Epstein-Barr Virus-Encoded Latent Membrane Protein 2A Downregulates GCNT3 via the TGF-β1/Smad-mTORC1 Signaling Axis

LMP2A inhibited GCNT3 expression and mTORC1 pathway activation.

Based on our previous experiments, we observed lower GCNT3 protein expression in EBVaGC cells and tissues compared with EBVnGC, and high expression is closely related to lymph node metastasis (13). SGC7901 cells with strong GCNT3 expression were selected to transfect LMP2A (SGC7901 cells with pcDNA3.1-LMP2A plasmids [SGC7901-LMP2A]) by Geneticin screening. Microscopic fluorescence showed that the plasmids were successfully transfected into the cells (Fig. 1A). As shown in Fig. 1B, compared to SGC7901 cells with pcDNA3.1 expression vector (SGC7901-NC) (transfection control plasmid), GCNT3 expression in SGC7901-LMP2A cells stably expressing LMP2A was significantly suppressed. LMP2A transiently transfected into SGC7901 cells was used to further study the time-dependent effect of LMP2A on GCNT3 inhibition, and GCNT3 was significantly inhibited at 48 and 72 h after LMP2A transfection (Fig. 1C). GCNT3 was significantly increased after knockdown of LMP2A expression in GT38 EBV-infected cells (Fig. 1D). These findings confirmed the involvement of LMP2A in the inhibition of GCNT3 expression.

The mammalian rapamycin target protein (mTORC1) pathway is the most important energy sensor in the cell and regulates downstream signals to affect cell metabolism, cell cycle progression, and cell growth. Previous reports have confirmed that LMP2A regulates the mTOR pathway in NPC (17). We investigated the relationship between LMP2A and mTORC1 signaling in gastric cancer. As shown in Fig. 1E, inhibition of mTORC1 signaling was clearly detected in EBVaGC cells rather than EBVnGC as indicated by the decreased phosphorylation of mTOR at Ser²⁴⁴⁸, p-p70s6K (Thr³⁸⁹), and p-4EBP1 (Thr⁷⁰), which are downstream targets of mTORC1. mTORC1 signaling was clearly inhibited in SGC7901 cells stably expressing LMP2A by the decreased phosphorylation of mTOR (Ser²⁴⁴⁸), p70s6K (Thr³⁸⁹), and 4EBP1 (Thr⁷⁰) (Fig. 1F). When LMP2A was transiently transfected into SGC7901 cells, LMP2A also inhibited the phosphorylation of mTOR (Ser²⁴⁴⁸), p70s6K (Thr³⁸⁹), and 4EBP1 (Thr⁷⁰) at 48 and 72 h (Fig. 1G). In EBV-infected epithelial cells, mTORC1 signaling was activated after LMP2A was knocked down with small interfering RNA (siRNA) (Fig. 1H). The above-described results confirmed that LMP2A inhibited mTORC1 pathway activation in GC.

LMP2A downregulated GCNT3 expression via mTORC1 signaling.

Experiments were conducted to verify whether the mTOR pathway regulated GCNT3 in GC. Effective concentrations of 2 μM MHY1485 (mTOR activator) and 100 nM rapamycin (mTOR inhibitor) were selected. As shown in Fig. 2A, GCNT3 expression was significantly increased after MHY1485 was added to GT38 cells for 24 and 36 h. Rapamycin effectively inhibited mTORC1 pathway as well as GCNT3 expression in SGC7901 cells (Fig. 2B). When we extended the action time to 48 h, GCNT3 expression was positively correlated with mTORC1 signaling activation (Fig. 2C). Immunofluorescence was used to further verify that the mTORC1 pathway is a key factor in regulating GCNT3 expression (Fig. 2D). At the same exposure time intensity, GT38 with MHY1485 exhibited stronger fluorescence intensities of GCNT3; SGC7901 with rapamycin exhibited weaker fluorescence intensities of GCNT3. The inhibitory effect of LMP2A on GCNT3 expression was reversed after the addition of MHY1485 (Fig. 2E). mTOR is the catalytic subunit of the mTORC1 complex, and Raptor is a protein related to mTOR regulation, which combines with the TOR signal motif to promote the recruitment of the mTORC1 substrate (36). Decreased GCNT3 levels were observed following Raptor or mTOR inhibition (Fig. 2F). The above-described results indicated that GCNT3 was a downstream target of the mTORC1 pathway and that LMP2A inhibited GCNT3 expression by inhibiting mTORC1 pathway activation.

The 4EBP1-eIF4E complex interacted with GCNT3 to maintain GCNT3 stability.

GCNT3 was a downstream target of the mTORC1 pathway, and we investigated whether eIF4E regulates GCNT3 in GC. As shown in Fig. 3A, eIF4E expression was reduced in EBVaGC cells compared with EBVnGC cells. LMP2A suppressed eIF4E expression (Fig. 3B). Furthermore, we observed decreased GCNT3 expression following eIF4E inhibition in BGC823 and SGC7901 cells (Fig. 3C). These findings confirmed that LMP2A inhibited GCNT3 expression by inhibiting eIF4E. The SGC7901 cell extract was immunoprecipitated with antibodies against eIF4E, 4EBP1, or GCNT3, and then Western blotting was performed. Coimmunoprecipitation experiments showed that eIF4E, 4EBP1, and GCNT3 formed a complex in the cell (Fig. 3D). LMP2A inhibited the interaction between eIF4E and GCNT3, and complex formation was impaired (Fig. 3E). These data indicate that LMP2A inhibits the formation of the eIF4E-GCNT3 complex and GCNT3 expression by inhibiting eIF4E.

We hypothesized that eIF4E may regulate the protein turnover of GCNT3 in GC. To investigate whether eIF4E affects the stability of GCNT3 protein, we detected GCNT3 protein levels in the presence of cycloheximide (CHX), a protein biosynthesis inhibitor. GCNT3 degradation increased with the extension of CHX treatment time; when eIF4E was knocked out, the degradation rate increased (Fig. 3F and G). Further studies showed that in SGC7901 cells treated with the proteasome inhibitor MG132, knockdown of eIF4E could not induce GCNT3 degradation (Fig. 3H). This finding reflected the participation of the ubiquitination degradation pathway. Our results demonstrate that eIF4E binds to GCNT3 and maintains its stability to participate in the degradation of GCNT3, and LMP2A inhibits the above-described effects. In summary, these results provide evidence for LMP2A-eIF4E to regulate GCNT3 protein stability in GC.

LMP2A-inhibited TGF-β1/Smad signaling was dependent on NF-κB signaling.

Cross talk of the TGF-β/Smad and mTORC1 signaling pathways has been reported (37). Our previous experiments demonstrated that LMP2A inhibited Smad2 expression via the NF-κB/miR-155a axis (38). As shown in Fig. 4A, LMP2A not only inhibited the expression of Smad2, Smad3, and Smad4 but also inhibited the expression of p-Smad2 and p-Smad3. In addition, after transfection with LMP2A siRNA, the expression of the above-described genes was upregulated in GT38 EBV-infected epithelial cells (Fig. 4B). These results indicated that LMP2A inhibits the TGF-β1/Smad pathway activation in GC. LMP2A activates NF-κB signaling to mediate a variety of malignant phenotypes, thereby promoting tumorigenesis (39, 40). NF-κB signaling upregulates miR-146a and miR-155a in GC, and miR-146a and miR-155a target Smad4 and Smad2, respectively (38, 41). We hypothesized that LMP2A activated NF-κB signaling to upregulate microRNAs (miRNAs), thereby inhibiting the expression of Smad family proteins. The NF-κB signal inhibitor BAY11-7082 reversed the inhibitory effect of LMP2A on the TGF-β1/Smad pathway (Fig. 4C). Immunofluorescence showed that the fluorescence intensity of the above-described genes increased in SGC7901 cells after treatment with BAY11-7082 and that the signal in the nucleus was stronger (Fig. 4D and E). Based on the above-described results, we hypothesized that LMP2A activated NF-κB signaling to mediate its downstream signaling and ultimately inhibited the TGF-β1/Smad pathway.

The LMP2A/TGF-β1/Smad axis inhibited the mTORC1 pathway and downregulated GCNT3 transcription.

TGF-β1/Smad and mTORC1 signaling mediates a variety of malignant phenotypes, thereby affecting tumorigenesis. Since LMP2A inhibited the TGF-β1/Smad and mTORC1 signaling pathways in GC, we hypothesized that a functional interaction exists between these two signaling pathways. Western blots showed that TGF-β1 activated the mTORC1 signal and upregulated GCNT3 expression (Fig. 5A). SB431542, a specific inhibitor of TGF-β receptor I (TGF-βRI), inhibited Smad2 and Smad3 phosphorylation. As shown in Fig. 5B, SB431542 (10 μM) completely prevented TGF-β1-mediated phosphorylation of mTOR, p70S6K, and 4EBP1 and blocked activation of the mTORC1 signal. TGF-β1 reversed the inhibition of LMP2A on the mTORC1 signaling (Fig. 5C). Next, the specific mechanism by which TGF-β1/Smad regulates mTORC1 signaling was verified. In SGC7901 and BGC823 cells, siRNA-mediated knockdown of Smad2 and 3 or Smad4 only inhibited the phosphorylation of mTOR and p70S6K induced by TGF-β1 (Fig. 5D and E). Interestingly, immunofluorescence indicated that knockdown of Smad2/3 or Smad4 in SGC7901 cells inhibited the phosphorylation of mTOR and p70S6K, and the signal in the nucleus was stronger than that in the cytoplasm (Fig. 5F). The expression of Smad2/3 or 4 was increased by transfecting plasmids overexpressing Smad2/3 or 4, which reversed the inhibitory effect of LMP2A on mTOR and p70S6K phosphorylation (Fig. 5G). These results demonstrated that LMP2A inhibited the phosphorylation of mTOR and p70S6K by inhibiting the expression of Smad2/3/4. Coimmunoprecipitation experiments showed that Smad2/3 or Smad4 interacted with mTOR, and LMP2A might inhibit the phosphorylation of mTOR by inhibiting this binding. It is worth noting that LMP2A inhibited the binding of Smad2/3 and Smad4, impairing complex formation and thereby inhibiting the TGF-β1/Smad pathway (Fig. 5H).

Smad2/3/4 are important molecules in the TGF-β1/Smad pathway and act as important transcription factors to activate target gene transcription. We assessed whether GCNT3 is the direct target gene of Smad2/3/4. siRNA-mediated knockdown of Smad2/3 or Smad4 inhibited GCNT3 mRNA expression in SGC7901 cells. In addition, LMP2A inhibited GCNT3 mRNA expression; overexpressing Smad2/3 or 4 reversed the inhibitory effect of LMP2A on GCNT3 mRNA level (Fig. 5I). We further used the prediction software JASPAR to detect the promoter region of GCNT3 and found one of the most conserved putative Smad2/3/4 binding sites located at –1719 to –1707 bp in the promoter region of GCNT3 (Fig. 5J). To further confirm the direct binding of Smad2/3/4 to the consensus sequence of the GCNT3 promoter, we performed chromatin immunoprecipitation (ChIP) analysis using antibodies to pull down the components bound by Smad2/3 or Smad4. ChIP analysis revealed a significantly increased binding of Smad2/3 to the putative binding region of the GCNT3 promoter in cells with high expression of Smad2/3 compared with control cells; we obtained the same conclusion using Smad4 antibodies to pull down the Smad4 binding components (Fig. 5K). In summary, GCNT3 was a direct target gene of Smad2/3 or Smad4, and LMP2A downregulated GCNT3 transcription by inhibiting Smad2/3 or Smad4.

GCNT3 mediated autophagy and malignant properties in gastric cancer cells.

To further study the biological function of GCNT3 in GC, we reduced the expression of endogenous GCNT3 in SGC7901 cells by siRNA transfection. The proliferation ability of SGC7901 cells was significantly inhibited when GCNT3 was inhibited, and the inhibitory effect was weakened by MHY1485 (Fig. 6A). The siRNA target sequence to GCNT3 mRNA (siGCNT3) significantly inhibited migration and induced G₀/G₁ cell cycle arrest after transfection in SGC7901 cells, and MHY1485 reversed the reduction in migration and G₀/G₁ cell cycle arrest (Fig. 6B and D and E). CCND1 expression decreased after siGCNT3 transfection, and MHY1485 reduced the effect of siGCNT3 (Fig. 6C). These results confirmed that knockdown of GCNT3 inhibited cell proliferation and migration and induced G₀/G₁ cell cycle arrest, whereas the mTOR pathway enhanced these effects of GCNT3.

Rapamycin is a known activator of autophagy, and the mTORC1 pathway inhibits the occurrence of autophagy. The MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) protein exists in two forms. LC3-I can be partially converted to LC3-II by lipidation, which can then be recruited into the precursor of the autophagosome. As shown in Fig. 6F, after transfecting GCNT3 siRNA in SGC7901 cells, the expression of LC3-II was significantly increased, whereas LC3-I had no obvious change. Using immunofluorescence to monitor autophagy in cells, confocal laser scanning microscopy revealed an increased level of punctate LC3B staining after transfection of GCNT3 siRNA (Fig. 6G). In addition, an electron microscope was used to observe the formation of autophagosomes. Compared with control cells, the number of autophagosomes was significantly increased after GCNT3 siRNA transfection (Fig. 6H).

In addition, proteins related to autophagy were tested. After transfection of GCNT3 siRNA, the expression of mTOR, p-mTOR, and p-ULK1 was significantly reduced, and ULK1 expression was significantly increased. ATG7 expression was significantly reduced, whereas the expression of ATG12, ATG16, ATG5, and ATG3 was not significantly different (Fig. 6I and J). This finding suggested that knocking down GCNT3 activated autophagy by inhibiting the mTORC1 pathway and its downstream pathway. Immunoprecipitation showed that GCNT3 interacted with mTOR to form a complex, inducing the expression of mTOR and the activation of mTOR (Fig. 6K). The GCNT3-mTOR complex phosphorylated ULK1, prevented ULK1 from forming complexes with ATG and FIP200, and prevented autophagy in GC cells. In addition, given that EBV is a known activator of autophagy (42), we assessed whether LMP2A affects the formation of autophagosomes in GC. The number of autophagosomes increased in cells stably transfected with LMP2A (Fig. 6L). The above results indicated that knocking down GCNT3 promoted autophagy by downregulating the mTORC1 pathway, and LMP2A might promote autophagy by downregulating GCNT3 and the mTORC1 pathway.

The mTORC1 pathway mediated malignant properties in gastric cancer cells by regulating GCNT3.

The mTORC1 pathway is typically activated in cancer and regulates downstream signals to affect cell proliferation, apoptosis, migration and cell cycle progression. The cell proliferation ability was significantly enhanced after MHY1485 treatment and weakened after rapamycin treatment (Fig. 7A). Given that cancer cell metastasis is a key indicator of tumor progression, we found that the migration ability was stronger with MHY1485 treatment and much weaker with rapamycin treatment, and siGCNT3 inhibited the effect of MHY1485 (Fig. 7E and F). After the mTORC1 pathway was inhibited, N-cadherin, vimentin, β-catenin, Snail, and ZEB1 expression were downregulated, whereas E-cadherin was significantly increased. The opposite result was obtained when the mTORC1 pathway was activated (Fig. 7G). The mTORC1 pathway regulated important proteins involved in EMT to regulate migration. The mTORC1 pathway promoted the proliferation and migration of EBVaGC and EBVnGC cells, whereas these effects were suppressed after transfection with siGCNT3.

Flow cytometry assays were performed to identify the stages of cell cycle progression. Rapamycin resulted in a significant increase in the percentage of G₀/G₁ phase cells and a significant decrease in S phase cells. In addition, rapamycin downregulated CCND1 expression. Opposite results were obtained after treatment with MHY1485, and the percentage of G₀/G₁ phase cells increased after transfection with siGCNT3 (Fig. 7B to D). Under different conditions, the G₂/M phase was not significantly affected (Fig. 7B and C). Treatment with MHY1485 and rapamycin had no significant difference in apoptosis (Fig. 7H). The mTORC1 pathway promoted proliferation and migration and inhibited G₀/G₁ cell cycle arrest in EBVaGC and EBVnGC cells, whereas transfection with siGCNT3 inhibited these effects.

TGF-β1 activated the mTORC1-GCNT3 pathway to promote cell migration in EBVaGC and EBVnGC cells, and TGF-β1 promoted apoptosis and G₀/G₁ cell arrest in EBVnGC cells.

TGF-β1 activates a variety of signals to mediate cell proliferation, apoptosis, and migration to regulate tumor progression. For example, TGF-β activates the mTORC1 signaling pathway to induce EMT (23). The effect of TGF-β1 on the malignant characteristics of GC cells was tested. The migration ability of cells after TGF-β1 treatment was significantly higher; after adding rapamycin or transfecting siGCNT3 to TGF-β1-added cells, the cell migration ability was lower than for TGF-β1-added cells (Fig. 8A and B). TGF-β1 upregulated the expression of N-cadherin, vimentin, β-catenin, Snail, ZEB1, Twist1, and Slug but downregulated E-cadherin expression (Fig. 8C). This finding indicated that TGF-β1 promoted cell migration by activating the mTORC1-GCNT3 pathway.

TGF-β1 promotes apoptosis in GC cells (32), whereas LMP2A resists TGF-β1-mediated growth inhibition and apoptosis in EBVaGC (43). Consistent with the reported results, TGF-β1 induced apoptosis in SGC7901 cells, whereas TGF-β1 did not significantly induce apoptosis in GT38 cells. When LMP2A was knocked down in TGF-β1-stimulated GT38 cells, apoptosis increased (Fig. 8D and E). This finding suggested that TGF-β1 inhibited cell growth and promoted apoptosis in EBVnGC, whereas LMP2A inhibited TGF-β1-mediated growth inhibition and apoptosis in EBVaGC. We demonstrated that TGF-β1 promoted G₀/G₁ phase cell arrest in SGC7901 cells using flow cytometry (Fig. 8F). TGF-β1 exhibited different effects on cell proliferation and apoptosis in EBVaGC and EBVnGC, potentially because LMP2A inhibited the formation of Smad2/3/4 complexes and inhibited the activation of the TGF-β1/Smad pathway. This finding prompted our further assessment of the regulation of EBV on the TGF-β1 pathway.

Cell lines and treatment.

A total of six cell lines were used, three EBVaGC cell lines, i.e., GT38, GT39, and SNU719, and three EBV-negative gastric cancer (EBVnGC) cell lines, i.e., AGS, BGC823, and SGC7901. All cell lines (GT38, GT39, SNU719, BGC823, AGS, and SGC7901) were grown in Dulbecco modified Eagle medium (DMEM/F-12; Life Technologies, Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. All cells were maintained at 37°C in a 5% CO₂ incubator. GT38 and GT39 cell lines were sourced from T. Sairenji (Biosignal Division, Department of Biomedical Sciences, Tottori University, Japan).

TGF-β1 was purchased from Pepro Tech EC Ltd. MHY1485, BAY11-7082, SB431542, MG132, and cycloheximide were obtained from ApexBio (Houston, TX, USA). Rapamycin was purchased from Beyotime Biotechnology Corporation (Shanghai, China). The following primary antibodies were purchased from Abcam (Cambridge, UK): anti-LMP2A, anti-GCNT3, anti-p-4EBP1, anti-p-mTOR, anti-CCND1, and anti-β-actin. The following primary antibodies were purchased from Cell Signaling Technology (Chicago, IL, USA): anti-Smad2, anti-Smad3, anti-Smad4, anti-p-Smad2, anti-p-Smad3, anti-mTOR, anti-p70S6K, anti-4EBP1, anti-p-p70S6K, anti-eIF4E, anti-ULK1, anti-p-ULK1, anti-ATG3, anti-ATG5, anti-ATG7, anti-ATG12, anti-ATG16, anti-N-cadherin, anti-E-cadherin, anti-Vimentin, anti-ZEB1, anti-Snail, anti-Slug, anti-β-Catenin, and anti-Twist1. Anti-rabbit and anti-mouse antibodies conjugated to horseradish peroxidase were purchased from Cell Signaling Technology.

RNA extraction and quantitative reverse transcription PCR (qRT-PCR).

Cells were harvested and RNA was extracted using TRIzol reagent (Ambion, USA) according to the manufacturer’s instructions. qRT-PCR was performed using the LightCycler 96 system (Roche, Switzerland) to determine GCNT3 mRNA expression. The comparative cycle threshold (C_T) method was used to assess differences in LMP2A transcript levels using GAPDH as an internal loading control. Experiments were performed in triplicate. The following forward and reverse primers were used: GAPDH, 5′-CAAATTCCATGGCACCGTCA-3′ and 5′-ATCGCCCCACTTGATTTTGG-3′; GCNT3, 5′-TGTTCACATTTGCTGCCACG-3′, and 5′-GTGGAGGAGGACACAATCCTTT-3′.

Immunofluorescence assay.

The cells were placed on slides and fixed with 4% paraformaldehyde for 10 min. Then, slides were washed with phosphate-buffered saline (PBS), blocked, and permeabilized with buffer (1% bovine serum albumin [BSA], 0.15% Triton X-100, 22.52 mg/ml glycine in PBS) at room temperature for 1 h. The slides were incubated with primary antibody overnight at 4°C. The secondary antibody (1:1,000) was incubated for 1 h in the dark at room temperature following three washings with PBS with Tween (PBST), and the cells were then counterstained with Hoechst 33342 (Beyotime, China) for 5 min. The localization of GCNT3 in the cells was observed with a fluorescence microscope (Nikon, Japan).

Western blotting.

All cells were lysed using radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor, phenylmethanesulfonyl-fluoride (PMSF), and a phosphatase inhibitor mixture. These samples were transferred to polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany) using SDS-PAGE gel with a 10% mass concentration, blocked with bovine serum albumin (BSA) with a 5% mass concentration at room temperature for 2 h, incubated with the primary antibody overnight at 4°C, cultured with the second antibody the following day, and held at room temperature for 2 h. Following incubation, the membranes were visualized by using an ECL detection kit (Millipore Corporation, Billerica, MA, USA).

Transfection with small interfering RNA (siRNA) and overexpression of LMP2A and Smad2/3/4.

Specific small interfering RNAs were designed and synthesized at GenePharma (Shanghai, China). The sequences of siRNAs were as follows: GCUCAGUGCCGUGGAAAUATT for siGCNT3-1, GCUACUGCGAGCUGUGUAUTT for siGCNT3-2, GAACGAUGAGGAACGUGAAT for siLMP2A, CCUCUUGCUCAGAUGCCUUTT for siRaptor, CCACCCGAAUUGGCAGAUUTT for simTOR, GCUAACCCAGAACACUAUATT for sieIF4E, GGUGUUCGAUAGCAUAUUATT for siSmad2, GCCUGGUCAAGAAACUCAATT for siSmad3, GCCAUCGUUGUCCACUGAATT for siSmad4, and UUCUCCGAACGUGUCACGUTT for control siRNA. Cells were transfected with the RNA oligoribonucleotides using Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, Germany) for 48 h at 50 nM per the manufacturer’s recommendations. Smad2, Smad3, and Smad4 plasmids were purchased from Genechem (China). LMP2A was cloned into the pcDNA3.1-EF1a-mcs-3flag-CMV-EGFP vector. Three micrograms of LMP2A (pcDNA3.1-LMP2A) or a negative-control vector (pcDNA3.1-vector) plasmid was transfected into SGC7901 cells using Lipofectamine 2000. Cells were harvested for assay at the indicated times. Stable transfection of LMP2A was selected by G418 and observed under a fluorescence microscope.

Immunoprecipitation.

Protein complexes were precipitated from whole-cell lysates with anti-eIF4E (Santa Cruz Biotechnology), anti-4EBP1 (Santa Cruz Biotechnology), anti-GCNT3 (Santa Cruz Biotechnology), anti-Smad2/3 (Santa Cruz Biotechnology), or anti-Smad4 antibodies (Cell Signaling Technology) and then precipitated with protein A/G PLUS-Agarose (Santa Cruz Biotechnology). Following the instructions, SDS-PAGE and Western blotting were performed after the sample was boiled.

Chromatin immunoprecipitation.

A chromatin immunoprecipitation (ChIP) kit was purchased from Cell Signaling Technology, and ChIP was performed according to the manufacturer’s recommendations. The antibodies used were normal rabbit IgG against Smad2/3 and Smad4. The primer pairs used for the GCNT3 promoter region were as follows: 5′-GAGAGGATGTGGAGCTCTAAGG-3′ (F), 5′-AACTGGTTTCTAATCAGGGTTGC-3′ (R).

Cell proliferation assay.

Cell proliferation was assessed using cell counting kit-8 (CCK8; Boster Biological Technology, Wuhan, China). For growth curve analysis, cells were seeded in sextuplicate at a density of 8 × 10³ cells per well in 96-well plates. After 12 h of incubation, different reagents were added to the experimental wells, and the cells were allowed to grow for another 24, 48, or 72 h. A total of 10 ml of CCK-8 solution was added to each well for 2 h, and viability was measured using a microplate reader at a wavelength of 450 nm.

Cell cycle assay.

Cells were seeded into six-well plates at a density of 1 × 10⁶ cells per well and incubated overnight. After treatment for 36 h, the cells were digested by trypsin and washed gently with PBS. The collected cells were fixed in 66% ethanol at 4°C overnight. Then, the cells were incubated with propidium iodide (PI) and RNase A for 25 min in the dark. The cell cycle was analyzed by using flow cytometry (BD Biosciences).

Migration assays.

The migration ability of the cells was measured using Transwell plates with 8-μm pores (Corning, NY, USA). Cells were digested with trypsin and resuspended in serum-free medium. A total of 1 × 10⁵ cells in 200 μl serum-free medium were seeded into the upper chambers of Transwell plates. Cells attached to the bottom of the chamber membrane were stained with crystal violet after incubation at 37°C for 36 h. Five fields were randomly photographed under each microscope, and the experiments were repeated thrice.

Cell apoptosis detection.

Cell apoptosis analyses were detected by flow cytometry analysis using a fluorescein isothiocyanate (FITC)-annexin V apoptosis detection kit I (BD Biosciences, San Jose, CA). Cells were seeded in six-well plates at a density of 1 × 10⁶ cells per well. After treatment for 36 h, the cells were digested and washed twice with cold PBS. Next, 5 μl FITC-annexin V was added, and the cells were protected from light for 15 min at room temperature. Then, 5 μl propidium iodide (PI) was added. All experiments were repeated in triplicate.

Electron microscopy.

To identify autophagosomes at the ultrastructural level, cells were fixed with 2.5% glutaraldehyde and then dehydrated and embedded. The number of autophagosomes was observed in a randomly selected field of view in each sample.

Statistical analysis.

All data are expressed as the means ± standard error of the mean (SEM). The experimental data were analyzed using a t test (P < 0.05 was considered significant; ns, nonsignificant; *, P < 0.05; **, P < 0.01). Statistical analyses were conducted using SPSS 19.0 statistical software (Chicago, IL).