Novel long non‐coding RNA CYB561‐5 promotes aerobic glycolysis and tumorigenesis by interacting with basigin in non‐small cell lung cancer

Abstract Abnormally expressed long non‐coding RNAs (lncRNAs) have been recognized as potential diagnostic biomarkers or therapeutic targets in non‐small cell lung cancer (NSCLC). The role of the novel lnc‐CYB561‐5 in NSCLC and its specific biological activity remain unknown. In this study, lncRNAs highly expressed in NSCLC tissue samples compared with paired adjacent normal tissue samples and atypical adenomatous hyperplasia were identified by RNA‐seq analysis. Lnc‐CYB561‐5 is highly expressed in human NSCLC and is associated with a poor prognosis in lung adenocarcinoma. In vivo, downregulation of lnc‐CYB561‐5 significantly decreases tumour growth and metastasis. In vitro, lnc‐CYB561‐5 knockdown treatment inhibits cell migration, invasion and proliferation ability, as well as glycolysis rates. In addition, RNA pulldown and RNA immunoprecipitation (RIP) assays show that basigin (Bsg) protein interacts with lnc‐CYB561‐5. Overall, this study demonstrates that lnc‐CYB561‐5 is an oncogene in NSCLC, which is involved in the regulation of cell proliferation and metastasis. Lnc‐CYB561‐5 interacts with Bsg to promote the expression of Hk2 and Pfk1 and further lead to metabolic reprogramming of NSCLC cells.

tumour cells, showing tumour-suppressive or -promoting (oncogenic) functions. [11][12][13] In addition, tumour-associated chronic inflammation is a hallmark of cancer that fosters progression to a metastatic stage, and lncRNAs may interfere with inflammation in tumoral microenvironment thus promote tumour growth. 14,15 Recently, Yu and co-workers identified 64 lncRNAs significantly dysregulated in 57 NSCLC tumour samples compared with 16 normal tissues and determined that multiple lncRNAs were associated with tumorigenesis and histological differentiation. 16 However, the specific biological activity of many lncRNAs that are closely related to tumorigenesis and their specific biological activities remains unknown. Therefore, conducting more research to elucidate the detailed mechanism of action of lncRNAs will have far-reaching significance for improving lung cancer diagnosis and treatment strategies.
In this study, we identified a novel lncRNA, namely lnc-CYB561-5 (Gene ID: ENSG00000233635), which is markedly upregulated in malignant lung cancer and potentially correlated with the prognosis of NSCLC patients in The Cancer Genone Atlas (TCGA) database. Further studies suggested that the high expression of lnc-CYB561-5 in lung cancer cells significantly promotes cell proliferation, migration and invasion in vitro and in vivo. Therefore, we propose that lnc-CYB561-5 may be closely involved in the pathogenesis and progression of NSCLC and has the potential to be used as a biomarker of poor prognosis.
The overall purposes of this study are as follows: (1) to investigate the role of lnc-CYB561-5 in NSCLC. (2) to elucidate the detailed mechanism of action of lnc-CYB561-5 in regulating the initiation and progression of NSCLC.

| Clinical samples
A series of human NSCLC tissue samples were obtained from the pathology department of Xuzhou Cancer Hospital. All specimens were pathologically confirmed as NSCLC, and the patients had not received radiotherapy or chemotherapy before surgery. After resection, the tumour and adjacent tissues were frozen in liquid nitrogen, and the specimens were immediately stored at −80°C. The specimens were collected under the guidance of the U.S. Health Insurance Portability and Accountability Act (HIPAA) protocol and supervised by the ethics committee of the hospital.

| RNA sequencing assay and data analysis
For human NSCLC tissue samples, high-throughput RNA sequencing analysis was performed by Shanghai Aksomics Biotech Company Ltd. Briefly, total RNA was extracted from each sample and the concentration and quality of the total RNA were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc. Similarly, total RNAs from the H1299 cells treated with sh-lnc-CYB561-5 treatment and control cells were isolated and quantified. Gene Ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed based on the top 50 differentially expressed mRNAs.

| Preparation of plasmids, lentivirus and stable cells
Short hairpin RNAs (shRNAs) were designed and synthesized by GenePharma. The shRNA-ctrl vector and shRNA-lnc-CYB561-5 were transfected into H1299 and H292 cells using Lipofectamine 2000 transfection reagent (Invitrogen). Recombinant lentivirus (Bsg-LV, Vector-LV) were prepared by Genechem Co., Ltd. The titre of the concentrated virus suspension was 5 × 10 11 Tu/L. Cells were infected with lentivirus for 48 h and then selected with 2 ng/ml puromycin for 2 weeks, with the medium refreshed every 3 days.

| Reverse transcription-PCR and qRT-PCR
TRIzol reagent was used to extract the total RNA from human lung tissue and cell lines. The cDNA was generated with random primers using the Reverse Transcription System (Promega Corporation).
Beta-actin was used for the normalization of the qRT-PCR data.
Primer sequences used in this study are listed in Table S1.

| Cell proliferation, invasion and migration assays
Cell proliferation ability was determined using a Cell Counting Kit-8 (CCK-8; KeyGen Biotech) and a colony formation assay as previously reported. 19 Briefly, 5 × 10 3 cells were seeded in 96-well plates, and recommended volume of CCK-8 solution was added at 24, 48, 72 and 96 h, respectively. For the colony formation assay, 1 × 10 3 cells were cultured in 6-well plates at 37°C for 10 days. The visible colonies were fixed with 4% paraformaldehyde and stained with crystal violet. The area of colony formation was measured using ImagePro Plus 6.0 software (Media Cybernetics, Silver Spring).
The migration and invasion assays were performed as previously described. 19 For the migration assay, 4 × 10 4 H1299 cells or H292 cells were seeded in serum-free medium in the upper chamber and incubated for 24 h at 37℃. Cells that traversed the membrane were stained with crystal violet (0.04%) and counted under a microscope.
For the invasion assay, the Transwell filter inserts were coated with Matrigel (Cat No.356237; 1:10; BD Biosciences), and 1 × 10 5 H1299 cells or H292 cells were seeded in serum-free medium in the upper chamber and incubated for 48 h at 37℃.

| Analysis of apoptosis by flow cytometry
The Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (KeyGen Biotech) was used for the detection of apoptotic cells with corresponding treatment. Briefly, 0.5 ml of binding buffer was added into 1 × 10 5 cells. Sequentially, the cells were stained with PI at room temperature for 15 min and then analysed by flow cytometry on a FACSCanto ™ II flow cytometer (BD Biosciences).

| Western blotting analysis
For Western blot analysis, an equal amount of protein was separated on a 12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) pre-cast gel (KeyGen Biotech Co.,

| Extracellular flux measurements
The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured by the Seahorse XFe96 Analyzer (Seahorse Bioscience/Agilent Technologies). Glycolytic rates were measured with the Seahorse XF glycolytic rate assay (S7805A, Seahorse Agilent).

| TCGA data analysis
This study was performed using TCGA publication guidelines. The clinical data and expression level of lnc-CYB561-5 in NSCLC (including 533 cases with lung adenocarcinoma (LUAD) and 502 cases of lung squamous carcinoma (LUSC)) and normal tissue were downloaded and extracted from the TCGA database.

| Gene co-expression network analysis
The gene co-expression network was performed as previously reported. 20 Briefly, the Pearson correlation coefficients between the expression profiles of lnc-CYB561-5 and differentially expressed mRNA were calculated to determine the co-expression relationships of lnc-CYB561-5 and mRNA expression. The protein-coding genes (PCGs) with (Pearson correlation coefficients > 0.8, p < 0.05, FDR < 1) were treated as the potential lncRNAs-related mRNA.

| RNA pulldown assay
RNA pulldown assays were performed as previously described. 21 Briefly, biotin-labelled RNAs (antisense RNA) were transcribed using Biotin RNA Labeling Mix (Promega Corporation) and T7 RNA polymerase treated with RNase inhibitor and purified with a Clean-up kit (Promega Corporation). The biotinylated lnc-CYB561-5 probes were dissolved in binding and washing buffer and incubated with streptavidin agarose resin (Thermo Fisher Scientific Inc.). Then, H1299 cell lysates were incubated with probe-coated streptavidin beads, and the pulled-down proteins were run on SDS-PAGE gels. Samples were prepared for Western blotting analysis, as described above.

| RNA immunoprecipitation
The RIP assay was performed using the EZ-Magna RIP Kit (Millipore) according to the manufacturer's protocol using 5 mg of antibody.
H1299 cells were lysed in complete RIP lysis buffer, and the cell extract was incubated with protein A/g agarose beads conjugated with the antibody against Bsg or control IgG for 2 h at 4°C. Beads were washed and incubated with Proteinase K to remove proteins.
Eventually, the purified RNA was subjected to qRT-PCR analysis.

| Statistical analysis
In this study, all of the data are expressed as the means ± SD and were processed using the SPSS 20.0 software (IBM Corporation).
Multiple group comparisons were performed using one-way analysis of variance (ANOVA) test followed by the least significant difference t test for post hoc analysis. Data between two independent groups were compared using a two-tailed Student t-test. p < 0.05 was considered as a significant difference.

| RE SULTS
3.1 | Lnc-CYB561-5 is upregulated and associated with a poor prognosis in NSCLC.
The hierarchical clustering in Figure 1A shows differentially expressed lncRNAs in 6 NSCLC tissues and tumour-adjacent tissue and 6 atypical adenomatous hyperplasia (fold change <0.5 or >2, and p < 0.05). Among the top 100 genes (ranked by p value and fold change), 11 lncRNAs were significantly upregulated in NSCLC compared with benign tumours and tumour-adjacent tissue.
In addition, using PCR primers designed for lnc-CYB561-5, we confirmed by qRT-PCR analysis that lnc-CYB561-5 was significantly upregulated in both LUAD and LUSC tissues by qRT-PCR analysis (p < 0.05) ( Figure 1C, Figure S1A). However, analysis of TCGA data sets showed that lnc-CYB561-5 was only significantly expressed in LUAD, but did not appear to play a significant role in biological functions of LUSC. The discrepancy between our data and TCGA database may be due to the different ethnicity of the samples investigated. Correlation analysis demonstrated that high levels of lnc-CYB561-5 were associated with tumour stage (p < 0.05) and lymph node metastasis (p < 0.05). However, there was no significant relationship between lnc-CYB561-5 expression and other factors, including age (p > 0.05) and sex (p > 0.05) (Supplementary Table S, Figure S1B and Figure S1C).
The data from TCGA database showed that the expression of lnc-CYB561-5 in LUAD tissues was upregulated and reduced survival, but there was no difference in the level of lnc-CYB561-5 between LUSC and adjacent tissues ( Figure 1D

| Lnc-CYB561-5 promotes aerobic glycolysis in vivo
To determine the detailed mechanism of action of lnc-CYB561-5 in regulating lung cancer proliferation and metastasis, we further performed RNA-seq analysis on lnc-CYB561-5 knockdown treated H1299 cells ( Figure 4A). GO term enrichment analysis revealed that the significantly upregulated (fold change>2, p < 0.05, top 50) genes were enriched for 'nucleosome positioning', 'cell motility' and 'regulation of glycolytic process' (Figure 4B). Heatmap analysis showed that known glycolysis-related genes, such as pfk1, g6pi, pck2 and hk2, were upregulated after sh-lnc-CYB561-5-#2 treatment ( Figure 4C). We then measured both oxygen consumption rate (OCR), an indicator of aerobic respiration, and extracellular acidification rate (ECAR), a readout of lactic acid produced from aerobic glycolysis, to determine whether the lnc-CYB561-5-induced changes in metabolic phenotypes in lung cancer cells. H1299 cells with shlnc-CYB561-5-#2 treatment showed decreased basal and maximum EACR but increased OCR relative to the control group ( Figure 4D, E). The glycolysis rates (OCR/EACR ratio) were approximately 50% lower in the control H1299 cells than in sh-lnc-CYB561-5-#2 treated H1299 cells ( Figure 4F). In addition, the analysis of gene and protein  Representative lung sections of in vivo metastasis assays, bar = 500 μm. (J) Quantitative statistics of tumour burden. *p < 0.05, **p < 0.01, ***p < 0.001, NS, p > 0.05 vs. the sh-Ctrl group, n = 5. Data are presented as means ± SEM. Multiple group comparisons were performed using one-way ANOVA followed by Tukey's post hoc test. Two-group comparisons were performed using unpaired t-test and qRT-PCR analysis showed that lnc-CYB561-5 knockdown could reduce the expression level of basigin (Bsg), but had no effect on the expressions of delta-sterol reductase (Lbr) and seizure 6-like protein 2 (Sez6l2), which have been associated with lung cancer as previously reported ( Figure 5B-D, Figure S3). Further Western blot analysis on RNA pulldown material revealed that lnc-CYB561-5 was associated with the expression of Bsg but not Lbr or Sez6l2 ( Figure 5E). Moreover, the results of RIP assays confirmed the significant interaction of lnc-CYB561-5 with Bsg in H1299 cells ( Figure 5F). In addition, Bsg overexpression significantly reversed the downregulation of the expressions of Hk2 and Pfk1, as well as the lnc-CYB561-5 knockdowninduced ECAR, and glycolysis rate ( Figure 5G

| DISCUSS ION
Currently, lung cancer is still the leading cause of cancer-related deaths worldwide. Recently, the crucial role of lncRNAs in lung cancer development has been extensively investigated. However, although many researchers believe that lncRNAs have great potential as biomarkers or therapeutic targets for lung cancer, but few have been experimentally validated and functionally annotated. [24][25][26] In this study, we found that lnc-CYB561-5 was highly expressed in NSCLC and showed a close correlation with the proliferation, metastasis and metabolic reprogramming of lung cancer cells, suggesting that it might represent an independent prognostic biomarker in NSCLC.
Although most lncRNAs lack protein-coding ability, they are closely involved in cell proliferation, differentiation and cell metabo-  [30][31][32] To facilitate sustained cell survival, excessive proliferation, metastasis and escape from the immune system, cancer cells would actively choose glycolysis as the main way to obtain energy, even in an oxygen-rich environment, a process known as the Warburg effect. 33 Besides providing ATP, aerobic glycolysis generates less reactive oxygen species and more antioxidants, lipids and nonessential amino acids which can promote increased survival of cancer cells. 34 Increasing evidence supports the view that some lncRNAs can serve as 'metabolic regulators' by participating in cellular energy programming. Wang and co-workers proposed that lnc-LINRIS could promote aerobic glycolysis in colorectal cancer by stabilizing IGF2BP2. 35 Another study by Wang et al. 36 also provided compelling evidence that lnc-HULC directly binds to lactate dehydrogenase A (LDHA) and pyruvate kinase M2 (PKM2), leading to the phosphorylation of these two enzymes, thereby promoting glycolysis. In this study, upon knockdown of lnc-CYB561-5, gene enrichment analysis identified a significant decrease in metabolic pathways including energy metabolism and glycolytic process genes upon lnc-CYB561-5 knockdown, such as Together, these results suggest that lnc-CYB561-5 is an important metabolic regulator in the context of NSCLC.
Mechanistically, interacting with proteins is a crucial way for ln-cRNAs to exert their biological effects. In this study, we predicted that Bsg may be an interacting protein of lnc-CYB561-5 and further confirmed this prediction by RNA pulldown and RIP assays in vitro.
Bsg, also known as CD147, is expressed in a variety of cell types and is involved in the processes of organ development, wound healing, inflammation and tumour progression. 37,38 Recent studies show that Bsg is highly expressed in several cancer cell types and is associated with tumour size, tumour stage, progression and prognosis. [39][40][41] Zhou et al. 42 determined that Bsg recycling is required for lung cancer cell migration and invasion, and targeting Bsg recycling may be a rational strategy for lung cancer therapy. Albrechtsen et al. 43 also found that Bsg is proteolytically shed from the cell surface and high concentrations of soluble Bsg in the blood indicates poor prognosis in cancer patients. Our results showed that knockdown of lnc-CYB561-5 led to decreased expression of Bsg in H1299 cells.
Overexpression of Bsg can effectively block the downregulation of the glycolysis level, as well as the decrease of cell proliferation, migration and invasion activity caused by lnc-CYB561-5 knockdown in H1299 cells. Therefore, the involvement of lnc-CYB561-5 in the regulation of lung cancer cell metabolism, proliferation and metastasis is Bsg dependent.
Despite the significance of the findings in this study, there are several limitations that warrant discussion. Firstly, although our evidence supports the conclusion that lnc-CYB561-5 promotes the metastasis of lung cancer cells, further experimental metastasis assay, such as in vivo bioluminescence imaging of mice, is needed to be performed to confirm its role in lung cancer metastasis. Secondly, increased analysis of the function of Bsg on cell proliferation, migration and invasion is necessary to further prove that BSG is the target of lnc-CYB561-5.
In conclusion, we found that lnc-CYB561-5 can promote NSCLC cell proliferation and metastasis in vitro and in vivo. Lnc-CYB561-5 interacted with Bsg to promote the expression of HK2 and Pfk1 and further led to aerobic glycolysis in NSCLC. These data provided important insights into the use of lncRNA as a biomarker or therapeutic target for NSCLC.

ACK N OWLED G EM ENT
None.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All relevant data have been presented in the manuscript. All requests for or questions about the data can be initiated by contacting 2010692@tongji.edu.cn.