MicroRNA-124 suppresses proliferation and glycolysis in non–small cell lung cancer cells by targeting AKT

Abstract

Non–small cell lung cancer accounts for 85% of all types of lung cancer and is the leading cause of worldwide cancer-associated mortalities. MiR-124 is epigenetically silenced in various types of cancer and plays important roles in tumor development and progression. MiR-124 was also significantly downregulated in non–small cell lung cancer patients. Glycolysis has been considered as a feature of cancer cells; hypoxia-inducible factor 1-alpha/beta and Akt are key enzymes in the regulation of glycolysis and energy metabolism in cancer cells. However, the role of miR-124 in non–small cell lung cancer cell proliferation, glycolysis, and energy metabolism remains unknown. In this research, cell proliferation was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; furthermore, glucose consumption and lactic acid production were assessed; adenosine triphosphate content and NAD⁺/NADH were also detected. These tests were conducted using the normal non–small cell lung cancer cell line A549, which was transfected variedly with miR-mimics, miR-124 mimics, miR-124 inhibitor, pc-DNA3.1(+)-AKT1, and pc-DNA3.1(+)-AKT2 plasmid. Here, we show that miR-124 overexpression directly decreased cell growth, glucose consumption, lactate production, and energy metabolism. MiR-124 also negatively regulates glycolysis rate–limiting enzymes, glucose transporter 1 and hexokinase II. Our results also showed that miR-124 negatively regulates AKT1 and AKT2 but no regulatory effect on hypoxia-inducible factor 1-alpha/beta. Overexpression of AKT reverses the inhibitory effect of miR-124 on cell proliferation and glycolytic metabolism in non–small cell lung cancer. AKT inhibition blocks miR-124 silencing–induced AKT1/2, glucose transporter 1, hexokinase II activation, cell proliferation, and glycolytic or energy metabolism changes. In summary, this study demonstrated that miR-124 is able to inhibit proliferation, glycolysis, and energy metabolism, potentially by targeting AKT1/2–glucose transporter 1/hexokinase II in non–small cell lung cancer cells.

Keywords

Non–small cell lung cancer miR-124 AKT1/2 hypoxia-inducible factor 1-alpha/beta glycolysis

Introduction

Lung cancer is believed to be one of the most common causes of cancer-related deaths worldwide. Despite advances in the detection and treatment of lung cancer, the overall 5-year survival for patients with this disease has not been substantially reduced during the past years.¹ Approximately, 85%–90% of all lung cancer deaths were resulted by non–small cell lung cancer (NSCLC). The molecular basis of NSCLC is complex and heterogeneous. Surgical resection remains the most effective treatment of early-stage NSCLC, but many patients with NSCLC still develop tumor metastasis and recurrence after pulmonary resection. However, the molecular pathways underlying each step still remain obscure.^2,3

Normal somatic cells use mitochondrial oxidative phosphorylation as their main metabolic pathway. Cancer cells, however, preferentially use a far less efficient pathway, aerobic glycolysis, to metabolize glucose. This phenomenon, termed as the Warburg effect, is characterized by increased glycolysis and lactate production, even in the presence of oxygen.^4,5 The AKT pathway and hypoxia-inducible factor 1 (HIF-1) are major signaling cascade that regulates glucose metabolism as well as controls cellular growth.^6,7 They could enhance glucose transporter gene expression, increase phosphofructokinase activity, and stimulate the glucose to lactate metabolic pathway,^8,9 AKT mainly enhances hexokinase II (HKII) gene expression, thus promoting glucose capture.¹⁰

MicroRNAs (miRNAs) are single-stranded, small non-coding RNAs with 18–25 nucleotides in length. The miRNAs may act as oncogenes, tumor suppressors, and modulators of tumor proliferation, invasion, apoptosis, and therapy resistance. MiR-124 is generally considered as a tumor suppressor in several types of cancer, such as hepatocellular carcinoma, gastric cancer, and cervical cancer.^11,12 Previous study showed that low miR-124 expression is associated with higher vasculogenic mimicry, migration, and invasion of NSCLC cells.^13–15 Furthermore, the molecular mechanisms utilized by miR-124 to modulate the malignant phenotype of NSCLC cells are not fully understood. This study aimed to elucidate the role of miR-124 in mediating proliferation and glycolysis as well as the underlying mechanisms in NSCLC cells.

Materials and methods

Cell culture

Human NSCLC cell lines (A549) was grown in RPMI-1640 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA). All cells were cultured in a humidified atmosphere containing 5% CO₂ at 37°C.

MiRNA and transient transfection

All transient transfections were carried out using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) as described previously according to the manufacturer’s protocol. Transient transfection of miR-124 mimics, miR-124 inhibitor, scramble, pc-DNA3.1(+)-AKT1, and pc-DNA3.1(+)-AKT2 plasmids were obtained from Shanghai GenePharma Co., Ltd (Shanghai, China) and were transfected at a final concentration of 20 nM with lipofectamine 2000 according to the manufacturer’s protocol. After 48 h of miRNA transfection, the cells were harvested for further studies. MiR-124 mimics: 5′-UAAGGCACGCGGUGAAUGCCCAUUCACCGCGUGCCUUAUU-3′; miR-124 inhibitor: 5′-GGCAUUCACCGCGUGCCUUA-3′.

Cell proliferation assay

For all groups, 10,000 cells per well were plated on a 96-well plate. Following treatment, the plates were incubated for 0, 24, 48, or 72 h at 37°C in 5% CO₂. To assess cell proliferation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed according to the manufacturer’s instructions. In brief, 10 µL MTT reagent (5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) was added to each well and incubated for 4 h at 37°C in 5% CO₂. The supernatant was removed and 100 µL dimethyl sulfoxide (Beyotime Institute of Biotechnology, Shanghai, China) was added. The absorbance was detected at 490 nm using an Infinite M200 Pro type absorbance reader (Tecan, Switzerland).

Detection of glucose consumption and lactate production

Glucose-uptake experiments were conducted with each group according to the manufacturer’s instructions (BioVision, Milpitas, CA, USA). To measure lactate production, A549 cells from different treatment groups were collected and measured for lactate concentration using a Lactate ELISA Kit (BioVision) according to the manufacturer’s instructions with Infinite M200 Pro type absorbance reader (Tecan).

Measurement of intracellular NAD⁺/NADH

NAD⁺ and NADH levels were measured using a commercially available fluorimetric assay kit (BioVision). NAD⁺ and NADH were specifically recognized and detected using an enzyme cycling reaction and according to the manufacturer’s instructions.

Adenosine triphosphate assay

The level of adenosine triphosphate (ATP) in A549 cell lines was determined using the ATP Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol. Briefly, harvested cultured cells were lysed with a lysis buffer, followed by centrifugation at 10,000g for 2 min at 4°C. The level of ATP was determined by mixing 50 µL of the supernatant with 50 µL of luciferase reagent, which catalyzed the light production from ATP and luciferin. The emitted light was linearly related to the ATP concentration and measured using a microplate illuminometer.

Western blot analysis

The expression quantity of various proteins was determined in human lung cancer A549 cells using Western blotting according to the standard procedures. The antibodies against HIF-α/β, phosphoinositide 3-kinase (PI3K), Akt, glucose transporter 1 (GLUT1), HKII, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Abcam. In short, total protein from untreated or treated cells was extracted in radioimmunoprecipitation assay (RIPA) lysis buffer. The same amount of protein (25 µg) from each group were separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA). Each membrane was incubated with a specific primary antibody (1:1000) at 4°C overnight after blocking with 5% skim milk at room temperature for 1 h. After three washes with washing buffer (20 mmol/L Tris-HCl, 500 mmol/L NaCl, and 0.1% Tween 20), each suitable secondary antibody was incubated in the membrane at room temperature for 2 h. An ECL Advanced Western Blot Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to visualize the specific protein bands.

Statistical analysis

Data are expressed as the mean ± standard deviation (SD) of triplicate samples. Statistical analysis for multiple comparisons was analyzed by a one-way analysis of variance (ANOVA). p values below 0.05 were considered to be statistically significant.

Results

MiR-124 negatively regulates the proliferation and glycolysis of NSCLC cells

To investigate the effect of miR-124 on the proliferation and glucose metabolism of NSCLC, two groups of NSCLC cells were transfected with miR-124 mimics and its inhibitors, respectively. MTT assay results showed that miR-124 significantly influenced NSCLC cell proliferation. Specifically, the overexpression of miR-124 via the transfection of target miRNA mimics led to a decrease in absorbance (optical density (OD) value) at 490 nm in A549 cells (Figure 1(a)). In contrast, miR-124 knockdown via the transfection of target miRNA inhibitors led to an increase in the OD value at 490 nm in cells.

Figure 1.

MiR-124 negatively regulates the proliferation and glycolysis of NSCLC cells. (a) MTT assay was used to evaluate the results of miR-124 mimics/miR-124 inhibitors transfection that led to the absorbance (OD value) at 490 nm. (b) Glucose consumption and lactate production in miR-124 mimic/miR-124 inhibitor–transfected A549 cells was also evaluated. Data are presented as mean ± SEM (n = 3; *p < 0.05 vs control).

Glucose consumption and lactate production were the main index to evaluate the cellular glycolysis function. As shown in Figure 1(b), overexpression of miR-124 decreased the consumption of glucose and the production of lactate. However, after the inhibition of miR-124, the downregulation effect was inhibited and the cellular glucose consumption and lactate production were significantly promoted.

MiR-124 negatively regulates the energy metabolism of NSCLC cells

Cancer cells could provide the energy supplement through the Warburg effect to maintain their proliferation requirements. As Figure 2 showed, ATP content decreased in miR-124 overexpressing NSCLC cells but rose when miR-124 was knockdown. Although, the statistical results had no significant difference to control, the change trend of ATP is the same as miR-124. Moreover, the production of key metabolic products NAD⁺/NADH significantly decreased as miR-124 expression was silenced and raised in miR-124 overexpressing NSCLC cells.

Figure 2.

MiR-124 negatively regulates the energy metabolism of NSCLC cells. (a) ATP content was detected in both miR-124 mimic and miR-124 inhibitor–transfected cells. (b) NAD⁺/NADH in miR-124 mimic/miR-124 inhibitor–transfected A549 cells were also evaluated. Data are presented as mean ± SEM (n = 3; *p < 0.05 vs control).

MiR-124 negatively regulates glycolysis rate–limiting enzymes

MiR-124 plays a key role in the regulation of glycolysis and energy metabolism in NSCLC cells, as per the results shown in glycolysis and energy metabolism experiments. GLUT1 and HKII are the crucial speed-limiting enzymes in catalyzing specific reactions in glycolysis. So the influences of miR-124 to the speed-limiting enzymes were also evaluated. As demonstrated in Figure 3, the protein expression of HKII and GLUT1 was upregulated when miR-124 was silenced. Similarly, GLUT1 and HKII were all downregulated in miR-124 mimic–transfected cells; this indicates that miR-124 is involved in the regulation of HKII and GLUT1 and thus contributed to the activation of key enzymes in the Warburg effect.

Figure 3.

MiR-124 negatively regulates GLUT1 and HKII expression. The protein levels of GLUT1 and HKII in A549 cells (pretreatment with miR-124 mimics or miR-124 inhibitor) were determined by Western blotting.

MiR-124 has negative regulation effect on AKT1 and AKT2 and no effect on HIF-1α/β

Akt and HIF play a key role in the regulation of Warburg effect. We hypothesized that miR-124 might play a role in the Warburg effect by targeting Akt and HIF and negatively regulating their expression. AKT is the most frequently activated onco-protein in human cancers. There are three members in AKT isoforms (AKT1, AKT2, and AKT3). HIF-1 as a heterodimeric transcription factor has HIF-1α as the oxygen responsive subunit and HIF-1β as the constitutively expressed subunit. HIF-1α accumulates and binds to HIF-1β forming the active HIF-1 complex. To test our hypothesis, we employed miRNA mimics and inhibitors to specifically overexpress and knockdown endogenous expression of miR-124 in A549 cells. As shown in Figure 4, the expression of p-Akt1 and p-Akt2 was significantly decreased after the cells were being transfected with miR-124 mimics, but the protein levels were significantly increased after administration of miR-124 inhibitors. Meanwhile, no change in p-Akt3 and HIF-α/β was observed, indicating that p-Akt3 and HIF are not involved in miR-124 regulation of the Warburg effect in NSCLC cells.

Figure 4.

MiR-124 negatively regulates AKT1 and AKT2, no regulatory effect on HIF-α/β. Western blotting results show that miR-124 inhibition increased AKT1 and AKT2 levels, and miR-124 overexpression decreased AKT1 and AKT2 levels in A549 cells. While no change in AKT3 and HIF-α/β levels was observed.

Overexpression of AKT reverses the inhibitory effect of miR-124 on cell proliferation and glycolytic metabolism in NSCLC

In view of miR-124 could affect the protein expression of Akt, we examined the cell proliferation and glycolytic metabolism in Akt overexpressing NSCLC cells. The MTT assay results (Figure 5(a)) showed that overexpression of AKT1 and AKT2 could enhance cellular proliferation in NSCLC cell lines, but the proliferation experiment resulting in simultaneous overexpression of miR-124 and AKT1/2 group demonstrated no significant change. Akt participates in cancer cell glycolytic metabolism. The result showed that overexpression of AKT1/2 significantly increased glucose consumption and lactate production in A549 cells (Figure 5(b) and (c)). In addition, ATP content had also significantly increased, and the NAD⁺/NADH ratio was decreased (Figure 5(d) and (e)). However, these effects were significantly reduced after miR-124 overexpression.

Figure 5.

Overexpression of AKT reverses the inhibitory effect of miR-124 on cell proliferation, glycolytic metabolism, and energy metabolism in NSCLC. (a) MTT assay results reveal that both AKT1 and AKT2 overexpression led to an increase in absorbance (OD value) at 490 nm in A549 cells, while simultaneous overexpression of AKT1/2 with miR-124 led to decreased absorbance (OD value) at 570 nm. (b) AKT1/2 overexpression led to increased glucose consumption in NSCLC cells, while pretreatment with miR-124 led to no significant change in glucose consumption. (c) AKT1/2 overexpression led to increased lactate production, but pretreatment with miR-124 led to no significant change in lactate production. (d and e) AKT1/2 overexpression negatively regulates ATP and NAD⁺/NADH metabolism, but pretreatment with miR-124 led to no significant change. Data are presented as mean ± SEM (*p < 0.05 vs control and ^#p < 0.05 vs pcDNA 3.1 Akt1/2).

AKT inhibition blocks miR-124 silencing-induced AKT1/2 activation and glycolytic metabolic changes

To confirm the involvement of the miR-124-AKT axis in the regulation of the Warburg effect in NSCLC cells, we next silenced miR-124 and simultaneously employed AKT inhibitors to knockdown AKT expression. Cell proliferation and glycolytic metabolism in NSCLC were also evaluated. As demonstrated in Figure 6, two AKT inhibitors (A-674563 AKT1 inhibitor and CCT128930 AKT2 inhibitor) have been used. These AKT inhibitors could specifically inhibit AKT1 or AKT2 and thus selected for subsequent experiments. Western blotting showed that A-674563 could block the miR-124 silencing-induced activation of AKT1 and CCT128930 could block the activation of AKT2. The expression of HKII and GLUT1 was downregulated in miR-124 silenced cells when treated with AKT1/2 inhibitors. So this result indicated that the miR-124-AKT axis regulates the activity of key glycolytic enzymes in NSCLC cells (Figure 6(a)). Moreover, as shown in Figure 6(b)–(d), compared to treating the cells with miR-124 inhibitors alone, simultaneous treatment with AKT1 and AKT2 inhibitors resulted in significant change in cell proliferation and glycolytic metabolism. Pretreatment of either Akt1 or 2 inhibitor with miR-124 silenced cells also had significantly changed the cellular proliferation and glycolytic metabolism. Treatment of AKT1 and AKT2 inhibitors simultaneously also enhanced the NAD⁺/NADH ratio as well as decreased ATP production in NSCLC cells. These results indicate that Akt1/2 inhibitors blocked the miR-124 silencing-induced glycolytic metabolic changes and thus provide additional evidence of the function of the miR-124-AKT axis in regulating the Warburg effect in NSCLC cells.

Figure 6.

AKT inhibition blocks miR-124 silencing-induced AKT1/2, GLUT1, HKII activation, cell proliferation, and glycolytic metabolism or energy metabolism changes. (a) The protein levels of GLUT1 and HKII in A549 cells were determined by Western blotting. AKT inhibition blocks miR-124 silencing-induced AKT1/2, GLUT1, and HKII activation. (b) MTT assay results reveal that both AKT1 and AKT2 inhibitors led to a decrease in absorbance (OD value) at 490 nm in miR-124 silenced-A549 cells, while simultaneous inhibition of AKT1 and 2 led to significantly decreased absorbance (OD value) at 570 nm; AKT1 inhibitor (A-674563, abbreviated as A in Figure) or AKT2 inhibitor (CCT128930, abbreviated as CCT in Figure) led to decreased glucose consumption in NSCLC cells, while simultaneous inhibition of AKT1 and 2 led to significant change in glucose consumption. AKT1 or AKT2 inhibitor led to decreased lactate production, while simultaneous inhibition of AKT1 and 2 led to significant change in lactate production. AKT1 or AKT2 inhibitor led to negative regulation of energy metabolism, while simultaneous inhibition of AKT1 and 2 led to significant change. Data are presented as mean ± SEM (*p < 0.05 vs miR-124 inhibitor+A+C and ^#p < 0.05 vs miR-124 inhibitor).

Discussion

The Warburg effect, which was described as the propensity of cancer cells and tissues to take up glucose avidly and convert it almost exclusively to lactate (aerobic glycolysis), was the central tenet of cancer cell metabolism.^4,5 This effect occurs even in the presence of oxygen. It is a common feature of tumor cells. In the last decade, numerous miRNAs have been identified, and they play important roles in diverse cellular processes and in the development of disease by modulating gene expression at the post-transcriptional level. In addition, it has also been reported to be involved in aerobic glycolysis (Warburg effect) of tumor cells.

MiR-124, a brain-enriched miRNA, was first found to be involved in stem cell regulation and neuro-development. Previous studies indicated that miR-124 is epigenetically silenced in various types of cancer and play important roles in tumor development and progression. Studies have shown that miR-124 was significantly downregulated in NSCLC patients. Also, the methylation of miR-124-2 and miR-124-3 has been associated with an advanced tumor status of NSCLC.^13–15 Whether miR-124 is involved in regulating tumor glucose metabolism has not been reported yet. The regulatory mechanism of miR-124 in NSCLC remains to be studied.

In this study, the effect of miR-124 on cell viability of NSCLC was measured using the MTT assay at first. The growth of NSCLC cells was inhibited when treated with miR-124 mimics and significantly increased in miR-124 inhibitors group. Then, we focused on the effects of miR-124 on glycolysis of NSCLC cells. The glucose consumption and lactate production are key indicators of the glycolysis. In addition, the cancer cells promote the production of ATP through the Warburg effect to provide energy for the cell proliferation. In this study, we demonstrated that (1) miR-124 reduced glucose consumption and lactate production in NSCLC cells and (2) miR-124 suppressed ATP formation and increased NAD⁺/NADH ratio, revealing that miR-124 suppresses energy metabolism in cancer cells.

In order to engage in the Warburg effect, the cancer cells use much more glucose than normal cells, which harvests most of its energy from aerobic metabolism. GLUT1 is a glucose transporter that as the glucose influx into a cell is the first rate-limiting process involved in glucose metabolism.^16,17 HKII is a key enzyme that catalyzes the first step in the glycolysis pathway.^18,19 These two enzymes are key enzymes in the regulation of cellular glycolysis and energy metabolism. Our results demonstrated that miRNA-124 acts as a molecular switch to regulate glycolysis-related protein (GLUT1 and HKII). By giving NSCLC cells miR-124 mimics, the expression of GLUT1 and HKII was significantly decreased. But the GLUT1 and HKII expression was increased when NSCLC cells pretreated with miR-124 inhibitors.

The HIF or PI3K/AKT pathway is a major signaling cascade that regulates glucose metabolism as well as controls cellular growth. The upregulation of glucose transporter proteins (GLUTs) in cancer cells is an important aspect regulated by the HIF-1.^16,20 Furthermore, the activation of HIF-1 is able to upregulate the levels of HK1 and HKII in tumor cells.^21,22 Akt activation is known to regulate cell death, cell cycle progression, and cell growth, as well as controlling rates of glucose uptake into the cell via the GLUT1 transporter.^23,24 Previous research had shown that Akt was known to further influence glycolysis by HKII.^10,25 In this research, PI3K/Akt and HIF-1α/β were both detected in miR-124 mimics and miR-124 inhibitor groups. We found that (1) miR-124 was involved in the regulation of AKT1 and AKT2 expression, not in AKT3.and (2) miR-124 has no significant effect on HIF-1α/β.

To confirm the involvement of miR-124-AKT axis in the regulation of Warburg effect in NSCLC cells, AKT1/AKT2 was overexpressed in NSCLC cells. As a result it has been shown that (1) cellular proliferation and glycolytic metabolism were enhanced by overexpression of AKT1 and AKT2, while simultaneous overexpression of miR-124 and AKT1/2 led to no statistical change in cellular proliferation; (2) the overexpression of AKT1/2 increased ATP content and decreased NAD⁺/NADH, but this effect was inhibited by miR-124 mimics; (3) cell proliferation and glycolytic metabolism in NSCLC cells were increased in silenced miR-124 cells, this effect was disappeared when simultaneously employed with AKT1/2 inhibitors to knockdown AKT phosphorylation, while inhibition with either AKT1 or 2 could not completely offset this effect; and (4) the expression of HKII and GLUT1 was downregulated in miR-124 silenced cells when treated with AKT1/2 inhibitors.

Cancer cells employ aerobic glycolysis to generate biosynthetic materials that support cancer cell proliferation and build a tumor-favoring microenvironment that facilitates cancerous invasion and metastasis. We demonstrated in our study that miR-124 suppresses glycolysis by targeting AKT–GLUT1/HKII in NSCLC cells. In addition, the identification of miR-124 downregulation in NSCLC cells may increase the scope of potential therapeutics for NSCLC.

Footnotes

Acknowledgements

X.Z. and C.L. contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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MicroRNA-124 suppresses proliferation and glycolysis in non–small cell lung cancer cells by targeting AKT–GLUT1/HKII