The human ESCC lines KYSE150 and KYSE410 (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum and 1% antibiotic-antimycotic solution (all Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. shRNA sequences were as follows: shRNA-DNMT1 forward, 5-GAT CCC CGC AGG CGG CTC AAA GAT TTGTTC AAG AGA CAA ATC TTT GA GCC GCC TGC TTT TTC-3 and reverse, 5-TCG AGA AAA AGC AGG CGG CTC AAA GAT TTG TCT CTT GAA CAA ATC TTT GAG CCG CCT GCG GG-3 were constructed by Shanghai GenePharma Co., Ltd. (Shanghai, China). The KYSE150 and KYSE410 cell lines were transfected with shRNA-DNMT1 (50 µM) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 72 h. Negative control shRNA (shRNA-NC; 50 µM) was used for comparison.

For in vivo tumor formation, KYSE150 and KYSE410 cells (1.5×10⁷) were isolated using trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA tetrasodium salt; Gibco; Thermo Fisher Scientific, Inc.). Subsequently, 0.3 ml Protanal^®LF 10/60 sodium alginate solution (1.5%; FMC Health and Nutrition, Philadelphia, PA, USA) and 40 µl CaSO4 (21%) were added. Following 72 h posttransfection with shRNA-DNMT1 or shRNA-NC, cell clusters were subcutaneously injected into the dorso-lumbar area of 10 male nude mice (7-week-old; body weight, 20±2 g; Japan SLC, Inc., Hamamatsu, Japan). Subsequently, the 10 mice were divided into two groups (n=5). Food and water were offered ad libitum under a pathogen-free condition at 26–28°C with 12 h dark/light cycles. The animals were sacrificed with an overdose of sodium pentobarbital anesthetic (cat. no. P3761; dosage, 100 mg/kg; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) 14 days following transplantation of cells. Tumors (50–150 mm³) were excised using a scalpel and surgical forceps. Excised tumor samples were froze in liquid nitrogen and stored in a freezer at −80°C for subsequent western blotting analysis and methylation-specific polymerase chain reaction (MSP) analyses. Furthermore, harvested tumors were fixed in paraformaldehyde for subsequently use in immunohistochemistry. These experiments were approved by the Use Committee for Animal Care of the Second Affiliated Hospital of Guilin Medical University (Guilin, China), and conducted according to the Guide for the Care and Use of Laboratory Animals (NIH publication no. 80–23, revised 1996).

Total RNA was isolated from the KYSE150 and KYSE410 cells using a UNIQ-10 column and TRIzol^® Total RNA Isolation kit (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturer's protocol. A 1 µg sample of total RNA was used for reverse transcription in a reaction volume of 20 µl [RNA, 10.0 µl (0.2 µg/µl); 5X RT Buffer, 4.0 µl; Reverse Transcriptase Enzyme mix, 1.0 µl; Primer mix, 1.0 µl; diethyl pyrocarbonate H2O 1.0 µl; total volume, 20 µl] using cloned avian myoblastosis virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 10 min at 50°C; 10 min at 80°C; and then the reactions were cooled to 4°C. Oligo d (T) 20 (#18418012; Invitrogen; Thermo Fisher Scientific, Inc.) were used as the reverse transcription primer. A total of 2 µl cDNA was used for qPCR using an ExTaq RT-PCR version 2.1 kit (Takara Bio, Inc., Otsu, Japan). Gene-specific PCR primers for p16 and GAPDH are listed in Table I, and PCR signals were detected using a DNA Engine Opticon^® 2 Continuous Fluorescence Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Thermocycling conditions for the qPCR analysis were performed as follows: 94°C for 2 min; 94°C for 20 sec; 58°C for 20 sec; followed by 40 cycles of 72°C for 20 sec. At the end of the PCR cycles, melting curve analysis was performed using fluorescent quantitative PCR (Stratagene, Mx3000P; Agilent Technologies, Inc., Santa Clara, CA, USA). Agar gel electrophoresis (2%) was performed to assess the purity of the PCR products. Negative control reactions (lacking template) were routinely included to monitor potential contamination of reagents. Relative amounts of p16 mRNA were normalized to GAPDH mRNA as described by Livak and Schmittgen (15). Experiments were performed in triplicate.

To evaluate the change in target gene expression in vivo and in vitro as a result of DNMT1 silencing, proteins of cell lines and tumor samples from nude mice were extracted using a Total Protein Extraction kit (#AR0103; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Subsequently, protein concentration was determined using a BCA assay kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 20 µg protein lysate was separated using SDS-PAGE on a 10% gel, followed by transfer on to nitrocellulose membranes. Western blot analysis was performed as previously described (16), and the signal was detected using an RapidStep™ ECL detection reagent (EMD Millipore, Billerica, MA, USA) according to the manufacturer's protocol. The primary antibodies used were anti-human p16 (#sc-68393; dilution, 1:4,000), anti-mouse p16 (#sc-68393; dilution, 1:2,000) and anti-GAPDH (#sc-32233; dilution, 1:2,000) (all Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The secondary antibody bovine anti-rabbit immunoglobulin G-horseradish peroxidase was provided by Santa Cruz Biotechnology (#sc-2374; dilution, 1:2,000).

Immunohistochemistry was used to detect the distribution of p16. Esophagus' of nude mice were dissected and fixed overnight in 4% paraformaldehyde using a 0.1 M phosphate buffer solution (pH 7.4) at room temperature. Sections were subsequently embedded in paraffin and sliced into 5-µm-thick serial sections using a microtome (Leica Microsystems, Inc., Buffalo Grove, IL, USA). The sections were deparaffinized by xylene at room temperature followed by washing with deionized water. Subsequently, samples were treated with sodium citrate buffer (pH 6.0; 100°C). Then, slides were rinsed twice for 90 sec each with PBS (pH 7.4) at room temperature followed by blocking with 3% bovine serum albumin (#V900933; Sigma-Aldrich; Merck Millipore) for 30 min. The sections were incubated with the primary anti-mouse p16 antibody (#ab151303; dilution, 1:1,000 dilution; Abcam, Cambridge, UK) at 4°C overnight, followed by washing three times with PBS (pH 7.4). Secondary antibody anti-rabbit immunoglobulin G (#ab98489; dilution, 1:200; Abcam) was added to the sections, which were incubated at 37°C for 50 min. Following washing three times with PBS (pH 7.4), immunoreactivity was visualized with 3,3-diaminobenzidine (#D8001; Merck KGaA, Darmstadt, Germany). Three images of the identified adjacent areas were captured (magnification, ×400).

The methylation status of the p16 (NCBI reference sequence of p16, NG_1029) promoter region was determined using MSP. Primers were designed using the MethPrimer design tool (www.urogene.org/methprimer). Primers illustrated in Table II, which distinguished between unmethylated (U) and methylated (M) alleles, were designed to amplify the specific sequence. Each PCR sample contained 20 ng of sodium bisulfite-modified DNA, 250 pmol of each primer, 250 pmol of deoxynucleoside triphosphate, 1X PCR buffer (#EM101-01; Tiangen Biotech Co., Ltd., Beijing, China) and 1 U of ExTaq Hot Start polymerase (Takara Bio, Inc.) in a final reaction volume of 20 µl. Thermocycling conditions were as follows: Initial denaturation at 95°C for 3 min; 40 cycles of 94°C for 30 sec, 65°C (M) or 63°C (U) for 30 sec and 72°C for 30 sec. For each set of MSPs, H2O was used as a control. PCR products were separated on 4% agarose gels, stained with ethidium bromide and visualized under UV illumination. For cases with borderline results, PCR analyses were repeated using three independent experiments.

Values are expressed as the mean ± standard deviation. Statistical significance was assessed using Student's t-test and one-way ANOVA followed by a Tukey's post hoc test using SPSS software (version 19.0; IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to demonstrate the effect of silencing DNMT1 on the expression of the tumor suppressor gene p16 in ESCC cells, the p16 mRNA level was evaluated using RT-qPCR. RNA interference (RNAi) of DNMT1 was conducted successfully with significantly decreased DNMT1 mRNA expression in KYSE150 (P=0.0014) and KYSE410 (P=0.001) cell lines following shRNA-DNMT1 transfection compared with the negative control (paired Student's t-test; Fig. 1A). Furthermore, the mRNA level of p16 was detected in the KYSE150 and KYSE410 cell lines following RNAi. p16 mRNA was significantly increased compared with the control group (KYSE150, P<0.0001; KYSE410, P=0.001; paired Student's t-test; Fig. 1B).

Western blot analysis was used to investigate the effect of DNMT1 silencing on p16 protein expression in vitro and in vivo. The p16 protein level in the KYSE150 and KYSE410 cell lines (Fig. 2A) and in tumor tissues from nude mice transplanted with modified KYSE150 and KYSE410 cell lines (Fig. 2B) were increased compared with the control group. These results are consistent with the results of the p16 RT-qPCR (Fig. 1B). These data suggest that silencing DNMT1 upregulates expression of the tumor suppressor gene p16.

Using immunohistochemistry, it was identified that p16 expression was increased in tumor tissues from nude mice transplanted with the shRNA-DNMT1-treated KYSE150 and KYSE410 cells respectively (Fig. 3) compared with the group transfected with the shRNA-NC.

In order to demonstrate the underlying molecular mechanisms of DNMT knockdown-induced p16 upregulation in ESCC cells, the methylation status of p16 was investigated using MSP assays. It was demonstrated that unmethylated p16 levels were increased in the KYSE150 and KYSE410 cell lines following shRNA-DNMT1 transfection compared with their corresponding controls, and no methylated p16 was revealed when RNAi of DNMT1 was performed (Fig. 4A). These results were consistent with results obtained from tumor tissues. Methylation of p16 was inhibited in tumors isolated from nude mice injected with the DNMT-knockdown KYSE150 and KYSE410 cells compared with their corresponding controls (Fig. 4B). These results suggest that silencing DNMT1 downregulates the methylation status of the tumor suppressor gene p16.