The primary HDEC and CRL-2586 EOMA cells used in these experiments were from the Institute of Biochemistry and Cell Biology (Shanghai, China). The lentivirus vector Lv-siIGF2R, negative control vector (scramble-siRNA) and virion-packaging elements were from Genechem (Shanghai, China). The IGF2R primer was synthesized by ABI (Framingham, MA, USA) and all antibodies were from Santa Cruz Biotechnology (Dallas, TX, USA).

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Thermo Fisher Scientific Inc. (Waltham, MA, USA); TRIzol reagent and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA, USA); M-MLV reverse transcriptase was from Promega (Madison, WI, USA); SYBR Green Master Mixture was from Takara (Otsu, Japan). ECL-Plus/kit was from GE Healthcare (Piscataway, NJ, USA). Cell cycle analysis kit and apoptosis kit [propidium iodide (PI), RNase A, Annexin V-FITC] were from KeyGen Biology (Nanjing, China).

Twenty-seven freshly resected human HAs and 18 cases of adjacent normal skin tissue samples were collected at the Department of General Surgery, and were classified according to Mulliken criteria. Tissues and clinical information were obtained as part of an approved study at Shanghai Jiao Tong University School of Medicine. There were 15 cases of proliferating phase HAs and 12 cases of involuting phase HAs. A portion of each tissue sample was fixed with 10% formalin for histopathological and IHC examination. All HA tissues were diagnosed by two independent pathologists.

IHC staining for IGF-II (rabbit polyclonal, ab9572, Abcam, MA, USA), IGF2R (rabbit polyclonal A0545, Sigma, USA) and PCNA (mouse monoclonal PC10, Sigma) using polyclonal and monoclonal antibodies was performed. Unstained sections were deparaffinized and incubated overnight at 4°C with primary antibodies against the proteins, then with biotinylated secondary antibody (1:200) at room temperature for 1 h, followed by incubation with ABC peroxidase and 3,3′ diaminobenzidine (DAB; 30 mg dissolved in 100 ml Tris-buffer containing 0.03% H₂O₂). Sections were counterstained with hematoxylin. Digital images were acquired and the integrated optical density (IOD) and positive expression rate (cell positive area/cell total area) of semi-quantitative analysis was performed.

The proliferating phase HDEC and CRL-2586 EOMA cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 100 U/ml of penicillin and 100 μg/ml of streptomycin. They were all placed in a humidified atmosphere containing 5% CO₂ at 37°C. Lv-siIGF2R and negative control viruses were transfected into proliferating phase HDEC and CRL-2586 EOMA cells. Cells were subcultured at a 1:5 dilution in 300 μg/ml G418-containing medium. On the day of transduction, HA cells were replated at 5×10⁴ cells/well in 24-well plates containing serum-free growth medium with polybrene (5 mg/ml). When the cells reached 50% confluence, they were transfected with recombinant experimental virus or control virus at the optimal MOI (multiplicity of infection) of 50, and cultured at 37°C and 5% CO₂ for 4 h. Then supernatant was discarded and serum containing growth medium was added. At 4 days of post-transduction, transduction efficiency was measured by the frequency of green fluorescent protein (GFP)-positive cells. Positive stable transfectants were selected and expanded for further study. The clones transfected with the Lv-siIGF2R vectors were named as the Lv-siIGF2R group, and the clones transfected with the negative control vectors was named as the NC group.

To quantitatively determine the mRNA expression level of IGFII and IGF2R in proliferating phase HA tissues and cells, Real-time PCR was used. Total RNA of each clone was extracted with TRIzol according to the manufacturer’s protocol. Reverse-transcription was carried out using M-MLV and cDNA amplification was carried out using SYBR Green Master Mix kit according to the manufacturer’s protocol. The IGF2R gene was amplified using specific oligonucleotide primer and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control. The PCR primer sequences were listed as follows: IGFII, 5′-AGAGAGGCCAAACGTCATCGT-3′ and 5′-TCACC CCACCTTGCAGAATTA-3′; IGF2R, 5′-GAAAACACAAG AATGAAGGTCAA-3′ and 5′-CCACCAGCTCTCCTCCAC ATA-3′; GAPDH, 5′-CAACGAATTTGGCTACAGCA-3′ and 5′-AGGGGTCTACATGGCAACTG-3′. Data were analyzed using the comparative Ct method (2^−ΔΔCt). Three separate experiments were performed for each clone.

The proliferating phase HDEC and CRL-2586 EOMA cells were harvested and extracted using lysis buffer (Tris-HCl, SDS, mercaptoethanol, glycerol). Cell extracts were boiled for 5 min in loading buffer and then an equal amount of cell extracts from each sample was separated on 15% SDS-PAGE gels. Separated protein bands were transferred onto polyvinylidene fluoride (PVDF) membranes and the membranes were blocked in 5% skim-milk powder dissolved in PBST. The primary antibodies against IGF-II (rabbit polyclonal ab9572, Abcam), IGF2R (rabbit polyclonal A0545, Sigma), PCNA (mouse monoclonal PC10, Sigma), Ki-67 (rabbit polyclonal ab888, Abcam), Bcl-2 (rabbit monoclonal SP66, Sigma), Bax (rabbit monoclonal ab32503, Abcam) and Cyclin D1 (rabbit monoclonal sc-718, Santa Cruz, USA) were diluted according to the manufacturer’s instructions of antibodies and incubated overnight at 4°C. Then, horseradish peroxidase-linked secondary antibodies were added at a dilution ratio of 1:1,000, and incubated at room temperature for 2 h. The membranes were washed three times with PBS for three times and the immunoreactive bands were visualized using ECL-Plus/kit according to the manufacturer’s instructions. The relative protein level in different cell lines was normalized to the endogenous level of β-actin protein. Three separate experiments were performed for each clone.

Cell proliferation was analyzed with the MTT assay. Briefly, cells infected with Lv-siIGF2R were incubated in 96-well-plates (8 wells each group) at a density of 1×10⁵ cells per well with DEME medium supplemented with 10% FBS. Cells were treated with 20 μl MTT dye at 0, 24, 48, and 72 h and then incubated with 150 μl of DMSO for 5 min. The color reaction was measured at 570 nm with enzyme immunoassay analyzer (Bio-Rad, USA). The proliferation activity was calculated for each clone.

To detect cell apoptosis, the proliferating phase HDEC and CRL-2586 EOMA cells were trypsinized, washed with cold PBS and resuspended in binding buffer according to the instruction of the apoptosis kit. FITC-Annexin V and PI were added to the fixed cells for 20 min in darkness at room temperature. Then, Annexin V binding buffer was added to the mixture before the fluorescence was measured on FACsort flow cytometer (Becton-Dickinson, Mountain View, CA, USA). The cell apoptosis was analyzed using CellQuest software (Becton-Dickinson). Cell apoptotic rate = the number of cell apoptosis/the number of apoptotic cells and the normal cells. Three separate experiments were performed for each clone.

To detect cell cycle variation, the proliferating phase HDEC and CRL-2586 EOMA cells were trypsinized, washed with PBS and fixed with 80% cold ethanol overnight at −20°C. After another wash with PBS, the fixed cells were stained with PI in the presence of RNase A for 30 min at room temperature in darkness. Each sample was filtered through a 50-μm nylon filter to obtain single-cell suspension. The samples were then analyzed on FACsort flow cytometer (Becton-Dickinson). ModFit3.0 software (Verity Software House, Topsham, ME, USA) was used for cell cycle analysis. Three separate experiments were performed for each clone.

Six-week-old female immune-deficient nude mice (BALB/c-nu) were bred at the laboratory animal facility (Institute of Chinese Academy of Sciences, Shanghai, China), and were housed individually in microisolator ventilated cages with free access to water and food. All experimental procedures were performed according to the regulations and internal biosafety and bioethics guidelines of Shanghai Jiaotong University and the Shanghai Municipal Science and Technology Commission.

Four mice were injected subcutaneously with 1×10⁷ HDEC cells in 50 μl of PBS pre-mixed with an equal volume of Matrigel matrix (Becton-Dickinson). Mice were monitored daily, and three out of four mice developed a subcutaneous tumor. When the tumor size reached ~5 mm in length, it was surgically removed, cut into 1–2 mm³ pieces, and re-seeded individually into other mice on the right flanks. When tumor size reached ~5 mm in length, the mice were randomly assigned as PBS group, negative control (NC) group and Lv-siIGF2R-treated group. In treatment group, 15 μl of Lv-siIGF2R was injected into subcutaneous tumors using a multi-site injection format (at least three sites). Mice in the PBS (HDEC) group and the NC group received 15 μl of PBS with or without negative control lentivirus, respectively. Injections were repeated every other day after initial treatment. The tumor volume was measured every three days with a caliper, using the formula volume = (length × width)²/2.

SPSS 20.0 was used for the statistical analysis. One-way analysis of variance (ANOVA) was used to analyze the differences between groups. The LSD method of multiple comparisons was used when the probability for ANOVA was statistically significant. Statistical significance was P<0.05. In the figures, the error bars depict standard deviation.

The protein expression of IGF-II, IGF2R and PCNA was evaluated using IHC analysis. As shown in Fig. 1A, the positive expression of IGF-II, IGF2R and PCNA was found increased in proliferating phase HAs, but decreased in involuting phase HAs. From the cell localization, the positive staining of IGF-II was mainly in the cytoplasm, that of IGF2R was in the membrane and cytoplasm, but PCNA was in the cellular nucleus in HA and normal tissue cells. The average IOD and positive rates of IGF-II, IGF2R and PCNA were significantly higher in proliferating phase HAs than those in involuting phase HAs or in normal tissues (Table I, each P<0.01). Spearman correlation analysis showed the positive correlation between the IGF-II, IGF2R and PCNA expression in HA tissues of different phases (r=0.625, P=0.015). Then, we examined the mRNA and protein expression levels of IGF-II and IGF2R in HA tissues by real-time PCR (Fig. 1B and C) and western blot assays (Fig. 1D and E), and further found that the expression of IGF-II and IGF2R was increased in proliferating phase HAs, but decreased in involuting phase HA.

To examine the Lv-siIGF2R knockdown efficiency for IGF2R expression in proliferative phase HDEC and CRL-2586 EOMA cells, we investigated the expression levels of IGF2R in Lv-siIGF2R-transfected HA cells using real-time PCR and western blot assays. As shown in Fig. 2, the knockdown efficiency for IGF2R expression was found to be significantly increased in Lv-siIGF2R-transfected HDEC (Fig. 2A and C) and CRL-2586 EOMA cells (Fig. 2B and D), respectively reaching >80 and 70% (each ^**P<0.01).

To investigate the effect of Lv-siIGF2R on cell proliferation in HA cells (HDEC and CRL-2586 EOMA), we examined cell proliferative activities using MTT assay. We found that Lv-siIGF2R could significantly diminish the proliferative activity of HA cells in a time-dependent manner compared with the NC group (Fig. 3A and B). In addition, to determine whether IGF2R knockdown suppressed the endogenous expression of Ki-67 and PCNA, we examined the protein expression of Ki-67 and PCNA by western blot assay. The amount of Ki-67 and PCNA proteins was significantly decreased in the Lv-siIGF2R group compared with the NC group (each ^**P<0.01) (Fig. 3C and D), suggesting that knockdown of IGF2R might inhibit HA cell proliferation through downregulation of Ki-67 and PCNA expression.

To determine whether IGF2R knockdown affected the apoptosis in HA cells (HDEC and CRL-2586 EOMA), flow cytometric analysis was performed. Cell apoptotic rates were markedly increased in Lv-siIGF2R group compared with the NC group (each ^**P<0.01) (Fig. 4A and B). To determine whether IGF2R knockdown influences the expression of Bax and Bcl-2, we examined the expression of Bax and Bcl-2 in HA cells by western blot assay. It was found that the amount of Bax was markedly increased, while that of Bcl-2 protein was decreased in the Lv-siIGF2R group compared with the NC group (each ^**P<0.01) (Fig. 4C and D), suggesting that knockdown of IGF2R might induce HA cell apoptosis through regulation of the expression of Bax and Bcl-2.

To determine whether IGF2R knockdown influenced cycle distribution in HA cells (HDEC and CRL-2586 EOMA), flow cytometry analysis was performed. We showed that the G₀/G₁ phase fraction was increased, while the S and G₂/M phase fractions were decreased, and more HA cells were arrested in the G₀/G₁ phase in the Lv-siIGF2R group compared with the NC group (Fig. 5A and B). In order to determine whether IGF2R knockdown suppressed the expression of Cyclin D1 and E, we examined the expression of Cyclin D1 and E by western blot assay. The amount of Cyclin D1 and E protein was markedly decreased in the Lv-siIGF2R group compared with the NC group (each ^**P<0.01) (Fig. 5C and D), suggesting that knockdown of IGF2R might induce cycle arrest in HA cells through downregulation of Cyclin D1 and E expression.

Many studies have demonstrated that the PI3K/AKT pathway is involved in regulating HA formation (20,21). Whether Lv-siIGF2R affects PI3K/AKT signaling transduction of PI3K/AKT needed to be explored. Western blot assays were performed to assess the expression of P-PI3K, P-AKT, PI3K and AKT in Lv-siIGF2R-transfected HA cells. We found that the activity of P-PI3K, P-AKT, PI3K and AKT proteins was reduced in the Lv-siIGF2R group compared with the NC group (each ^**P<0.01) (Fig. 6), suggesting that knockdown of IGF2R might block the PI3K/AKT signaling transduction in HA cells.

Our in vitro experiments demonstrated that knockdown of IGF2R could efficiently inhibit proliferation and induce apoptosis in HA cells. Therefore, we further investigated the antitumor effect of Lv-siIGF2R in vivo using the HDEC xenograft model and lentivirus-mediated gene therapy. The mean volume of tumors in all experimental mice before treatment was 78.30±8.20 mm³. During the first 3 days of recovery, the tumors in each group grew slowly, with no obvious difference in tumor size. However, from the 3rd day after the second treatment and beyond, the tumors in PBS and NC groups grew rapidly until the end of the observation period on the 15th day. Conversely, the tumors treated with Lv-siIGF2R maintained a slower growth rate (Fig. 7A and B), and there was a significant difference in tumor volume between Lv-siIGF2R group and PBS and NC groups over the two-week observation period (Fig. 6, P<0.01). After recovery of two weeks, the tumor samples were removed for western blot analysis of the protein expression of IGF2R, PCNA, Bcl-2 and Cyclin D1. It was found that the protein expression levels of IGF2R, PCNA, Bcl-2 and Cyclin D1 were remarkably downregulated in Lv-siIGF2R group compared with those in PBS and NC groups (Fig. 7C).