shRNA Glut-1 inhibits cell viability, apoptosis and migration of laryngeal carcinoma HEp-2 cells through regulating Beclin-1-mediated autophagy

Objective Glut-1 is a key regulator in the process of glucose uptake. Previous studies have shown that Glut-1 affects autophagy. However, it is unclear whether there is a correlation between Glut-1 and autophagy in the progression of laryngeal carcinoma. This study was performed to investigate the role of Glut-1 in the development of laryngeal carcinoma. Methods A stable HEp-2 cell model was constructed by Glut-1 and Beclin-1 shRNA lentiviral infection. The autophagosome was measured by transmission electron microscopy. Protein levels of LC3, ATG5, CyclinD1, Bcl-2, Caspase-3, and c-Myc were determined by Western blotting. CCK8 assay and Transwell assays were used to determine cell viability and migration rate of HEp-2 cells, respectively. Flow cytometry was performed to analyze the rate of apoptosis. Immunofluorescence was performed to determine the expression distribution of LC3. Results Glut-1 knockdown significantly promoted autophagosome formation by upregulating the ratio of LC3-II/LC3-I as well as the role of rapamycin (RAP) and Beclin-1 overexpression on autophagy flux in HEp-2 cells. Glut-1 inhibition also reduced the viability of HEp-2 cells followed by the decreases in expression of cyclinD1 and c-Myc. In addition, Glut-1 depletion increased the number of apoptotic HEp-2 cells accompanied by activation of caspase-3 and downregulation of Bcl-2. Glut-1 knockdown also reduced the migration rate of HEp-2 cells by promoting the expression of N-cadherin and inhibiting the expression of E-cadherin. Beclin-1 consumption significantly reversed Gult-1 knockdown-mediated autophagy activation, resulting in promotion of both proliferation and migration and inhibition of apoptosis. Conclusions Glut-1 knockdown-induced autophagy inhibits the proliferation and migration of HEp-2 cells, and promotes apoptosis of HEp-2 cells partly by regulating autophagy.


Introduction
Laryngeal squamous cell carcinoma (LSCC), which is related to many risk factors, including smoking, drinking alcohol, and laryngeal reflux, is one of the most common malignant tumors occurring in the head and neck [1]. Some studies have demonstrated that autophagy and glucose metabolism play important roles in the occurrence and development of cancer cells [2][3][4][5]. However, the mechanisms underlying autophagy and glucose metabolism in the process of LSCC remain unclear.
It has gradually become apparent that most tumor cells show a variable glycolysis phenotype, so increased glycolytic metabolism has been identified as a characteristic of malignant cells, known as the Warburg effect [4]. Previous studies confirmed the upregulation of members of the glucose transporter family (Gluts) during the process of glycolysis in tumor cells to meet the energy needs of cancer cells [6][7][8]. Glut-1, as an important carrier of glucose uptake and a major protein involved in the transport of glucose, plays a key role in maintaining the homeostasis of the body [9]. It has been reported that Glut-1 transporters are expressed at higher levels in human solid cancer cells compared to their normal counterparts [10][11][12]. Glut-1 also affects cell proliferation and chemoradiotherapy sensitivity of head and neck squamous cell carcinoma, including laryngeal carcinoma13-18. Our previous studies suggested that Glut-1 may affect the proliferation, glucose uptake, chemosensitivity, and radiosensitivity [8,[13][14][15][16][17]. Although we found that the mechanism may be mediated through the PI3K/Akt pathway, the role of the activated PI3K/Akt pathway in this process was not significant. Therefore, further studies are required to investigate the mechanism by which Glut-1 results in proliferation, migration, and invasiveness.
Autophagy through Glut-1 translocation has been shown to affect glucose uptake by cancer cells [19][20][21][22][23]. Autophagy is the process by which damaged proteins or organelles are degraded and recycled by eukaryotic cells, and it plays a dual role in promoting cell survival and inducing cell death during cancer growth and migration [19][20][21][22][23]. The energy supply of cancer cells in an anoxic environment is associated with high levels of Glut-1 expression and autophagy activation [24,25] suggesting that Glut-1-mediated energy supply and cell growth are involved in the process of autophagy.
Beclin-1 is a homolog of the yeast ATG6/Vps30 gene, which was first identified as a molecule mediating autophagy in mammals [26]. It plays a positive regulatory role in the process of autophagy [27,28]. During the process of autophagy, Beclin-1 and Class III PI3K kinase form a complex that promotes the formation of autophagic vacuoles [29]. Previous studies have shown that stable transfection of Beclin-1 promotes autophagy and reduces tumorigenicity, suggesting that Beclin-1-mediated autophagic cell death may be associated with inhibition of tumor cell growth [30].
Conversely, some studies showed that Beclin-1 upregulation in the same type(s) of cancer led to activation of autophagy and increased clonogenic survival, indicating an association between Beclin-1-mediated autophagy flux and tumorigenesis [34,35].
Although our previous study showed high levels of Glut-1and Beclin-1 expression in head and neck cancer and a correlation between Glut-1 and Beclin-1 expression, it remains unclear whether Glut-1-mediated autophagy flux in the process of oncogenesis is associated with Beclin-1-induced activation of autophagy in laryngeal carcinoma.
In this study, we explored the relationship between autophagy activity and Glut-1 in laryngeal carcinoma HEp-2 cells and provided a theoretical basis for understanding the role of Glut-1 in the growth and migration of LSCC.

Vector construction and cell transfection
Specific interference fragments targeting the Glut-1 and Beclin-1 genes were designed and synthesized with reference to the human Glut-1 and Beclin-1 cDNA sequences in

Transmission electron microscopy
After 48 hours of treatment with RAP or transfection with Glut-1 shRNA and lenti-Beclin-1, the cells were washed in PBS, fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, and gradually dehydrated in ethanol and acetone. After embedding with epoxy resin, they were cut into sections and stained with uranyl acetate and lead citrate. Autophagy of the cells was observed by transmission electron microscopy (TEM; Thermo Scientific).

CCK-8 assay
Cells were inoculated into 96-well cell culture plates at 5000/well and cultured for 24, 48, or 72 hours. Then, 10 μl of CCK-8 (Dojindo) was added to each well and culture was continued for 2 hours. The fluorescence was determined spectrophotometrically at a wavelength of 450 nm using a microplate reader (Thermo Scientific).

Transwell assay
Suspensions of 5×10 4 treated cells from each group in 500 μl of serum-free medium were inoculated into the upper chamber of a Transwell system for migration assay.
Cells were added to the lower chamber of the Transwell system in 750 μl of RPMI 1640 medium containing 10% FBS for 8 hours. After the chamber was removed, the upper layer was wiped to pass through the cells. The cells were dyed with crystal violet and the number of perforated cells was determined under a microscope.

Western blotting
Total protein was extracted with RIPA solution (Beyotime Biotechnology, Shanghai, China) and protein concentration was determined using a BCA kit. Protein samples were separated by SDS-PAGE. After blocking for 1 hour with 5% skim milk, the membranes were incubated with primary antibodies at 4°C overnight. The next day, the samples were incubated with HRP-conjugated goat anti-rabbit IgG (diluted 1:500) for 1 hour at 37°C. After washing three times with TBST (Tris-buffered saline and Polysorbate 20), a chemiluminescence system was used to measure the levels of specific proteins using an enhanced chemiluminescence (ECL) coloring solution.

Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was isolated according to the manufacturer's instructions. Briefly, 1 μg of RNA was reverse transcribed using a First Strand cDNA Synthesis Kit (K1622; Fermentas, Burlington, ON, Canada) and polymerase chain reaction (PCR) using a SYBR Green qPCR kit (Merck, Darmstadt, Germany) with incubation at 37°C for 60 minutes, 85°C for 5 minutes, and 4°C for 5 minutes, followed by storage at −20°C.
The 2 ΔΔCt method was used to calculate the relative expression levels of these genes.

Immunofluorescence
After incubation for 48 hours, the cells was washed three times with PBS and fixed for 15 minutes at room temperature with 4% paraformaldehyde. Then, cells were treated with 0.2% Triton X-100 for 15 minutes and blocked using 5% BSA (SH30574.03; Hyclone, Logan, UT) for 1 hour at room temperature. The cells were subsequently incubated with anti-LC3 antibody (1:500) at 4°C overnight. The next day, the cells were scrubbed and washed three times with PBS for 5 minutes each time. Finally, cells were stained with secondary antibodies for 1 hour and subsequently with DAPI for 5 minutes, and examined by confocal laser scanning microscopy (LSM 800; Zeiss, Oberkochen, Germany).

Statistical analysis
Each experiment was repeated at least three times. Data are presented as the mean ± standard deviation (SD) and were analyzed using SPSS 25.0 (SPSS Inc., Chicago, IL).
The least significant difference (LSD) or Dunnett's method in one-way analysis of variance ANOVA (LSD for variance and Dunnett's for variance inequality) was used to analyze and compare the data between groups. In all analyses, P<0.05 and P<0.01 were taken to indicate significance and high significance, respectively.

Glut-1 inhibition promotes autophagy in HEp-2 cells
To explore the role of Glut-1 in the autophagy process of laryngeal carcinoma, we first established stable Glut-1 knockdown cell lines. As shown in Fig. 1A, Glut-1 shRNA lentivirus was constructed successfully and showed very high efficiency of infection in HEp-2 cells (Fig. 1A). Western blotting and qRT-PCR experiments showed that Glut-1 protein and mRNA levels were significantly decreased in HEp-2 cells infected with Glut-1 shRNA lentivirus compared with control and negative control (NC) groups, indicating that Glut-1 was effectively knocked down in HEp-2 cells (Fig. 1B-1C). TEM experiments showed that there were few autophagosomes in the control and NC groups. However, the number of autophagosomes was markedly increased in HEp-2 cells transfected with Glut-1 shRNA, suggesting that Glut-1 may be involved in the autophagosome formation ( Fig. 2A). As an autophagy activator, rapamycin (RAP) promotes the formation of autophagosomes. In addition, Beclin-1 upregulation also promoted the autophagy process. The data presented here indicated that the acceleration effect of Glut-1 inhibition on autophagosome formation was similar to the effects of RAP exposure and Beclin-1 upregulation in HEp-2 cells. In addition, Glut-1 depletion plus RAP and Glut-1 depletion plus Beclin-1 overexpression showed additive effects on autophagosome formation compared to cells exposed separately to Glut-1 shRNA, RAP, and lenti-Beclin-1 ( Fig. 2A). The overexpression efficiency of Beclin-1 was confirmed by qRT-PCR (Fig. 3A-3B).
Furthermore, the expression of autophagy-associated protein LC3 was observed in the Glut-1 shRNA group. In the Glut-1 shRNA group, the ratio of LC3-II/LC3-I was significantly increased as well as in RAP and Lenti-Beclin-1 exposure groups. In addition, the groups with combination of Glut-1 shRNA plus RAP and Glut-1 shRNA plus lenti-Beclin-1 showed higher LC3-II/LC3-I ratios (Fig. 2B). These results suggested that Glut-1 knockdown directly promoted autophagy in HEp-2 cells.

Glut-1 consumption inhibits HEp-2 cell viability
Autophagy showed further reductions in the levels of cyclinD1 and c-Myc compared to individual treatments ( Fig. 4B-4C). Therefore, our results suggested that Glut-1 knockdown-mediated autophagy may inhibit the proliferation of HEp-2 cells.

Glut-1 knockdown increases the apoptosis rate of HEp-2 cells
We further examined the effects of Glut-1 knockdown-mediated autophagy on cell apoptosis. The results of flow cytometry analyses suggested that Glut-1 knockdown significantly promoted the apoptosis rate of HEp-2 cells similar to the effects of RAP treatment and Beclin-1 upregulation. Greater numbers of apoptotic cells were observed in the Glut-1 inhibition plus RAP treatment group or Glut-1shRNA plus Beclin-1-overexpressing cells (Fig. 5A). As shown in Figure 3B, there were no significant differences in expression of caspase-3 between the groups. However, Glut-1 knockdown significantly upregulated the expression of cleaved-caspase-3 protein and downregulated the expression of anti-apoptotic Bcl-2 protein and the corresponding mRNA expression level of Bcl-2 compared with the control and NC groups ( Fig. 5B-5C). The expression of cleaved-caspase-3 protein was further increased in the Glut-1 shRNA and RAP combination group and the Glut-1 shRNA and lenti-Beclin-1 combination group compared with the Glut-1 shRNA group ( Fig.   5B-5C). The expression of Bcl-2 protein and corresponding mRNA expression in the Glut-1 shRNA plus RAP combination group and the Glut-1 shRNA plus lenti-Beclin-1 combination group were lower than in the Glut-1 shRNA group (Fig.   5B-5C). These observations confirmed that Glut-1 inhibition-induced autophagy enhanced the apoptosis of HEp-2 cells and showed an additive effect on cell apoptosis along with RAP or lenti-Beclin-1 treatment.

Inhibition of Glut-1 decreases migration ability of HEp-2 cells
Next, we evaluated the effects of Glut-1 inhibition on migration ability of HEp-2 cells.
Transwell assay showed that Glut-1 knockdown significantly inhibited the number of migrating HEp-2 cells. The inhibitory effect of Glut-1 depletion on migration was markedly increased by combination with RAP treatment or Beclin-1 overexpression (Fig. 6A). Moreover, Glut-1 inhibition also downregulated N-cadherin protein and relative mRNA expression levels, but upregulated E-cadherin protein and relative mRNA expression compared with the control and NC groups (Fig. 6B-6C). The expression of N-cadherin was further decreased, while the expression of E-cadherin was significantly increased in the Glut-1 shRNA plus RAP group and the Glut-1 shRNA and lenti-Beclin-1 group compared with the Glut-1 knockdown alone group ( Fig. 6B-6C). These results suggested that Glut-1 inhibition-evoked autophagy potentially increases the migration ability of HEp-2 cells.
However, the levels of ATG5 and LC3-II expression were significantly decreased, while that of p62 was restored in the Glut-1 shRNA and Beclin-1 shRNA combination group compared to the Glut-1 shRNA group (Fig. 7A-7B). Fluorescence staining also showed that Glut-1 knockdown promoted the expression of mRFP-LC3 and eGFP-LC3, while Beclin-1 inhibition reversed these phenomena and reduced their expression (Fig. 7C). These results suggest that Beclin-1 knockdown inhibited Glut-1 inhibition-mediated autophagy.

Beclin-1 is required in the process of Glut-1 knockdown-mediated improvement of tumor biological characteristics
We also measured the effects of Beclin-1 on Glut-1 knockdown-mediated proliferation inhibition. Our results showed that Beclin-1 knockdown significantly increased the viability of HEp-2 cells at 24, 48, and 72 hours. The Glut-1 depletion-mediated reduction of cell proliferation was significantly enhanced in cells exposed to Beclin-1 shRNA (Fig. 8A-8D). The results of flow cytometric analyses indicated that Glut-1 knockdown-mediated promotion on apoptosis of HEp-2 cells was blunted in the presence of Beclin-1 shRNA (Fig. 9A-9B). In addition, the inhibitory effect of Glut-1 knockdown on migration ability was also blocked by administration of Beclin-1 shRNA (Fig. 9C). However, both inhibition of cell apoptosis and migration and the promotion of cell proliferation capability were weaker in the Glut-1 shRNA plus Beclin-1 shRNA group than the Beclin-1 shRNA alone group. Taken together, these results confirmed that the Glut-1 knockdown-induced improvement of tumor biological characteristics is partly mediated by Beclin-1-induced autophagy activation.

Discussion
Autophagy is the process of capture, degradation, and recycling of organelles and proteins in lysosomes [36]. Previous studies indicated that autophagy activation has a dual effect on the occurrence of tumors [37]. In most cases, it was shown that activated autophagy of cancer cells can induce autophagic cell death, which may represent a promising approach for cancer treatment [19][20][21][22][23][24][25][26][27][28][29][30][31]. In the present study, we showed that Glut-1 knockdown evoked autophagy activity in HEp-2 cells leading to subsequent cell apoptosis, suggesting that Glut-1 may be a novel inducer of cell autophagy and useful for the treatment of laryngeal carcinoma.
Preliminary studies have shown that autophagy through Glut-1 translocation affects glucose uptake of cancer cells [19][20][21][22][23]. In comparison with normal cells, cancer cells preferentially produce energy by glycolysis in anaerobic environments [4]. The energy supply of cancer cells in hypoxic environments is related to the high-level expression of Glut-1 and autophagy. Many studies have shown that inhibition of Glut-1 in malignant tumors can effectively limit malignant tumor growth by inhibiting cell proliferation and migration, inducing cell cycle arrest and apoptosis [38,39]. In our previous study, the detection of 2-fluoro-2-deoxyglucose (FDG) concentration in head and neck cancer was associated with increased Glut-1 expression [31]. We also showed previously that inhibition of Glut-1 using antisense oligonucleotides reduced glucose uptake and promoted cell death in HEp-2 cells 8. Another study showed that Beclin-1 expression was negatively correlated with HIF-1α and Glut-1 expression and positively correlated with increased E-cadherin expression in human gastric cancer tissues, suggesting that autophagy deficiency promotes glycolysis and metastasis of gastric cancer in patients [40]. These studies suggested that Glut-1-mediated energy supply acts as a key regulator of tumor growth by affecting autophagy. Here, following knockdown of Glut-1 in HEp-2 cells, autophagy flux was significantly activated resulting in proliferation and migration inhibition and promotion of apoptosis compared to control cells. Based on the effect of autophagy on tumor cell death, we postulated that Glut-1 depletion-mediated autophagy activity was responsible for the death of HEp-2 cells, suggesting that targeting Glut-1 may be useful in the treatment of laryngeal carcinoma by regulating autophagy.
The mTOR (mechanical target of rapamycin) signaling pathway is considered to be one of the most important pathways regulating autophagy [41]. Rapamycin (RAP) can relieve the inhibition of ATG1 and promote autophagy by inhibiting the activity of mTOR42. Furthermore, activated ATG1 can form a protein complex through a series of ubiquitination-like ligation reactions with other autophagy-related factors, such as ATG3 and ATG5, in a manner dependent on the activity of phosphorylated Beclin-1 [43]. Beclin-1 is a key protein in the process of autophagy in mammalian cells and is a direct mediator of autophagy [20,28,29,43]. Beclin-1 protein complex mediates autophagosome formation at the start of autophagy [20,28,29,43]. In this process, the free form of LC3 (LC3-I) in the cytoplasm forms LC3-II by binding to the phosphatidylethanolamine group and integrates into the autologous bilayer membrane structure to complete closure of the autophagosome [20,28,29,43]. In addition, the level of autophagy-related gene Beclin-1 expression in head and neck cancer tissues was shown to be significantly lower than that in adjacent tissues, indicating that Beclin-1-mediated autophagy participates in the progress of tumor formation [20]. In the present study, RAP markedly induced autophagy in HEp-2 cells.
We also confirmed that Beclin-1 upregulation promoted, while Beclin-1 downregulation inhibited, autophagy followed by alteration of LC3-II/LC3-I rate, ATG5 level, and tumor characteristics, suggesting that Beclin-1 is critical for autophagy flux in laryngeal carcinoma development. Furthermore, Beclin-1 downregulation significantly restored the promotion effect of Glut-1 knockdown on autophagy activation and improvement of oncogenesis. However, the inhibitory effect on tumor growth was stronger with Beclin-1 inhibition alone than with Glut-1 shRNA plus Beclin-1 shRNA, suggesting that the mechanism of Glut-1 depletion-induced inhibition of tumor growth may be related to Beclin-1-mediated autophagy activation.

Conclusion
This study was performed to explore the relationship between Glut-1 knockdown and Beclin          The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/jQ5DSw