of Naloxone and MOR siRNA to treat human liver cancer HepG2 cells, the cell’s chromatin concentrated with the brightness obviously higher than the normal control group (Fig. 4A). The apoptosis rate was 18.36 and 22.99%, respectively, which are both higher than the 6.6% of the control group (Fig. 4B and C). This indicates that downregulation of MOR can promote apoptosis of human hepatoma carcinoma cell to inhibit the growth of human hepatoma carcinoma cells in turn.

In order to study whether downregulating MOR has any impact on the progress of the cycle of human hepatoma carcinoma cells, we used flow cytometry to analyze the cell cycle. The results indicated that after HepG2 cells were treated with the administration of Naloxone and MOR siRNA, the number of cells remaining in the G0/G1 stage increased significantly and was higher than that of the control group (Fig. 4D). Therefore, downregulation of MOR functions inhibiting the progress of the human hepatoma carcinoma cell cycle, so as to inhibit cell growth.

We used the nude mouse model to establish the in vitro tumors. After 4 weeks, the five nude mice of the normal control group all had tumors with the tumor formation rate of 100%. Four nude mice in the MOR siRNA group had tumors with the tumor formation rate of 80%. In addition, the tumors of the nude mice in the control group grew faster than those in the MOR siRNA group (Fig. 4E and F). These results evidenced that downregulation of MOR can distinctly inhibit the tumor progress in vivo.

In order to verify the molecular mechanism for downregulating MOR to inhibit the growth of human hepatoma carcinoma cells, we used RT-PCR and western blotting, respectively, to test the MKK7 mRNA and MKK7 proteins and their phosphorylation levels. We found that after MOR was downregulated, the total protein expression level of MKK7 mRNA and MKK7 was not obviously changed, while the MKK7 phosphorylation level was significantly increased and was higher than that of the control group. Moreover, we found JNK and its phosphorylation level also increased drastically (Fig. 5A–C). These results show that downregulating MOR can increase the expression level of phosphorylation MKK7 (but not for all MKK7), which results in JNK and its phosphorylation level. This may be its molecular mechanism to inhibit the growth of hepatoma carcinoma cells.

In order to demonstrate whether MKK7 plays a key role in the inhibition of the growth of human hepatoma carcinoma cells via downregulation of MOR, we used RNA interference to silence MKK7 expression and observed the growth of human hepatoma carcinoma cell. The results showed that after MKK7 was silenced, A490-nm value of the hepatoma carcinoma cells was apparently on the increase as compared to that in the group with downregulating MOR. At the same time, JNK and its phosphorylation level dropped significantly (Fig. 6A–C). Our data indicated that the inhibition of the growth of human hepatoma carcinoma cells via downregulation of MOR may be through the JNK pathway mediated by the MKK7 pathway.