Long Non-coding RNA MEG3 Activated by Vitamin D Suppresses Glycolysis in Colorectal Cancer via Promoting c-Myc Degradation

Colorectal cancer (CRC), a common tumor, is characterized by a high mortality rate. Long non-coding RNA maternally expressed gene 3 (MEG3) serves a regulatory role in the carcinogenesis and progression of several types of cancer; however, its role in CRC remains largely unknown. The aim of this study was to explore the regulatory role and mechanism(s) of MEG3 in CRC. The Warburg effect or aerobic glycolysis is characteristic of the metabolism of tumor cells. To determine the effect of MEG3 on glycolysis of CRC cells, we used an XF analyzer to perform glycolysis stress test assays and found that overexpression of MEG3 significantly inhibited glycolysis, glycolytic capacity, as well as lactate production in CRC cells, whereas knockdown of MEG3 produced the opposite effect. Mechanistically, overexpression of MEG3 induced ubiquitin-dependent degradation of c-Myc and inhibited c-Myc target genes involved in the glycolysis pathway such as lactate dehydrogenase A, pyruvate kinase muscle 2, and hexokinase 2. Moreover, we found that MEG3 can be activated by vitamin D and vitamin D receptor (VDR). Clinical data demonstrated that MEG3 was positively associated with serum vitamin D concentrations in patients with CRC. We found that 1,25(OH)2D3 treatment increased MEG3 expression, and knockdown of VDR abolished the effect of MEG3 on glycolysis. These results indicate that vitamin D-activated MEG3 suppresses aerobic glycolysis in CRC cells via degradation of c-Myc. Thus, vitamin D may have therapeutic value in the treatment of CRC.


INTRODUCTION
Colorectal cancer (CRC), the third most commonly diagnosed cancer, has a high cancer-related mortality rate worldwide (1,2). In China, CRC is the fourth most common cancer, with 376,000 new patients diagnosed in 2015, leading to about 191,000 deaths (3). However, the molecular mechanisms underlying CRC progression are not fully understood. More research is needed to discover and develop effective biomarkers and targets for diagnosis and treatment of CRC. Recently, accumulating evidence has shown that long non-coding RNAs (lncRNAs), non-coding RNA transcripts longer than 200 nucleotides (4), are expressed differentially in various cancers including CRCs, suggesting that lncRNAs have roles in tumorigenesis and tumor metastasis (5-7).
Among them, maternally expressed gene 3 (MEG3) has been reported to be aberrantly expressed in CRC and may act as a tumor suppressor (8)(9)(10). However, the mechanism of action of MEG3 in CRC requires further investigation.
It has been demonstrated that most cancer cells have altered energy metabolism characterized by glycolysis with lactate production and a higher uptake of glucose as the main source of energy even in the presence of oxygen, well-known as the "Warburg effect" (11,12). Under normoxic conditions, glycolysis is commonly driven by c-Myc (13,14), which upregulates glycolytic enzymes such as lactate dehydrogenase A (LDHA) and hexokinase 2 (HK2) (15)(16)(17). Many non-coding RNAs have been reported to be involved in the regulation of cancer metabolism (18). For example, lncRNA PVT1 promotes glycolysis and tumor progression by regulating the miR-497/HK2 axis in osteosarcoma (19), whereas lncRNA-MIF inhibits aerobic glycolysis and tumorigenesis by suppressing c-Myc and miR-586 in cancer cells (20).
In this study, we demonstrate that MEG3 activated by vitamin D and vitamin D receptor (VDR) suppresses activation of glycolysis by promoting c-Myc degradation under normoxic conditions.

Clinical Samples
A total of 80 CRC tissue samples and corresponding adjacent normal mucosal samples were collected at the First Affiliated Hospital of Nanjing Medical University. All samples were snapfrozen in liquid nitrogen immediately after collection and stored at −80 • C until total RNA was extracted. The clinicopathological characteristics of the patients with CRC from whom the samples were obtained are summarized in Table 1

Cell Culture
The human CRC cell lines DLD-1 and RKO were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China), and were maintained in RPMI-1640 (HyClone, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Gibco). All cell lines were cultured in a humidified incubator of 5% CO 2 at 37 • C. Cells were collected for further studies after treatment with cycloheximide (CHX) and MG132 (MedchemExpress, Monmouth Junction, NJ, USA) for various periods of time.

RNA Extraction and Real-Time Polymerase Chain Reaction
Total RNA was extracted from frozen tissues with TRIzol reagent (Invitrogen, Grand Island, NY, USA) and reverse transcribed to complementary DNA (cDNA) by using a PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan) according to the user's manual. Real-time polymerase chain reaction (RT-PCR) was performed with gene-specific primers to determine the relative expression of genes of interest using SYBR green reagents (Takara Bio) in an ABI 7300 sequence detector (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or ACTIN mRNA was used for normalization. The PCR primers used in this study are listed in Table S1.

CCK8 Assays
Cell proliferation was detected using a Cell Counting Kit 8 (Donjindo, Kumamoto, Japan). Briefly, cells (3 × 10 3 ) transfected with either siRNA or plasmid were seeded into 96-well plates, and cell growth was determined every 24 h for 4 days in accordance with the manufacturer's protocol. The absorbance at a wavelength of 450 nm was measured using a microplate reader (Bio-Tek, Winooski, VT, USA).

Colony Formation Assays
Transfected cells with siRNA or plasmid were seeded in six-well plates at 600 cells per well and 1,000 cells per well, respectively, and cultured for 10-15 days with replacement with new medium every 3 days. Colonies were fixed, stained with crystal violet, and photographed. For each treatment group, wells were counted in triplicate.

Transwell Assays
Cell migration and invasion assays were carried out using 24well Transwell chambers with 8-µm pore size polycarbonate membrane (Corning, NY, USA). A total of 6 × 10 4 or 8 × 10 4 cells in 200 µl serum-free medium were seeded in the upper chambers coated without or with Matrigel (BD Biosciences, NY, USA) for migration or invasion assays, respectively, after which 600 µl medium with 10% fetal bovine serum (FBS) (20% FBS for invasion assays) was added to the lower chambers. The cells on the upper surfaces of the membranes were removed 36-48 h later. Cells on the bottom surfaces of the membranes were fixed, stained with 0.1% crystal violet, and counted in five fields using a Zeiss microscope (Melville, NY, USA).

Glycolysis Stress Test
The extracellular acidification rate (ECAR) was measured using a Seahorse XF96 Analyzer Glycolysis (Seahorse Bioscience, Santa Clara, USA) according to the manufacturer's protocol. Briefly, DLD-1 or RKO cells in 10% FBS RPMI-1640 were seeded in XF 96-well plates and incubated at 37 • C in a 5% CO 2 humidified atmosphere overnight. The cells were then incubated in the glycolysis stress test medium without glucose, and the ECAR was measured. Following this, D-glucose (10 mM), oligomycin (1 µM), and 2-deoxyglucose (100 mM) were added into the wells at the indicated time points; meanwhile, corresponding ECARs were assessed. The ECAR values are presented as the mean ± SD of experimental triplicates. The key variables of glycolysis and glycolytic capacity were analyzed using XF Glycolysis Stress Test software.

Lactate Production Assay
CRC cells transfected with siRNA or plasmid or treated with vitamin D were cultured, and the culture medium was collected 24 h later. Lactate production was quantitated using a lactate assay kit (Jiancheng, Nanjing, China) according to the manufacturer's instructions. Total viable cell numbers were used for normalization.

Serum Vitamin D Measurement
Serum 25(OH)D 3 concentrations were measured by chemiluminescence using an ADVIA Centaur Vitamin D Total (VitD) Assay kit (Siemens Healthineers, Erlangen, Germany) according to the manufacturer's instructions.

In vivo Ubiquitination Assay
In vivo ubiquitination assays were performed according to a previously described protocol (7)

Statistical Analysis
All data are presented as mean ± SEM. Student's t-test was used to compare data between two groups. Overall survival curves were plotted using the Kaplan-Meier method and analyzed with the log-rank test. Correlations were analyzed with Pearson r analysis. The clinicopathological characteristics of the 80 patients with CRC were subjected to logistic regression analysis. P < 0.05 was considered to denote statistical significance.

MEG3 Is Downregulated in CRC and Associated With Tumor Prognosis
Downregulation of MEG3 has been observed in CRC tissues (8,21). To further confirm this, we used RT-qPCR to measure MEG3 expression in 80 CRC samples and the matched samples of adjacent normal mucosa. Consistent with others' findings, we found that there was significantly lower expression of MEG3 in CRC tissues (P < 0.05) than the corresponding adjacent normal mucosa ( Figure 1A). Low expression of MEG3 was significantly associated with advanced CRC clinical stage (P < 0.05) ( Figure 1B). However, in our study, there was no significant association between the level of MEG3 expression and  various other clinical variables such as sex, age, and tumor size ( Table 1). We also examined the relationship between MEG3 expression and clinical outcomes, including overall survival. We divided the patients into two groups on the basis of the fold difference in expression of MEG3 in the tumor and the corresponding adjacent normal tissue: MEG3-high group (fold difference >1, n = 22) and MEG3-low group (fold difference <1, n = 58) and then performed a Kaplan-Meier survival analysis and log-rank tests. We found that overall survival was significantly poorer in the MEG3-low group than that in the MEG3-high group (P < 0.05) (Figure 1C). These results indicate that MEG3 expression is associated with cancer progression and poorer prognosis in patients with CRC.

MEG3 Inhibits Cell Proliferation and Invasion
We next investigated the effect of MEG3 in CRC cells. We first transfected pcDNA-MEG3 vector or specific MEG3 siRNAs into DLD-1 and RKO cells. As shown as Figure 2A, pcDNA-MEG3 transfection increased the expression of MEG3, whereas siRNAs  (Figure 2C). The effects of MEG3 on migration and invasion were then investigated. The results of the Transwell assays showed that overexpression of MEG3 significantly impaired CRC cell migratory and invasive ability, whereas knockdown of MEG3 strengthened these abilities compared with control cells (Figures 2D,E).
Taken together, the above data suggest that MEG3 inhibits CRC cell proliferation and invasion.

MEG3 Influences Glucose Metabolism in Colorectal Cancer Cells
Proliferation of tumor cells is reportedly usually accompanied by metabolic changes (11). Glycolysis is a hallmark of cancer cells.
To investigate the role of MEG3 in glycolysis in CRC cells, we performed glycolysis stress test assays by using an XF analyzer to measure the ECAR. We found that MEG3 overexpression significantly inhibited glycolysis and glycolytic capacity in DLD-1 and RKO cells (Figure 3A), whereas MEG3 knockdown produced the opposite effect ( Figure 3B). Moreover, we assessed cellular lactate production and found that MEG3 overexpression inhibited lactate production (Figure 3C), whereas knockdown of MEG3 by siRNA boosted lactate generation ( Figure 3D). These data indicate that MEG3 inhibits glucose metabolism in CRC cells.

MEG3 Reduces c-Myc Protein Stability
Previous studies have demonstrated that c-Myc is primarily responsible for metabolic reprogramming of cancer cells (13). We therefore next investigated whether c-Myc participates in MEG3-mediated inhibition of glycolysis. As shown in Figure 4A, we found that MEG3 overexpression inhibited amounts of c-Myc protein and its targeted genes (HK2, PKM2, LDHA), whereas MEG siRNA did the opposite. The results of RT-qPCR indicated that amounts of mRNA of HK2, PKM2, and LDHA decreased in MEG3-overexpressed cells, whereas c-Myc did not change (Figure 4B), suggesting that MEG3 decreases c-Myc expression in the protein level. To verify this, we performed CHX treatment and found that the half-life of c-Myc in pcDNA3.1 is longer than that in pcDNA-MEG3 (59.19 ± 3.39 min for DLD-1/pcDNA3.1 vs. 37  ( Figure 4C). These data showed that MEG3 overexpression reduced the c-Myc stability. To determine whether MEG3 affects proteasomal degradation of c-Myc, we performed In vivo ubiquitination by transfecting pcDNA-Ub-HA and pcDNA-c-Myc together with pcDNA-MEG3 into DLD-1 or RKO cells in which we immunoprecipitated ubiquitin with anti-HA antibody and detected it by anti-Flag. We found that overexpression of MEG3 increased polyubiquitination of c-Myc (Figure 4D). Treatment with MG132, a proteasome inhibitor to inhibit protein degradation through proteasome-dependent pathway, rescued c-Myc protein expression (Figure 4E), suggesting that MEG3 affects c-Myc stability in proteasome-dependent degradation. Numerous studies have shown that E3 ubiquitin ligase of FBXW7 controls proteasome-mediated degradation of c-Myc (22). We therefore measured the expression of FBXW7 in MEG3 overexpression and MEG3-knockdown cells and found that MEG3 induced the expression of FBXW7 (Figure 4F). Meanwhile, knockdown of MEG3 reversed the FBXW7 level induced by MEG3 overexpression in CRC cells (Figure 4G).
These results indicate that MEG3 may contribute to inhibiting glycolysis through promoting c-Myc degradation by increasing the amounts of FBXW7.

MEG3 Activated by Vitamin D Through Vitamin D Receptor
A previous study has shown that vitamin D can promote MEG transcription through binding VDR (10). To further investigate this, we first measured serum concentrations of vitamin D [25(OH) 2 D 3 ] in samples from CRC patients and found that these concentrations correlate positively with MEG3 expression in CRC tissues (Figure 5A). To further determine the effect of vitamin D on MEG3 expression, qRT-PCR analysis was performed and showed that amounts of MEG3 increased with vitamin D treatment and VDR overexpression but decreased with VDR knockdown (Figures 5B-D). Above all, our clinical and cellular data suggest that MEG3 expression is indeed positively regulated by vitamin D through VDR.

Vitamin D Receptor Knockdown Abolishes the Effect of MEG3 on Glucose Metabolism
To further determine whether vitamin D or VDR affects the effects of MEG3 on glucose metabolism, we performed glycolysis stress assays and lactate production assays. As shown in Figure 6A, both in DLD-1 and RKO cells, VDR knockdown greatly reversed MEG3 overexpression-induced inhibition of glycolysis and glycolytic capacity. Lactate production also increased consistently after VDR knockdown in MEG3-overexpressed cells (Figure 6B). Previous studies have shown that vitamin D decreases glycolysis in various cancers, including CRC (23)(24)(25). We speculated whether MEG3 mediated vitamin D-induced inhibition of glycolysis. To investigate this possibility, we treated MEG3-knockdown cells with vitamin D and performed glycolysis stress assays and lactate production assays. The results showed that vitamin D treatment inhibited significantly the glycolysis and glycolytic capacity and decreased the lactate production of CRC cells (Figures 6C,D). These inhibitions are partially rescued by MEG3 knockdown (Figures 6C,D). These data implicate that vitamin D inhibits glycolysis in CRC cells partially through the VDR/MEG3 pathway.

DISCUSSION
CRC had the third highest annual age-standardized incidence (2009-2013) worldwide, being 40.7 per 100,000 people with a mortality rate (2010-2014) of 14.8 per 100,000 persons (1,2). However, the molecular mechanisms that underlie CRC tumorigenesis are yet to be fully elucidated. There is increasing evidence that lncRNAs acting as oncogenes or anti-oncogene factors have functions in tumorigenesis and tumor metastasis in CRC cells (5,8,26,27).
MEG3 is aberrantly expressed in multiple types of cancers and is assumed as a tumor suppressor (10,28,29). MEG3 participates in carcinogenesis and cancer progression by regulating gene expression through chromatin modification, transcription, and posttranscriptional procession. For example, MEG3, as a competing endogenous RNA, reduces the invasiveness of human bladder cancer cells by competing with PHLPP2 mRNA for miR-27a (30); MEG3 inhibits the proliferation of epithelial ovarian cancer cells by regulating ATG3 activity and inducing autophagy (31); and MEG3 inhibits the proliferation and invasion of gallbladder cancer by associating with EZH2 and promoting its ubiquitination (32). Some recent studies have reported that the amounts of MEG3 are significantly reduced in tissue and serum from patients with CRC and that this can serve as a prognostic marker in such patients (8,28,33). However, the role of MEG3 in CRC remains largely unknown. In the present study, we also found that MEG3 expression was lower in tumor tissue than adjacent normal tissues from patients with CRC ( Figure 1A). We identified downregulation of MEG3 in advanced stages of CRC, and this was closely associated with poor overall survival of the patients (Figures 1B,C). Functional studies demonstrated that MEG3 inhibited CRC cell proliferation, migration, and invasion (Figure 2), which is consistent with previous studies. Moreover, we found that MEG3 inhibited glycolysis in CRC cells (Figure 3).
Increased glycolysis is the main source of energy in cancer cells, which use this metabolic pathway to generate ATP (34), this being known as the Warburg effect because it was first described by Otto Warburg in the 1920s (35). Many studies have found that lncRNAs can regulate the Warburg effect to influence the growth and survival of cancer cells (36)(37)(38). The mechanism of lncRNA-mediated regulation is complex; however, many studies have found that lncRNAs can affect genes involved in glucose metabolism. It has been extensively documented that the oncogene of c-Myc is a key regulator of the Warburg effect by directly activating several glycolytic genes, including LDHA, PKM2, and others (39)(40)(41). Several studies have shown that lncRNAs regulate processes of glycolysis in cancer by altering amounts of c-Myc and, more specifically, by modifying the transcriptional patterns on c-Myc target genes. For example, lncRNA GLCC1 promotes glucose metabolism in CRC cells by protecting c-Myc from ubiquitination via direct interaction with heat shock protein (HSP)90 chaperone (42). LINC01123 promotes non-small-cell lung carcinoma (NSCLC) cell proliferation and aerobic glycolysis by increasing c-Myc mRNA expression with sponging miR-199a-5p (43). In the current study, we found that MEG3 inhibited c-Myc expression and its target glycolytic genes including HK2, PKM2, and LDHA (Figures 4A,B). Expression of c-Myc is largely posttranslationally regulated by E3 ubiquitin ligase, which binds c-Myc to promote its degradation. In the current study, we found that MEG3 inhibited the expression of c-Myc by promoting its ubiquitination, further increasing its degradation (Figures 4C-E). Moreover, the increase in ubiquitination of c-Myc that was induced by MEG3 may result from upregulation of FBXW7 ( Figure 4F). However, how MEG3 regulates FBXW7 is not clear and requires further study.
Multiple studies have consistently shown an inverse association between serum vitamin D concentrations and risk of CRC (44)(45)(46). Our previous study also showed that vitamin D-deficient mice develop colonic inflammation (47). In the current study, we found a positive correlation between serum vitamin D concentration and MEG3 expression in patients with CRC ( Figure 5A); to the best of our knowledge, we are the first to document a correlation between MEG3 and serum vitamin D. In addition, we showed that MEG3 can be induced by vitamin D via VDR transcription factor (Figures 5B-D), which is consistent with previous research (10). Moreover, VDR knockdown and MEG3 knockdown retard inhibition of glycolysis induced by MEG3 or vitamin D, respectively (Figures 6A-D). Interestingly, we found that glycolysis and glycolytic capacity and lactate production were consistently lower in VD + siRNA-MEG3 compared to vehicle + siRNA-MEG3 cells. Considering that vitamin D could regulate many genes by binding VDR, it might be possible that additional inhibitory mechanisms are involved in the glycolysis inhibited by vitamin D. Our data suggest that MEG3 activated by vitamin D is partly responsible for the anti-CRC effect of vitamin D.
In conclusion, in this study, we found that MEG3 is significantly decreased in CRC tissues and is positively associated with serum vitamin D concentrations in patients with CRC, indicating that it is a potential prognostic biomarker and therapeutic target for CRC. Vitamin D-activated MEG3 suppresses aerobic glycolysis of CRC cells via degrading c-Myc, suggesting that vitamin D may have therapeutic value in the treatment of CRC.
Thus, the present data indicate that the anticancer function of vitamin D may be executed via the VDR/MEG3 pathway.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Research Ethics Committee of Nanjing Medical University [Approval ID: (2016)640]. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
XY designed the study and wrote the manuscript. SZ and LW developed the methodology and performed the analyses. YW collected the data.