HTM and GTM cells were kindly provided by Yishui Central Hospital (Shandong, China). HTM and GTM cells were grown in an adherent cell culture with Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C, 5% CO₂ and 100% humidity in a controlled incubator. Cells were subcultured in a humidified 5% CO₂ incubator at 37°C when they reached a confluence of 70–90%, then washed twice with D-Hanks solution.

Plasmids expressing siRNA targeting Nrf2 (siNrf2), overexpressing Nrf2 and control siRNA (non-silencing) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Cells were seeded in 6-well plates and cultured in DMEM overnight at 37°C in a humidified atmosphere of 95% air and 5% CO_2. Subsequently, cell transfections were conducted using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Total mRNA was isolated from the transfected cell groups and untransfected cells, as previously described (30). Complementary DNA (cDNA) was produced by reverse transcription using an iScript™ cDNA Synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's instructions. Levels of mRNA expression were measured by SYBR-Green-based qPCR using a SYBR^® Green Master mix (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The primer sequences used were as follows: Nrf2, forward 5′-ATGGATTTGATTGACATACTTT-3′ and reverse 5′-ACTGAGCCTGATTAGTAGCAAT-3′; and GAPDH, forward 5′-TCCTGCACCACCAACTGCTTAG-3′ and reverse 5′-ATGGGCAGTGATGGCATGGACT-3′. qPCR conditions were as follows: Initial denaturation was performed at 95°C for 15 sec, followed by 30 cycles of 95°C for 30 sec, 61°C for 5 sec, 72°C for 15 sec, and a final extension at 72°C for 10 min. GAPDH gene expression was used as a reference. mRNA expression levels were quantified using the 2⁻ΔΔ^Cq method (31).

The transfected HTM and GTM cells during the logarithmic growth phase were cultured in 96-well plates with DMEM (5×10⁴ cells/ml) at 37°C. Four repeat cultures were completed for each cell transfection group. Following culture for 24, 48, 72 and 96 h, 20 µl fresh DMEM supplemented with 0.5 mg/ml MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to each well and incubated for 4 h at 37°C. A total of 200 µl dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) was added to each well. Cells incubated with just DMSO were used as controls. Optical densities of each well were subsequently measured at an absorbance of 492 nm.

Cells transfected with siNrf2 for 28 h were seeded in a 6-well plate at a density of 2×10⁵ cells/well in DMEM. Cell apoptosis was assayed using the FITC Annexin V Apoptosis Detection kit (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), according to the manufacturer's instructions. Stained cells were analyzed with a fluorescence-activated cell sorting Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). The data were analyzed using FlowJo v. 9.0 software (Tree Star, Inc., Ashland, OR, USA). The percentage of total apoptotic events was defined as the sum of cells in the early (Annexin V positive/PI negative) and late (Annexin V positive/PI positive) stages of apoptosis, as described previously (32).

Protein was extracted from cells transfected with Nrf2, siNrf2 or control using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein samples (30 µg/lane) were separated by 10–12% SDS-PAGE, blotted onto polyvinylidene difluoride membranes, blocked in 5% fresh non-fat milk in phosphate-buffered saline (PBS)-Triton X-100 (0.1% Triton in PBS) for 1 h at room temperature. Subsequently, the membranes were incubated with primary antibodies: Nrf2 (ab31163), BCL2-Associated X (Bax; ab32503), B-cell lymphoma (BCL)-2 (ab32124), p53 (ab1101), phospho (p)-p53 (ab1431) and GAPDH (ab8245; all Abcam, Cambridge, USA; all 1:1,000) overnight at 4°C. Thereafter, membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (ab6721; ab6788; 1:5,000; Abcam) for 1 h at room temperature. Immunoreactive protein bands were developed by enhanced chemiluminescence western blotting substrate (Pierce; Thermo Fisher Scientific, Inc.) and analyzed using Image Gauge v. 4.0 software (FujiFilm Science Lab, Tokyo, Japan).

All experiments were repeated three times. Results are presented as the mean ± standard deviation. Statistical analyses were performed using SPSS 19.0 software (IBM SPSS, Armonk, NY, USA). P-values were calculated using one-way analysis of variance and P<0.05 was considered to indicate a statistically significant result.

Western blotting and RT-qPCR were performed to detect Nrf2 expression. Results demonstrated that the expression of Nrf2 was significantly upregulated in HTM cells compared with GTM cells (P<0.05; Fig. 1). This was demonstrated to be significant at the mRNA level by subsequent analysis of HTM and GTM cells transfected with siNrf2 to downregulate Nrf2 expression. As depicted in Fig. 2, the mRNA expression levels of Nrf2 were effectively regulated by siNrf2, with significant decreases in Nrf2 observed in siNrf2 transfectants compared with the control (P<0.05). In addition, the mRNA expression levels of Nrf2 overexpression plasmid together with siNrf2 were significantly upregulated in HTM and GTM cells compared with cells transfected with siNrf2 alone (P<0.01).

To determine the effect of Nrf2 expression on GTM and HTM cell viability, cells were transfected with Nrf2 overexpression plasmid alone and Nrf2 overexpression together with siNrf2. As demonstrated in Fig. 3A, regulation of Nrf2 expression significantly enhanced the viability of GTM and HTM cells. In HTM cells, overexpression Nrf2 significantly improved cell viability (P<0.05), while Nrf2 overexpression together with siNrf2 demonstrated no significant change compared with the respective non-transfected control. In GTM cells, overexpression of Nrf2 and Nrf2 overexpression together with siNrf2 significantly promoted cell viability (P<0.05; Fig. 3B). These results indicated that the greatest increases in GTM and HTM cell viability were observed in Nrf2 overexpression plasmid alone. Interestingly, the viability of untransfected GTM cells decreased after 48 h, while all other cell groups remained in a proliferative phase throughout the 96-h assay period.

To determine the effect of Nrf2 expression on GTM and HTM cell apoptosis, cells were transfected with Nrf2 overexpression plasmid alone and in combination with siNrf2. As depicted in Fig. 4, overexpression of Nrf2 significantly decreased the rate of apoptosis in GTM and HTM cells (P<0.001). This effect was most prominent in GTM cells, due to their higher starting rate of apoptosis, when compared to HTM cells. In turn, transfection overexpression Nrf2 together with siNrf2 significantly reversed the lowered rate of apoptosis than Nrf2 overexpressing cells (P<0.01). These results suggest that Nrf2 may regulate GTM and HTM cells through induction of apoptosis.

BCL-2, bcl-2-like protein 4 (Bax), tumor suppressor protein p53 and phosphorylated (p)-p53 are all apoptosis-related proteins, with BCL-2 considered to be a key anti-apoptotic factor (33,34). Therefore, to determine whether Nrf2 regulates the expression of apoptosis-related proteins, levels of BCL-2, Bax, p53 and p-p53 were evaluated by western blotting and densitometric analysis following Nrf2 overexpression in GTM cells. As depicted in Fig. 5, overexpression of Nrf2 significantly upregulated BCL-2 (P<0.01), while significantly downregulating Bax, p53 and p-p53 expression (P<0.01), relative to control cells. In turn, transfection with siNrf2 significantly reversed the altered expression of BCL-2 (P<0.01), Bax (P<0.01), p53 (P<0.05) and p-p53 (P<0.01) compared with Nrf2 overexpressing cells.