Upregulated miR‑378a‑3p expression suppresses energy metabolism and promotes apoptosis by targeting a GLUT‑1/ALDOA/PKM2 axis in esophageal carcinoma
- Authors:
- Published online on: August 10, 2023 https://doi.org/10.3892/ol.2023.14007
- Article Number: 421
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Copyright: © Qu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Esophageal cancer is an aggressive malignancy of the digestive system with incidence rates of >450,000 cases worldwide, and 5-year-survival rates ranging from 15–25% (1). Data from the Chinese Cancer Center in 2022 demonstrated that esophageal cancer was the sixth most common malignant tumor and the fifth leading cause of cancer-related death (2). There are two major histological subtypes of esophageal cancer; namely, esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (3). ESCC is the predominant histological classification of esophageal cancer in China with a 10-year survival rate of 14%, despite recent advances in therapeutics (4–6). Thus, further investigations into potential biomarkers for diagnosis and treatment, including oncogenes and tumor suppressor genes, are required.
MicroRNAs (miRNAs/miRs) are small RNAs that regulate the expression of complementary mRNAs (7). Numerous roles of miRNA have been identified in complex biological processes, such as metabolism (8), cancer (9), programmed cell death (10), cell proliferation, and differentiation (11). The results of a previous study demonstrated that there are a higher number of miRNAs in animals, and their regulatory impact is more pervasive than was previously suspected. Additionally, the same study showed that miRNAs can be used as diagnostic and prognostic markers for cancer (12). For example, miR-195 expression in ESCC is associated with a low survival rate (13). In colorectal cancer, the expression levels of miR-135b and miR-590-5p are associated with clinical stage and survival (7,14). miR-31-3p, −3676, −125a-5p, −100-5p, −125b-5p, −200a-5p, and miR-342 were significantly associated with the development of chemo- and radio-resistance in patients with locally advanced cervical cancer (15,16).
miR-378a is an intronic miRNA located in the peroxisome promoter-activated receptor γ activator 1-β gene and has been the focus of numerous studies (12,17–20). miR-378a-3p is the guide strand of miR-378a, and it is extensively associated with metabolism, while miR-378a-5p is regarded as the passenger strand (12). Notably, miR-378a-3p is the target gene of the oncogene Myc, which promotes tumor formation and angiogenesis in A549 adenocarcinoma cells (17). The results of further studies demonstrated that the expression of miR-378a-3p was reduced in ESCC tissues, which may lead to the overexpression of GLUT-1/SLC2A1 (21,22). Notably, the downregulation of miR-378a-3p induced decidual cell apoptosis, which contributed to early pregnancy loss (23). Therefore, the biological roles of miR-378a-3p in tumor cells remain contested. In addition, the association between functions, energy metabolism, and cell survival mechanisms in ESCC cells remains to be fully elucidated.
The energy metabolism of cancer cells, which exhibit increased metabolic requirements, is markedly different from healthy cells. In addition, cancer cells consume high quantities of glucose and produce lactic acid rather than catabolizing glucose through the tricarboxylic acid cycle. During this process, cancer cells generate higher levels of energy to further support their rapid proliferation (24). Genes associated with glycolysis, such as Aldolase A (ALDOA), Enolase 1 (ENO1), lactate dehydrogenase A (LDHA), phosphofructokinase L (PFKL), phosphoglycerate kinase 1 (PGK1), hexokinase-2 (HK2), 2,3-phosphoglyceraldehyde-3-phosphate dehydrogenation (GAPDH), and pyruvate kinase M2 (PKM2) participate in the energy metabolism of nucleosides, amino acids, and glucose, affect cell survival and apoptosis, and play a role in drug resistance and other biological mechanisms, to maintain the stability of tissue and the cellular environment (25,26).
The coding gene of Survivin, BIRC5, is located on human chromosome 17q25 and consists of three introns and four exons (27). Survivin is the smallest member of the inhibitor of apoptosis protein family. Wild-type Survivin is composed of 142 amino acids and often exists as a homodimer in the cytoplasm. Numerous previous studies have demonstrated that Survivin interacts with a variety of endogenous and exogenous apoptotic regulatory factors, such as Bad and Bcl-2, and significantly inhibits apoptosis via directly inhibiting the activities of Caspase-3 and Caspase-7, to block induced apoptosis (28–31).
In the present study, miR-378a-3p mimic and negative control were transfected into ECA-109 cells to detect and analyze the molecular regulatory process of miR-378a-3p. This interfered with its target genes, energy metabolism and the apoptosis of ESCC cells. The study will help us gain a deeper understanding of the regulatory mechanism of miR-378a-3p on tumor cells at the molecular biology level, and help us to understand the process of its intervention in tumor cells; it may also provide a novel theoretical basis for targeting miR-378a-3p as an interventional measure in the future.
Materials and methods
Cell culture
ESCC cell line ECA-109 was purchased from The BeNa Culture Collection (BNCC). The BNCC data showed that ECA-109 esophageal cancer cell line, established in 1973, originated from human esophageal middle squamous cell carcinoma tissue (ESCC). ECA-109 cells are considered one of the most representative models of ESCC (32–34). STR profiling was performed to confirm the identity of the cells. The cells were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin solution (penicillin 100 U/ml + streptomycin 100 mg/ml; all from Biological Industries). Cells were maintained in a humidified incubator at 37°C supplied with 5% CO2.
Determination of miR-378a-3p targets
First, the downstream target genes of miR-378a-3p in miRDB (https://mirdb.org) were screened. In view of its known interference in some energy metabolism related proteins, the 3′ non-coding regions of all glucose metabolism-related gene mRNAs were compared. Due to the phenomenon of incomplete matching of miRNA sequences that interfere with the target protein translation process (8,9), if the 6–8 nt at the 5′end of miRNA was complementary to the target gene, and the basic principle of ‘A’ at the first nucleotide of the miRNA corresponding to the target gene (8,9), the gene was considered a target gene (Fig. 1A).
qPCR analysis
Total RNA was extracted from the cells using TRIzol® reagent (cat. no. 15596026; Thermo Fisher Scientific, Inc.). The extracted Total RNA was reverse transcribed into cDNA using the Mir-X miRNA first strand synthesis kit according to the manufacturer's instructions (cat. no. 638315; Clontech; Takara Bio USA, Inc.), and qPCR was performed using SYBR Green I Master (cat. no. 4887352001; Roche Diagnostics), with U6 as an inner control. A ightCycler 480 II (Roche Diagnostics) was used for the qPCR. The qPCR conditions were as follows: Pre-denaturation at 95°C for 15 min, followed by 40 cycles of 10 sec of denaturation at 95°C, annealing at 60°C for 20 sec and extension at 72°C for 20 sec. The fluorescence signals were collected at 72–95°C after the reaction for melting curve analysis. The results were analyzed using a hyperbolic curve and the relative gene expression was determined. The forward primer sequences of miR-378a-3p were forward (F), 5′-ACTGGACTTGGAGTCAGAAGGC-3′ and reverse (R), mRQ 3′ primer in Mir-X miRNA First-Strand Synthesis Kit (cat. no. 638315; Clontech; Takara Bio USA Inc.). Primer sequences for U6 were F, 5′-GGAACGATACAGAGAAGATTAGC-3′ and R, 5′-TGGAACGCTTCACGAATTTGCG-3′. The comparative Cq method was used to quantify gene levels using the formula: ΔCq=CqmiRNA-CqU6. ΔΔCq=ΔCqApoE-ΔCqWT. The relative quantification for miRNA was calculated as 2−∆∆Cq. Expression of miRNA was normalized by U6. The specific primers/sequences for amplifying miRNAs and mRNAs are listed in Table I.
Overexpression of miR-378a-3p
miRNA mimics and their negative control counterparts, miRNA-sponge or miRNA-shRNA expression plasmids were obtained, and the information on their sequences and construction vectors are shown in Table I. Transient transfection with miRNA and DNA (expression plasmid) was performed using Lipofectamine™ 3000 (cat. no. L3000015; Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocols. Cells (2×105) were seeded into 6-well plates 1 day before transfection with 50 pmol miRNA mimics and negative control or 2.5 µg plasmid DNA using 5.0 µl Lipofectamine. After transient transfection (15 h), cells were washed with PBS (cat. no. C10010500BT; Thermo Fisher Scientific, Inc.) and used for experiments.
miR-378a-3p-shRNA overexpression and negative control plasmids (Table I) were transfected into cells during the logarithmic growth stage using electroporation (conditions: U, 280 V; C, 900 CF; R, 550 Ω) with Opti-MEM reduced serum medium (Gibco; Thermo Fisher Scientific, Inc.) with Gene Pulser XcellTM Electroporation System (Bio-Rad, USA).
Protein expression and cellular localization of TF-1 apoptosis-related gene-19 (TFAR19)
Prior to cell inoculation, a cover glass was placed in each well of a 6-well plate. Cells (1×105) were inoculated on the cover glass to make cell slides. miR-378a-3p mimic RNAs were transfected into cells in the logarithmic growth stage using Lipofectamine 3000. After 15 h of transfection, the medium was discarded, and the cells were rinsed twice with PBS buffer. Subsequently, cells were fixed with 4% paraformaldehyde (cat. no. G0528; GBCBIO Technologies, Inc.) for 30 min at room temperature (22–25°C), washed twice with PBS and sealed with blocking buffer for 1 h at room temperature.
Following blocking, cells were incubated overnight at 4°C with the anti-TFAR19 primary antibody (1:300; cat. no. K107297P; Beijing Solarbio Science & Technology Co., Ltd.). Following primary incubation, cells were washed with PBS for 5 min, and subsequently incubated with FITC-goat anti-rabbit secondary antibody (1:1,000; cat. no. A0562; Beyotime Institute of Biotechnology) for 1–2 h at room temperature. Cells were rinsed with PBS three times for 5 min each time, and stained with DAPI (Beyotime Institute of Biotechnology) in the dark for 3 min. Cells were rinsed again using PBS three times for 5 min each time. A drop of an anti-fluorescence quenching sealing agent was added to the slide and cells were subsequently observed using an inverted fluorescence microscope at ×400 magnification (IX83; Olympus Corporation).
Western blotting
Following 24/48 h transfection, cells were collected and lysed at 4°C for 30 min using RIPA lysate (Boster Biological Technology) supplemented with 1% protease inhibitor (Thermo Fisher Scientific, Inc.) and 1% phosphatase inhibitor (Thermo Fisher Scientific, Inc.). The supernatant was centrifuged (13,000 × g at 4°C for 15 min) to obtain the total protein. Protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). A total of 25 µg protein was loaded per lane on a 10% SDS-gel, resolved using SDS-PAGE, and transferred to PVDF membranes (Roche Diagnostics GmbH), which were subsequently blocked with TBS-Tween-20 (TBS-T) containing 5% skimmed milk at room temperature for 1 h. Following blocking, membranes were incubated overnight at 4°C with the following primary antibodies against GLUT-1 (1:100,000; Abcam, cat. no. ab115730), ALDOA (1:1,000; Abcam, cat. no. ab252953), PKM2 (1:1,000; Cell Signaling Technology, Inc., cat. no. 4053T), H2AX (1:1,000; Cell Signaling Technology, Inc., cat. no. 7631S), p-H2AX (1:1,000; Cell Signaling Technology, Inc., cat. no. 9718S), Caspase-3 (1:1,000; Cell Signaling Technology, Inc., cat. no. 9662S), cleaved Caspase-3 (1:1,000; Abcam, cat. no. AB32042), Bad (1:1,000; Cell Signaling Technology, Inc., cat. no. 9268S), Survivin (1:1,000; Cell Signaling Technology, Inc., cat. no. 2808S), Bcl-2 (1:1,000; Cell Signaling Technology, Inc., cat. no. 15071S), β-actin (1:1,000; Cell Signaling Technology, Inc., cat. no. 3700S), and TFAR19 (1:1,000; Beijing Solarbio Science & Technology Co., Ltd., cat. no. K107297P). The following secondary antibodies were used: anti-mouse antibody (Cell Signaling Technology, Inc., cat. no. 7076S, Lot#35, 1:4,000) anti-rabbit antibody (Cell Signaling Technology, Inc., cat. no. 7074S, Lot#30, 1:3,000). Following primary incubation, membranes were incubated at room temperature for 1 h with the secondary antibody, anti-mouse/rabbit HRP-conjugated antibodies. Following washing with TBS-T, signals were visualized using an enhanced chemiluminescence detection reagent (Thermo Fisher Scientific, Inc.). β-actin was used as the loading control. Image J version 1.53 was used for densitometry (National Institutes of Health).
Flow cytometry
A FITC Annexin V Apoptosis Detection kit (BD Biosciences) was used to detect the apoptosis of cells. Transfected cells in each group were collected, washed twice with pre-cooled PBS, and suspended in 1X binding buffer. A total of 5 µl FITC and 5 µl PI were added for incubation for 15 min at 25°C. Subsequently, 400 µl 1X binding buffer was added to each tube and analyzed using flow cytometry (FACSAria II) with BD FACSDiva software (version 8.0.2) (Becton, Dickinson and Company) within 1 h.
ELISA
Cells in the control, NC and miR378-mimics groups transfected for 24 or 48 h were collected and washed with pre-cooled PBS three times. A total of 250 µl NP40 lysate was added to cells for 30 min until cells were fully lysed, and the supernatant was collected. The ELISA kits (Human ALDOA ELISA Kit, cat. no. E-EL-H0309-96T, Lot# BRZUPTIBFG; Human GLUT-1 ELISA Kit, cat. no. E-EL-H1822-96T, Lot# 3MIHWZGF38; Human PKM2 ELISA Kit, cat. no. E-EL-H1089-96T, Lot# IUNGNXEV9W; Elabscience Biotechnology, Inc.) were used according to the manufacturer's instructions. The optical density of each well was measured using a microplate reader at a wavelength of 450 nm. The concentration of total protein was considered the level of the enzyme.
Detection of the dual-luciferase reporter assay
A Dual Luciferase Reporter Gene Assay kit (Beijing Solarbio Science & Technology Co., Ltd.) was used to assess the target genes of miR-378a-3p (Table I). Cells were co-transfected with the wild-type/mutant plasmid DNA of the respective firefly luciferase target genes + Renilla luciferase plasmid DNA ± miR-378a-3p plasmid DNA, and cultured for 24/48 h. Lysate was collected after cells (200 µl lysate/1×106 cells) were fully lysed at 4°C for 5 min. A total of 20 µl cell lysate was added to 100 µl 1X firefly/Renilla luciferase reaction solution, and luciferase activity was detected. Firefly luciferase activity/Renilla luciferase activity was measured as luciferase reporter gene activity.
ATP synthesis inhibition
Oligomycin (concentration, 200 nM) was used in ECA109 cells in the logarithmic growth phase for 12 or 24 h to inhibit ATP synthetase and decrease the ATP content in cells.
ATP detection
ATP Detection kit (Beijing Solarbio Science & Technology Co., Ltd.) was used to determine the ATP content of cells. Cells were suspended with ATP extract (1 ml/5×106 cells) and lysed using ultrasonication. Chloroform was mixed with 100 µl supernatant or the control and 900 µl ATP detection working solution, and the absorbance was measured at 10 sec and 3 min 10 sec with a wavelength of 340 nm (recorded as A1 and A2, respectively), using an ultraviolet spectrophotometer. DA=A2-A1; DA/(DA standard/C standard) × V extract/5 as ATP content (µmol/106 cells).
Detection of mitochondrial membrane potential
An enhanced mitochondrial membrane potential detection kit (JC-1) (cat. no. C2003S, Beyotime Institute of Biotechnology) was used to determine the mitochondrial membrane potential. For each well of a 6-well plate, the media was removed, cells were washed with PBS once, 1 ml cell culture medium was added, and 1 ml JC-1 dye working solution was added and mixed thoroughly. Next cells were incubated for 20 min. After incubation, the supernatant was aspirated, and cells were washed twice with JC-1 dye buffer. and 2 ml cell culture medium was added. Cells were observed using an inverted fluorescence microscope at ×100 and ×400 magnification (IX83; Olympus Corporation). When detecting JC-1 monomers, the excitation light wavelength was set to 490 nm and the emission light wavelength was set to 530 nm; when observing the JC-1 polymer, the excitation light wavelength was set to 525 nm and the emission light wavelength was set to 590 nm.
Statistical analysis
All data were analyzed in GraphPad Prism (version, 8.0.0, GraphPad Software, Inc.]. All quantitative data are presented as the mean ± standard deviation. A one-way ANOVA followed by Tukey's post hoc test was used to analyze the difference between multiple groups. P<0.05 was considered to indicate a statistically significant difference. All the experiments were performed at least three times.
Results
Baseline miR-378a-3p expression levels in ECA-109 cells
A miR-378a-3p-sponge overexpression vector was used to establish the baseline miR-378-3p expression levels (Table I). The results showed that the expression of miR-378a-3p was very low in ECA-109 cells (Fig. S1A and B), indicating that its expression as a tumor suppressor gene was suppressed. At low levels, miR-378a-3p had limited effect on the normal biological activity of ECA-109 cells as the miR-378a-3p-sponge had limited effect on GLUT-1, ALDOA, and PKM2 protein levels in ECA-109 cells (Fig. S1C and D).
ALDOA/GLUT-1/PKM2 are target genes of miR-378a-3p
To identify the target genes of miR-378a-3p, a dual-luciferase reporter assay was performed using a GV272 vector containing the wild-type or mutant miR-378a-3p-binding sequences in the 3′-untranslated region (UTR) of ALDOA, GLUT-1, PKM2, LDHA, and GAPDH. miR-378a-3p mimic and the reporter vector were co-transfected into ECA-109 cells. The results of the present study indicated that miR-378-3p markedly suppressed the dual-luciferase activity of key enzymes of glycolysis, including ALDOA, GLUT-1, and PKM2 (Fig. 1).
Expression levels of glycolysis-related enzymes are downregulated by miR-378a-3p mimic transfection
Based on the aforementioned findings that miR-378a-3p directly targeted glycolytic enzymes, the effects of miR-378a-3p on the glycolysis pathway were determined. Following miR-378a-3p transfection, the association between miR-378a-3p and energy metabolism was assessed. The major enzymes of the glycolysis pathway were detected using western blotting and ELISA. Protein expression levels and enzyme activities of PKM2, ALDOA, and GLUT-1 were inhibited between 24 and 48 h following transfection of miR-378a-3p (Fig. 2).
Survivin expression levels are downregulated, while Bad and Caspase-3 expression levels are upregulated by miR-378a-3p
To investigate the potential mechanism by which miR-378a-3p affected the apoptosis of ECA-109 cells at the protein level, protein expression levels of Bad, Caspase-3, and Survivin were investigated. The results showed that in the group of transfected with miR-378a-3p mimics, Survivin expression (an apoptosis inhibitor protein) was markedly decreased, while Caspase-3 and Bad expression (apoptosis-related proteins) were increased (Fig. 3). Cleaved caspase-3 expression was increased (Fig. S2). To confirm that there was no DNA damage involved in the early stage of apoptosis, histone variant H2AX phosphorylation was detected by western blotting, and it was shown that their protein levels were not increased following miR-378a-3p overexpression (Fig. S2).
Transfected with miR-378a-3p mimics promotes cell apoptosis
A cell apoptosis assay was performed to determine the biological function of miR-378a-3p. ECA-109 cells were transfected with miR-378a-3p mimics and negative control RNAs, and the cells were collected 24 and 48 h later. The results showed that in the miR-378a-3p mimic treatment group, the number of apoptotic cells in the early stage was significantly higher than that in the control or miR-NC negative control group (Fig. 4).
TFAR19 nuclear translocation is enhanced by miR-378a-3p
TFAR19, also known as programmed cell death 5 (PDCD5), cellular localization analysis was performed to verify the role of miR-378a-3p in promoting cell apoptosis. There was notable TFAR19 nuclear translocation in the miR-378a-3p mimics transfected group (Fig. 5A-D).
Energy metabolism is suppressed by miR-378a-3p
To further clarify the impact of miR-378a-3p on cancer cell apoptosis, the ATP content was determined. Following transfection with miR-378a-3p mimics or negative control miRNA for 24/48 h, it was shown that miR-378a-3p blocked energy metabolism, resulting in a decrease in ATP in the miR-378a-3p group (Fig. 5E). The mitochondrial membrane potential was also significantly decreased (Figs. S3 and S4).
Oligomycin leads to ATP loss, reduced expression of Survivin and Bcl-2, and increased cell apoptosis
To further verify the association between energy metabolism and apoptosis, oligomycin was used to inhibit ATP synthase. The results of the present study demonstrated that the ATP content, and the protein expression levels of Survivin and Bcl-2 were significantly decreased following treatment with oligomycin. These results indicated that inhibition of ATP synthesis may lead to apoptosis via alterations in the expression of Survivin and Bcl-2 (Fig. 6).
Discussion
miRNAs have been widely studied due to their anti-oncogenic functions. miRNAs are small non-coding RNA molecules that bind target mRNAs via complementary sequences in the 3′-UTR to inhibit their expression. Low expression of miR-378a-3p is closely associated with an unfavorable prognosis, which has been demonstrated in numerous different types of cancer cells, such as pancreatic cancer (35), ovarian cancer (36), and ESCC tissues (21). However, the results of previous studies involving the biological functions of miR-378 in tumor cells remain contested. Gao et al (37) demonstrated that miR-378 was involved in the inhibition of apoptosis of tumor cells by impacting the activity of p53 and Caspase-3 in lung cancer cells (37). In gastric cancer tissues, Yang et al (38) demonstrated that miR-378 may be a tumor suppressor gene, promoting cell apoptosis through BMP2 (38). Cui et al (39) also showed that miR-378a-3p/5p inhibited the tumor migration and invasion of oral squamous carcinoma cells through decreasing KLK4 expression (39). The miR-378a-3p/GLUT-1 regulatory model may be a novel therapeutic target in the metabolic remodeling of patients with ESCC (22). Survivin is an important inhibitor of apoptosis and plays a role in the survival of tumor cells (31). Survivin supports the survival of tumor cells by inhibiting the endogenous apoptosis pathway and the activity of apoptosis-related factors (30). In addition, Survivin is associated with the Akt/mTOR and NF-κB signaling pathways, which have previously been shown to be active in different cancer cell lines (40–47). Moreover, the results of a previous study demonstrated the positive cyclic regulation of Survivin and NF-κB (48). However, there are no studies assessing the association between miR-378a-3p and Survivin to the best of our knowledge. Thus, further investigations into the regulatory mechanisms of energy metabolism and apoptosis in ESCC cells may improve the current understanding of its regulation, and provide a novel experimental basis for determining the biological role and significance of miR-378a-3p in ESCC cells.
The glycolytic bioenergetics pathway is an important source of energy in tumor cells. The primary metabolic enzymes in this pathway include GLUT-1, HK2, PFKL, ALDOA, GAPDH, PGK1, ENO1, PKM2, and LDHA. Eichner et al (49) determined that miR-378a-3p decreased HK2 and LDHA expression, which may enhance oxidative phosphorylation, and suppress cell growth and tumorigenicity. Wang et al (50) also showed that miR-378a-3p repressed GLUT-1 expression by directly targeting its 3′UTR, and thus accelerated oral squamous cell carcinoma metastasis.
In the present study, it was first confirmed that ALDOA and PKM2 were the direct downstream target genes of miR-378a-3p. The results of the present study demonstrated that miR-378a-3p may block energy production in tumor cells by inhibiting the protein expression levels of GLUT-1, ALDOA, and PKM2. Following miR-378a-3p overexpression, ATP content, and mitochondrial membrane potential were reduced and apoptosis was promoted via Survivin, Bcl-2, Bad, and Caspase-3. Moreover, to determine the association between cell energy metabolism and apoptosis, an inhibitor of ATP synthesis, oligomycin, was used to treat cells. The results of the present study demonstrated that the levels of apoptosis inhibitory proteins (Survivin and Bcl-2) were reduced, and apoptosis was increased following treatment; thus, confirming that miR-378a-3p may induce apoptosis through the intervention of cell energy metabolism. TFAR19 was initially identified as a widely expressed apoptosis-accelerating protein. TFAR19 rapidly translocates from the cytoplasm to the nucleus in the early stage of apoptosis (30,51). The results of the present study demonstrated that TFAR19 nuclear translocation was significant in the miR-378a-3p transfection group. Moreover, the results of the present study demonstrated that miR-378a-3p promoted apoptosis of tumor cells was mediated by an endogenous apoptosis mechanism related to the impairment of mitochondrial membrane potential, particularly in the early stage. Survivin, Bcl-2, Bad, and Caspase-3 are common proteins in the apoptotic pathway. Notably, alterations in the expression levels of Survivin, Bcl-2, Bad, and Caspase-3 in the miR-378a-3p overexpression group demonstrated that the mode of cell death induced by miR-378a-3p was apoptosis. These results indicated that miR-378a-3p promotes the apoptosis of tumor cells by increasing the expression and the activity of Bad and Caspase-3, and reducing the expression of Survivin and Bcl-2. The results demonstrating the energy metabolism of ESCC cells provide a novel experimental basis to further understand the association between energy metabolism and apoptosis. Notably, the results of the present study are consistent with those of previous studies, demonstrating the decline of metabolism, including the reduction of glycolysis, ATP levels, and protein production, and the triggering of the apoptosis pathway (49,50). Compared with previous studies, it is hypothesized that miR-378a-3p may promote apoptosis through interfering with cell energy metabolism in ESCC cells (16,30,32).
As energy metabolism is vital for the survival of tumor cells, further investigations into the specific molecular mechanism of miR-378a-3p in the regulation of enzymes associated with energy metabolism are required.
The present study is limited, as the mechanism by which miR-378a-3p inhibited the enzyme activity of HK2 and LDHA in ECA-109 cells remains to be fully elucidated. Thus, whether miR-378a-3p induced cell apoptosis via Survivin and the Akt/mTOR/NF-κB signaling pathway requires further investigation. Additionally, the influence of miR-378a-3p on cell migration, invasion, and proliferation should be determined.
In conclusion, the results of the present study revealed a novel association between miR-378a-3p, glycolysis, and apoptosis. Specifically, miR-378a-3p may block energy production and promote the apoptosis of tumor cells by downregulating glycolytic enzyme expression in ECA-109 cells. miR-378a-3p inhibits ATP synthase, increases the expression levels and activity of Bad and Caspase-3, and decreases the expression levels of Survivin and Bcl-2. These results provide novel evidence demonstrating that miR-378a-3p is a tumor suppressor gene. In addition, the results of the present study demonstrated that Survivin, Bad, and Bcl-2 may be associated with the miR-378a-3p-induced inhibition of energy production, and the miR-378a-3p-induced promotion of apoptosis. The anti-tumor effect of miR-378a-3p may provide a novel treatment option for the management of ESCC.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
This study was supported by funding the National Nature Science Foundation of China (grant nos. 81460359 and 81460419).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
HL and HWL conceived and designed the experiments. YQ, SX, YJD, and LTH performed the experiments. YQ, YJZ and GPZ analyzed the data. YQ, YJZ and HWL confirm the authenticity of all the raw data. All authors reviewed and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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