Introduction
According to the latest annual global cancer statistics report, lung cancer is the most commonly diagnosed cancer (11.6% of the total cases) and the leading cause of cancer death (18.4% of the total cancer deaths) in the whole world [
1]. Primary lung cancers are usually classified into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for approximately 83% of all lung cancers, and nearly 80% of NSCLC patients are diagnosed with advanced or distant stages [
2]. NSCLC comprises multiple histological, genetical, and metabolical procedures. One predominant feature of NSCLC is the active glucose metabolism, which can be visualized by high
18F-FDG uptake on positron emission tomography/computed tomography (PET/CT) imaging [
3,
4]. Recently,
18F-FDG PET/CT has been recommended routinely used in the clinical staging of NSCLC according to the 8th edition of the TNM staging system [
5].
The underlying mechanism of
18F-FDG uptake is that cancer cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of abundant oxygen. This metabolic reprogramming, known as the Warburg effect or aerobic glycolysis, has been regarded as a new hallmark of cancer [
6,
7]. The Warburg effect facilitates cancer cells to rewire their metabolism to harness cellular stress to thrive and is an optimized way to favor their survival, progression, and metastasis [
8]. The aerobic glycolysis of cancer cells is regulated by several master transcription factors, most notably the c-Myc transcription factor [
9]. Recent studies have demonstrated that c-Myc, which is a frequently amplified human oncogene, functions as a key regulator of the Warburg effect by directly activating several glycolytic genes [
9‐
11]. Although the underlying mechanisms of the Warburg effect by protein-coding genes have been extensively studied, the potential functions and mechanism of the more recently identified lncRNAs in cancer metabolism remain largely unknown [
12].
With the development of RNA sequencing techniques, integrative genomic studies have shown that more than 90% of the DNA sequence is actively transcribed, but only < 2% of these transcripts encode protein, while the majority of the transcripts are referred to as non-coding RNAs (ncRNAs) [
13]. Among these ncRNAs, lncRNAs are a class of transcripts with lengths greater than 200 nucleotides. The lncRNAs are emerging as a significant regulator responsible for various biological processes, such as cell growth, cell migration, cell invasion, and metabolic rewiring [
14‐
16]. These abnormally expressed lncRNAs have been implicated as potential alternative biomarkers or therapeutic targets for NSCLC [
17,
18]. Although a growing number of lncRNAs have been annotated, the specific roles and molecular mechanisms of lncRNAs in regulating aerobic glycolysis of NSCLC remain poorly understood [
19].
In the current study, RNA sequencing analysis was performed between NSCLC tumor tissues with high 18F-FDG uptake and their corresponding noncancerous tissues. An upregulated lncRNA—LINC01123—was found to exert oncogenetic function in promoting proliferation as well as glycolysis. Moreover, LINC01123 was directly transcribed by c-Myc and inversely increased c-Myc expression level. This study indicated the existence of a positive feedback loop between LINC01123 and c-Myc, suggesting a novel mechanism in interpreting metabolic reprogramming and malignant progression of NSCLC.
Materials and methods
Clinical samples and RNA sequencing assay
Two independent cohorts were enrolled. Cohort 1: Fresh NSCLC tumor tissues and adjacent tissues (5 cm from the tumor edge) were obtained from three patients in November 2017 at Shanghai Chest Hospital. Cohort 2: Specimens from 92 patients who underwent surgery between January 2008 and July 2013 were acquired from the surgical specimen archives of Renji Hospital, School of Medicine, Shanghai Jiaotong University, and patients’ follow-up visit continue to June 2018. The data was censored at the last follow-up visit or at the time of the patient’s death without relapse. No recruited patients received any preoperative treatment. All patients were staged based on the criteria of the 8th Edition of the Lung Cancer Staging Manual [
5]. This research was approved by the institutional clinical research ethics committee of Shanghai Chest Hospital and Renji Hospital, School of Medicine, Shanghai Jiaotong University. Written informed consent was obtained from each patient, and the study was conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS).
Next-generation RNA sequencing assay was performed to detect the mRNA and ncRNA expression profiles at KangChen Bio-tech (Shanghai, China) using Illumina HiSeq 4000 (Illumina, San Diego, CA, USA). Solexa pipeline version 1.8 was used to align the reads to the genome, generate raw counts corresponding to each known gene (a total of 17,242 genes), and calculate the RPKM (reads per kilobase per million) values. The differential expression lncRNAs and mRNAs were identified through fold change/p value/FDR filtering (fold change ≥ 1.5, P value < 0.05, and FDR < 0.05).
Total RNA extraction and qRT-PCR
Total RNA was isolated by TRIzol Kit (Omega, Norcross, GA, USA), and the quantity of total RNA was measured by using a NanoDrop equipment (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using the cDNA Synthesis kit (Takara, Otsu, Japan). Real-time PCR was performed using SYBR Green PCR Master Mix (Takara) in a StepOnePlus RT-PCR system (Thermo Fisher Scientific). All target genes were normalized to the endogenous reference gene β-actin by using an optimized comparative Ct (2
ΔΔCt) value method. To measure the level of miR-199a-5p, miRNA extraction kit (Vazyme, Nanjing, China) and a stem-loop miRNA Synthesis Kit (Vazyme) were used according to the manufacturer’s instructions. U6 snRNA was used as the endogenous control. The sequences of primers used in this study are listed in Additional file
7: Table S1.
Subcellular RNA fractionation
Subcellular fractionation was performed with a PARIS™ Kit (Ambion, Austin, TX) according to the manufacturer’s instructions. The nuclear and cytoplasmic RNA was further analyzed by qPCR. β-Actin and U6 were used as cytoplasmic and nuclear controls, respectively.
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) analysis was performed according to a previously described method [
20]. Briefly, specific target probes were made for LINC01123. The hybridization was performed using RNAscope Fluorescent Reagent Kit (Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s instructions. Staining score was assessed by two independent reviewers as 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. Tumor cells in five fields were selected randomly and scored based on the percentages of positively stained cells (1 = 0–25%, 2 = 26–50%, 3 = 51–75%, 4 = 76–100%). The final scores were computed by multiplying the intensity score and the percentage score of positive cells. According to the score, samples were divided into two groups, the negative and low expression group (score 0–6) and high expression group (score 7–12).
Cell lines and culture condition
Five NSCLC cell lines, A549, H1299, H1650, H1975, and PC9, human normal lung epithelial cell line HBE, and human diploid fibroblast IMR-90 cells were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (GIBCO, Grand Island, NY, USA) at 37 °C with 5% CO2. All cells were tested for mycoplasma contamination and had no mycoplasma contamination.
Plasmids, oligonucleotides, siRNA, and transfection
LINC01123 pcDNA3.1 vector (1123-OE), c-Myc pcDNA3.1 vector (Myc-OE), and empty vector (vector) were subcloned into the expression vector pcDNA3.1 (Invitrogen, USA). MiR-199a-5p mimic, negative control oligonucleotides (mimics NC), miR-199a-5p inhibitor, negative control oligonucleotide (inhibitor NC), small interfering RNA of LINC01123 or c-Myc (si-1123, si-Myc), and scramble siRNA (si-NC) were purchased from GenePharma (Shanghai, China). The cells were seeded into six-well plates and cultured overnight until 70–80% confluence. Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. The transfection efficiency was determined using qRT-PCR and Western blot. Sh-1123 and sh-NC lentivirus were purchased from GenePharma (Shanghai, China) and constructed into A549 cell lines for further in vivo experiments. The transfection efficiency was evaluated by fluorescence microscopy by calculating the percentage of fluorescein-labeled cells.
Cell proliferation assay and colony formation assay
The relative cell viability at 24, 48, and 72 h after transfection was monitored using the Cell Counting Kit-8 (CCK-8, Bimake, Shanghai, China) according to the manufacturer’s protocol. Briefly, 5 × 103 cells were cultured in a 96-well plate at 37 °C. After 10 μL CCK-8 solution was added to each well, plates were incubated at 37 °C for 1 h. After that, the optical density at 450 nm (OD450) was measured for each sample.
As for the colony formation assay, a total of 500 cells were seeded in 6-well plates and cultured in a humidified atmosphere containing 5% CO2 at 37 °C for 2 weeks. Cell colonies were washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet (1 mg/mL) for 20 min. All the experiments were repeated in triplicate, and the mean was calculated.
Ethynyldeoxyuridine analysis
Ethynyldeoxyuridine (EdU) detection kit (RiboBio, Guangzhou, China) was used to assess cell proliferation according to the manufacturer’s instruction. Cells were cultured in 96-well plates at 5 × 103 cells/well. Ten microliters of EdU labeling media was added to the 96-well plates and then incubated at 37 °C under 5% CO2 for 2 h. After treatment with 4% paraformaldehyde and 0.5% Triton X-100, the cells were stained with the anti-EdU working solution and Hoechst 33342. Subsequently, the cells were visualized using a fluorescence microscope (Olympus, Tokyo, Japan). The EdU incorporation rate was calculated as the ratio of the number of EdU-positive cells (green cells) to the total number of Hoechst 33342-positive cells (blue cells).
Glucose uptake, lactate production, LDH enzyme activities, intracellular ATP, and ROS level
As an analog of glucose, 18F-FDG uptake assay could reflect the intracellular glucose uptake level of cells. The cells were seeded into 12-well plates at 1 × 105 cells/well and cultured overnight. After the culture medium was removed and washed twice with PBS, cells were incubated in 1 mL of glucose-free DMEM containing 74–148 kBq/mL (2–4 μCi/mL) 18F-FDG for 1 h at 37 °C. Then, wash the cells twice with PBS and add 1 mL 0.5 M NaOH per well to produce cell lysates. A well γ-counter was used to detect the radioactivity of lysates. The intracellular 18F-FDG uptake was radioactive readouts normalized to corresponding cell numbers. Three independent experiments were performed during our study.
For lactate production measurements and LDH enzyme activity, cell supernatant was collected to measure lactate concentration (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), while the cell pellets to be lysed and measured ATP level (Nanjing Jiancheng) according to the manufacturer’s instructions. ROS was measured by fluorescent 2′, 7′-dichlorofluorescin diacetate (DCF-DA) as described by manufacturer’s protocol of a commercial kit (Nanjing Jiancheng).
The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using the Seahorse XF 24 Extracellular Flux Analyzer (Seahorse Bioscience), according to a previously described method [
20]. ECAR and OCR were measured using Seahorse XF Glycolysis Stress Test kit and Seahorse XF Cell Mito Stress Test kit (Agilent Technologies), respectively.
Xenograft mouse model and micro-PET/CT
Tumorigenicity assays in nude mice were performed. Our study was approved by Renji Institutional Animal Care and Use Committee (Shanghai, China). The procedures were performed according to the guidelines and regulations of Renji Hospital, School of Medicine, Shanghai Jiaotong University (Shanghai, China). This study was carried out in accordance with the recommendations that cover all scientific procedures involving the use of live animal. Briefly, 4-week male BALB/c nude mice were purchased from Renji Hospital Experimental Animal Center (Shanghai, China). The mice were inoculated subcutaneously with 1 × 107 cells in the right flank with sh-1123, while sh-NC in the left flank.
After 3 weeks, a micro-PET/CT scanner (Super Nova® PET/CT, PINGSENG Healthcare Inc., Shanghai, China) was used to measure 18F-FDG uptakes in the mice. PET/CT scanner is ~ 0.6 mm, and the resolution of the CT is 0.2 mm. 18F-FDG (0.2 mL, 7.4 MBq) was injected into the tail vein of tumor-bearing mice. After 30 min, the animals were anesthetized with 2% isoflurane and immobilized during 20 min PET scan acquisition. PET images were reconstructed with the ordered-subsets expectation maximization (OSEM) algorithm using 16 subsets and 4 iterations. An irregular region of interest, which covered the entire tumor, was drawn on the CT and then copied to the co-registered PET using Avatar 1.2 software (Pingseng, Shanghai, China). 18F-FDG uptake by tumors was assessed by the maximum standard uptake value (SUVmax). After PET/CT scan, mice were sacrificed and tumors were excised and weighed.
Immunohistochemistry
Immunohistochemistry (IHC) was performed and measured as reported previously [
20].
Western blot analysis
Cells were lysed using radioimmunoprecipitation assay (RIPA) lysis solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, and complete protease inhibitor cocktail) for 30 min on ice, then centrifuged at 15,000×
g for 30 min at 4 °C. Cell extracts were boiled for 5 min at 100 °C, and the protein samples were analyzed by Western blot. Western blot analysis was cultured as previously described [
21]. c-Myc polyclonal antibody (Proteintech, Chicago, USA), HK2 and LDHA polyclonal antibody (Proteintech), and anti-β-actin monoclonal antibody (Proteintech) were used according to the manufacturer’s protocol. Immunoreactive bands were visualized using ECL Western blot kit (Amersham Biosciences, Buckinghamshire, UK).
Immunofluorescence (IF)
Cells were cultured in six-well plates on glass coverslips, fixed for 20 min with 4% formaldehyde, and permeabilized with 0.25% Triton X-100; the cells were treated with blocking buffer for 30 min and incubated overnight at 4 °C with the c-Myc polyclonal antibody (1:500, Proteintech), followed by incubation with the secondary antibody at room temperature for 1 h. Cell nuclei were counterstained with DAPI. Confocal laser scanning microscope (Olympus BX61) was used to observe the image.
Luciferase reporter assay
To determine the effect of c-Myc on LINC01123 promoter, pcDNA-c-Myc, pcDNA-vector, si-Myc, or si-NC was individually co-transfected into 293 T cells together with the pGL3-based construct containing LINC01123 WT or MUT promoter sequences plus Renilla luciferase reporter plasmid. Twenty-four hours after transfection, firefly and Renilla luciferase activity were measured by a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The ratio of firefly luciferase to Renilla activity was calculated for each sample. To evaluate the effect of miR-199a-5p on LINC01123 or c-Myc 3′UTR, 293 T cells were co-transfected with the pmirGLO-LINC01123-WT, pmirGLO-LINC01123-MUT, pmirGLO-c-Myc-3′UTR-WT, or pmirGLO-c-Myc-3′UTR-MUT reporter plasmids individually together with mimics NC, miR-199a-5p mimics, inhibitor NC, or miR-199a-5p inhibitor, respectively. To confirm the competing binding of miR-199a-5p between LINC01123 and c-Myc 3′UTR, pcDNA-LINC01123 or pcDNA-vector was co-transfected with miR-199a-5p mimics and pmirGLO-c-Myc-3′UTR-WT or pmirGLO-c-Myc-3′UTR-MUT, respectively. Si-1123 or si-NC was co-transfected with miR-199a-5p inhibitor and pmirGLO-c-Myc-3′UTR-WT or pmirGLO-c-Myc-3′UTR-MUT, respectively. Twenty-four hours after transfection, firefly and Renilla luciferase activity were measured by a Dual-Luciferase Reporter Assay System (Promega). Experiments were performed in triplicate, and the data are represented as mean SD.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) was performed with a ChIP assay kit (Beyotime, Haimen, Jiangsu, China) according to the manufacturer’s protocol. Briefly, A549 and H1299 cells were cross-linked with 1% formaldehyde for 10 min and sonicated to shear DNA to lengths between 200 and 1000 base pairs. Cell lysates were precleared with protein A/G beads before they were incubated at 4 °C overnight with protein A/G beads coated with the anti-c-Myc antibody (2 μg, Proteintech). Anti-rabbit immunoglobulin G (IgG) was also used as a negative control. After extensive washing, the bead-bound immunocomplexes were eluted using elution buffer. To reverse histone-DNA crosslinks, samples were added with 5 M NaCl and heated at 65 °C for 4 h, then treated with proteinase K and further incubated at 45 °C for 1 h. The bound DNA fragments were purified by DNA Extraction Kit (GeneMark, Shanghai, China) and subjected to real-time PCR using the specific primers.
RNA immunoprecipitation assay
RNA immunoprecipitation (RIP) was performed using a Magna RNA-binding protein immunoprecipitation kit (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. Briefly, cells were collected and lysed in complete RIPA buffer containing a protease inhibitor cocktail and RNase inhibitor. Next, the cell lysates were incubated with RIP buffer containing magnetic bead conjugated with human anti-Ago2 antibody (Proteintech) or control normal mouse IgG. The samples were digested with proteinase K to isolate the immunoprecipitated RNA. The purified RNA was finally subjected to real-time PCR to demonstrate the presence of the binding targets.
Statistical procedures
Statistical analysis was performed using SPSS 20.0 software (SPSS, Inc., Chicago, IL) and GraphPad Prism 7.0 (GraphPad Software, Inc., USA). The results are presented as mean ± SD. Comparison between two groups was assessed using Student’s t test (two-tailed, with P < 0.05 considered significant). The chi-square test, one-way analysis of variance, and Pearson’s correlation were also performed. The survival curves were calculated using the Kaplan-Meier method, and the differences were assessed by a log-rank test. Cox multivariate regression analysis was used to determine the independent factors that influenced survival and recurrence. All results were reproduced across triplicate experiments. One asterisk, two asterisks, and three asterisks indicate P < 0.05, P < 0.01, and P < 0.001, respectively.
Discussion
Multiple evidences have verified that lncRNAs are aberrantly expressed in various tumor types, where they hold promise utilization for cancer diagnosis, monitoring, prognosis, or prediction for therapeutic responsiveness [
18]. NSCLC is the most common type of lung cancer and is characterized by the dysregulation of gene networks including both protein-coding genes and non-coding RNAs [
2]. To date, numerous lncRNAs are identified in NSCLC, presenting new perspectives for exploring molecular pathways in NSCLC pathogenesis [
25]. In this study, we sought to search the aberrantly expressed lncRNAs by RNA-seq analysis of high
18F-FDG uptake NSCLC tissues. This current study found that LINC01123 was significantly upregulated in RNA-seq expression file and was associated with poor clinical outcomes in NSCLC patients, thus might represent as an independent prognostic biomarker in NSCLC.
As commonly recognized these years, cancer cells exhibit a unique metabolic phenotype with increased glucose uptake and lactate release to support their malignant biological processes [
26]. Many studies have indicated that lncRNAs are intimately connected to the regulation of Warburg effect to support growth and survival of cancer cells [
12,
27]. Elucidating the metabolic-related functions of lncRNAs provides a better understanding of the regulatory mechanisms of metabolism [
28]. For example, lncRNA PCGEM1, by directly interacting with c-Myc and being a coactivator for c-Myc, functions as a master regulator of metabolic reprogramming in cancer [
29]. Another notable example is lincRNA-p21, which is a hypoxia-responsive lncRNA. Being activated by HIF-1α, lincRNA-p21 in return stabilizes HIF-1α by disrupting the VHL-HIF-1α interaction, promoting glycolysis and OXPHOS downregulation [
30]. Consistent with the previously reported lncRNAs, our study indicated that LINC01123 functioned as an oncogene by facilitating tumor malignant phenotype, as well as mediating energy status. It regulated metabolic adaptation by upregulating glycolytic gene expression and enzyme activity, thus promoting glycolysis in vitro and
18F-FDG uptake of subcutaneous xenograft in vivo.
c-Myc is a human oncogene and contributes to multiple hallmarks of cancer. As a transcriptional factor, early studies identify that c-Myc transcriptional targets are involved in many biological processes, such as metabolism, cell growth, cell cycle regulation, and apoptosis [
31]. Besides a large number of protein-coding genes, many lncRNAs and microRNAs are newly proved downstream targets of c-Myc [
32]. Lu et al. reported Myc targeted lncRNA DANCR, which was overexpressed in a variety of tumor types, could promote cancer cell proliferation [
33]. LncRNA MINCR was another Myc-induced lncRNA able to modulate Myc’s transcriptional network in Burkitt lymphoma cells [
34]. We here showed that LINC01123 was a novel transcript by c-Myc, which further participated in tumor malignant transforming processes. Our study thus expanded the breadth of transcriptional roles of c-Myc underlying glucose dependence of NSCLC.
The expression of c-Myc is under the tight control of many regulatory mechanisms, which is exquisitely regulated at the transcription, translation, protein stability, and activity levels. In recent years, it has become clear that lncRNAs add a crucial additional layer to the regulation of Myc and its downstream effects [
35]. Zhang et al. reported that lncRNA-MIF (Myc inhibitory factor) had the ability to increase Fbxw7 mRNA expression, which was a characterized E3 ubiquitin ligase, thus causing c-Myc protein degradation [
36]. To the best of our knowledge, gene modulation at mRNA level is a fast-acting strategy for cancer cells to adapt to susceptible environment and maximum cell survival [
37]. LncRNA CCAT1 and SNHG3 were identified to modulate c-Myc mRNA expression via competing endogenous RNA (ceRNA) activity by sponging miR-155 or miR-182-5p, respectively [
38,
39]. Here, we proved the ceRNA crosstalk network that LINC01123 served as a decoy to sequester miR-199a-5p from binding c-Myc mRNA, relieving its inhibitory effect on c-Myc expression. Rescue experiments indicated that LINC01123 functioned as an oncogene in promoting glycolysis as well as tumor growth through c-Myc-dependent pathway. Additionally, previous studies have revealed miR-199a-5p as a tumor suppressor in many tumor types, and it could suppress the Warburg effect by targeting HIF-1α [
40‐
42]. In general, these results together illustrated the complicated and multi-dimensional interactions between LINC01123 and c-Myc, further elucidating the molecular mechanism of tumor progression and metabolic rewiring in NSCLC.
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