DMEM/F12 (HAM) 1:1 medium and fetal bovine serum (FBS) were from Biological Industries; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and cycloheximide (CHX) were obtained from Beijing Solarbio Science & Technology Co., Ltd. The RNAiso Plus was purchased from Takara Bio, Inc. All-in-one First Strand cDNA Synthesis Kit II and 2X SYBR Green qPCR Master Mix were from Sevenbio (http://www.7bio.com.cn/index.php). Turbofect was obtained from Thermo Fisher Scientific, Inc. Annexin V-FITC apoptosis detection kit was purchased from Nanjing KeyGen Biotech Co., Ltd. Antibodies for PKM2 (cat. no. 15822-1-AP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat. no. 10494-1-AP) were obtained from Proteintech Group, Inc. ATP assay kit (cat. no. S0026B) was obtained from Beyotime Institute of Biotechnology. Glucose assay kit (cat. no. F006-1-1) and pyruvate acid test kit (cat. no. A081-1-1) were purchased from Nanjing Jiancheng Bioenigeering Institute. HiPerFect Transfection Reagent were purchased from Qiagen GmbH. MG132 was obtained from MedChemExpress.

The CRC cell lines HT-29, DLD1, HCT116, SW620, SW480 and human colon epithelial cell line (FHC) were preserved in the authors' laboratory.. The cells were cultured in DMEM/F12 medium containing 10% FBS and placed in an incubator at 37°C with 5% CO₂.

Total RNA was extracted from cells with TRIzol^® reagent (Takara Bio, Inc.) and 500 ng of RNA was reverse-transcribed. The reverse transcription was performed according to the manufacturer's instructions. LncRNA and mRNA levels were quantified using RT-qPCR. The PCR reaction in the following conditions: one cycle at 94°C for 30 sec, followed by 40 cycles at 94°C for 5 sec, 60°C for 15 sec and 72°C for 10 sec. The quantitative analysis was performed using the 2^−ΔΔCq method (22). GAPDH was used as a reference gene. All primers used in the experiment are shown in Table SI.

Kaplan-Meier survival analysis for CRC patients with high or low gene expression level was performed through Gene Expression Profiling Interactive Analysis 2 (GEPIA 2; http://gepia2.cancer-pku.cn/#index). The coding potential of lncRNA was estimated by the coding-potential assessment tool (CPAT; http://lilab.research.bcm.edu/). LncRNA PhyloCSF value analysis was performed by the UCSC genome browser (http://genome.ucsc.edu/) with the PhyloCSF data hub. The metascape database (www.metascape.org) was used for gene enrichment analysis and the protein-protein interaction (PPI) network. The expression data of PKM2 and the correlation analysis of two genes in CRC tissues were performed through the GEPIA 2 (http://gepia2.cancer-pku.cn/#correlation). The interaction between lncRNA 495810 and PKM2 was predicted via RNA-Protein Interaction Prediction (RPISeq) (http://pridb.gdcb.iastate.edu/RPISeq/index.html). A potential PKM2-binding region of lncRNA 495810 (401–452) was predicted using catRAPI omics (http://s.tartaglialab.com/page/catrapid_omics_group).

The collected cells were lysed with hypotonic buffer (pH 7.4 25 mM Tris-HCl, 1 mM MgCl₂, 5 mM KCl and 1% NP-40), centrifuged (4°C, 5,000 g, 5 min), and the supernatant was received to obtain cytosolic fractions. Then, the aforementioned pellet was lysed with nuclear resuspension buffer (pH 7.9 20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF) and centrifuged (4°C, 13,000 g, 10 min) to obtain nuclear fractions. Nuclear and cytoplasmic RNA were extracted with TRIzol reagent. Finally, the expression levels of GAPDH, U6, and lncRNA 495810 in the nuclei and cytoplasm of CRC cells were detected using RT-qPCR assays.

To assay siRNA transfection, the cells were inoculated into a six-well plate, and 10 nM siRNA was transfected with HiPerFect Transfection Reagent for 48 h according to the manufacturer's protocol. For the overexpression assay, lncRNA 495810 overexpression plasmid and the control plasmid (pCDH; both from Miiaoling Biotech Science) were transfected into colon cancer cells using Turbofect reagent according to the manufacturer's instructions. After transfection for 48 h, the cells were collected for subsequent analysis or assay. The siRNA sequences (Shanghai GenePharma Co., Ltd) were as follows: lncRNA 495810-homo-112 (sense, 5′-CCAUACCAUCAAUGGUCAUTT-3′ and antisense, 5′-AUGACCAUUGAUGGUAUGGTT-3′).

The HT-29, SW620 and HCT116 cells (~5×10³) were seeded into 96-well plates and were incubated overnight to adhere to the wall. Cells were transfected with si-495810 or 495810 for 48 h. Then 10 µl of MTT (0.5 mg/ml) was added to each well for 4 h. After the formazan crystals formed, they were dissolved in DMSO and the OD value at 570 nm was evaluated. The cell survival rate was calculated with the following formula: Cell survival rate (%)=(OD570 treated/OD570 control) ×100.

Cells were resuspended with 500 µl binding buffer at the density of 1×10 ⁵ cells/ml, and then 5 µl connexin V-FITC and 5 µl propidium iodide were added. The cells were incubated at room temperature in the dark for 15 min. After that, the samples were detected and analyzed by flow cytometry (CytoFLEX; Beckman Coulter, Inc.).

The SW620 and HCT116 cells (~2×10⁴) were seeded into 24-well Transwell plates (cat. no. 3422; Corning, Inc.) and 200 µl serum-free DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) in the upper compartment and 660 µl DMEM medium supplemented with 15% FBS (Gibco; Thermo Fisher Scientific, Inc.) was added to the lower compartment. The plate was incubated at 37°C for 48 h. The migrated cells on the lower surface were detected.

Cells were seeded into the six-well plate, and medium and cells were collected after transfection with si-495810 or 495810 for 48 h. The medium was used to measure the contents of glucose and pyruvate acid, and the production of ATP was determined after cell lysis. The methods of measuring glucose, ATP and pyruvate acid were according to the manufacturer's instructions. Briefly, the medium was added to the reaction solution in the glucose and pyruvate acid kit, respectively, and its OD was measured at 505 nm to calculate its content. The cell lysate was added to the ATP assay solution, its fluorescence intensity was measured, and its content was calculated.

The templates for in vitro transcription were obtained from PCR. The primers containing the T7 promoter of 495810 are listed in Table SI. In vitro transcription was conducted using a TranscriptAid T7 High Yield Transcription kit (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol and the target RNA was labeled with biotin using Biotin RNA Labeling Mix 10X.

Cell lysates were prepared using IP lysis buffer. Next, the labeled RNA was incubated with streptavidin magnetic beads for 1 h at room temperature with agitation. Then, the bead-RNA complex and RNA-protein binding reaction master were incubated for 2 h at 4°C with rotation. Finally, the eluted interacting proteins were obtained.

Mass spectrometry was performed at Bioprofile. Mass spectrometry experiments were performed on a Q Exactive HF-X mass spectrometer that was coupled to Easy nLC (Thermo Fisher Scientific, Inc.). The full MS scans were acquired at a resolution of 60,000 at m/z 200, and 15,000 at m/z 200 for MS/MS scan. The maximum injection time was set to for 50 ms for MS and 50 ms for MS/MS. Normalized collision energy was 28 and the isolation window was set to 1.6 Th. Dynamic exclusion duration was 60 sec.

Western blotting was performed as previously described (21). Total protein was extracted by WBIP (Solarbio Science & Technology Co., Ltd.) and its concentration was determined by BCA assay. The same amount (60 µg) of protein was separated by 10% SDS-PAGE and transferred to the PVDF membrane. The membrane was blocked for 40 min at room temperature with 5% skim milk and incubated with primary antibodies against anti-PKM2 or GAPDH overnight at 4°C. The corresponding secondary antibodies (anti-Rabbit, ZB-2306/anti-Mouse, ZB-2305) were incubated for 2 h at room temperature the following day. The expression changes of target proteins were observed. Relative protein levels were analyzed using ImageJ software (National Institutes of Health). Expression of GAPDH was used as the internal control.

To observe the protein degradation process, HT-29, HCT116 and SW620 cells (~1×10⁵) were treated with CHX (20 µg/ml) for 0, 2, 4, 6 and 8 h, respectively. Total protein was extracted at indicated time points and analyzed by western blotting.

The data are presented as the mean ± SEM, data error bar indicating standard deviation. Single-variable comparisons were performed by independent samples t-test. Continuous variables (N≥3 groups) were analyzed by Single factor analysis of variance (ANOVA), followed by Tukey's post-hoc test. Cox proportional hazards regression models were applied to determine the independent factors that influence survival. Experimental data were executed using GraphPad Prism 9 software (GraphPad Software, Inc.). The data of TCGA was performed in the GEPIA 2. P<0.05 was considered to indicate a statistically significant difference.

To examine the clinical significance of lncRNA 495810 in colon cancer, the endogenous expression of lncRNA 495810 in normal colonic epithelial FHC and different CRC cell lines was first examined. It was found that lncRNA 495810 was highly expressed in most detected CRC cells compared with FHC (Fig. 1A). Then the expression of lncRNA 495810 was estimated in 68 paired samples of adjacent tissues and CRCs. The results revealed that lncRNA 495810 expression level was significantly increased in colon cancer tissues (Fig. 1B). Subsequently, the pathological and clinical value of lncRNA 495810 were assessed. The results revealed that high expression of lncRNA 495810 was associated with poor survival by survival analysis (Fig. 1C). Through COX regression analysis, it was identified that the expression level of lncRNA 495810 was a prognostic factor for CRC (Tables I and SII). The overall and disease-free survival curve for CRC patients with high or low lncRNA 495810 expression level further proved that high lncRNA 495810 expression levels indicated a poor prognosis in patients (Fig. 1D and E), and it was correlated with advanced clinical grade and stage (Fig. 1F).

To determine whether 495810 is a lncRNA, the characteristics of lncRNA 495810 were detected. The chromosomal coordinate ranges of lncRNA 495810 were chr7:26195712-26201301, and its size was 484 bp in length (Table SIII). Then, the CPAT displayed that 495810 had no coding ability (Fig. S1A). In addition, the PhyloCSF value of 495810, which was calculated to verify the conservation of the sequence, was minus (Fig. S1B). Nuclear and cytoplasmic fractions in CRC cells were also isolated and RT-qPCR was performed. The results demonstrated that 495810 was mainly localized in the nucleus (Fig. 1G). Collectively, the data indicated that 495810 is a lncRNA and exhibits oncogene potential.

The promotional effect of lncRNA 495810 on colorectal carcinogenesis was then characterized by functional assays. HCT116/SW620 cells with relatively lower or higher lncRNA 495810 expression were selected for lncRNA 495810 overexpression and knockdown. The HT-29 cells with moderate expression were used in both knockdown and overexpression experiments, which were used as parallel experimental groups (Figs. 1A, S1C and D). The results demonstrated that lncRNA 495810 knockdown significantly inhibited cell proliferation in SW620 and HT-29 cells, whereas overexpression of lncRNA 495810 increased CRC cell proliferation of HCT116 and HT-29 cells (Fig. 2A and B). Moreover, lncRNA 495810 knockdown significantly increased, whereas overexpression of lncRNA 495810 inhibited apoptotic levels (Fig. 2C and D).

Moreover, the migration ability of the si-495810 group was significantly reduced compared with the NC (non-specific control) group in SW620 cells (Fig. 2E). Whereas in the gain-of-function assays, 495810 overexpression significantly increased cell migration ability of HCT116 cells (Fig. 2F). The aforementioned results collectively indicated that lncRNA 495810 was involved in colorectal tumorigenesis and progression as an oncogene.

To determine the signaling pathway affected by lncRNA 495810 in promoting CRC, RNA pull-down combined with mass spectrometry (Bioprofile) was used to identify targets directly regulated by lncRNA 495810. The aforementioned target proteins were imported into the Metascape database (www.metascape.org) for gene enrichment analysis. The results demonstrated that the pathways closely related to lncRNA 495810 were glycolysis and gluconeogenesis (Fig. 3A-C). Furthermore, metabolic assays revealed that silencing lncRNA 495810 expression level declined, whereas ectopic lncRNA 495810 expression enhanced glucose consumption, pyruvate acid production and ATP production (Fig. 3D-I). These data indicated that 495810 is a glycolysis-related lncRNA and promotes aerobic glycolysis in CRC.

To identify targets that are directly regulated by lncRNA 495810, the proteins bound to the sense strand of lncRNA 495810 were used to construct a PPI network (Fig. 4A). At the same time, the MCODE algorithm built into the Metascape database was used to cluster these proteins (Table II). Glycolysis-related enzymes, including PKM2, LDHB, ENO1, PGK1, LDHA and TPI1, attracted the authors' attention (Fig. 4B). Among which, PKM2 was positively correlated with lncRNA 495810 (Fig. 4C), but not other proteins (Fig. S2A-E). Transcriptional and survival data for PKM2 were subsequently examined in the GEPIA 2 database. Consistent with lncRNA 495810, the expression of PKM2 was significantly upregulated in tumor tissues (Fig. 4D). The overall survival rates of the colon cancer patients with high levels of PKM2 were lower than those with low expression levels of PKM2 (Fig. 4E).

Next, the interaction between lncRNA 495810 and PKM2 was predicted via RPISeq analysis. The interaction probabilities of lncRNA 495810 and PKM2 were greater than 0.5 (RF=0.65, SVM=0.65). The data suggested that lncRNA 495810 may bind to PKM2 (Table SIV), which was also verified by RNA pull-down assay in HCT116 and HT-29 cells (Fig. 4F). In addition, a potential lncRNA 495810-binding region of PKM2 (401–452 nt) was predicted using catRAPI (Fig. 4G). To confirm the impact of lncRNA 495810 on PKM2, the expression of PKM2 was detected in CRC cells after depletion or overexpression of lncRNA 495810. The results identified that silencing lncRNA 495810 expression declined, whereas ectopic lncRNA 495810 expression enhanced the protein level of PKM2 (Fig. 4H and I).

Increasing evidence has demonstrated that lncRNAs regulate protein expression by post-translational modification (17,23–25). Its regulation mode of 495810 was then explored. The results showed that silencing lncRNA 495810 expression shortened, whereas ectopic lncRNA 495810 expression prolonged the half-life of PKM2 protein (Fig. 5A and B). In addition, suppression of proteasome activity by MG132, an inhibitor of proteasome, prevented the lncRNA-induced PKM2 expression (Fig. 5C and D), suggesting that PKM2 protein degradation was mediated by the ubiquitin-proteasome pathway.