Background
Colorectal cancer (CRC) is the most common malignancy of the digestive system, and the second leading cause of cancer-related mortality worldwide [
1]. Although significant progress has been made in surgical resection, chemotherapy, targeted therapy and immunotherapy for CRC, the prognosis of CRC patient is still unfavourable [
2‐
4]. CRC hallmarks include rapid proliferation, which requires nutrients and facilitates the removal of cellular waste in nutrient-poor environments [
5]. Research progress in the last decade has demonstrated that metabolic reprogramming is a pivotal general characteristic of cancer, that allow CRC cells to produce energy sufficient for rapid proliferation [
6]. Accumulating evidence has confirmed aberrant metabolic reprogramming as a critical contributor to CRC initiation, proliferation and metastasis [
7]. Hence, elucidating the regulatory mechanisms linking colorectal carcinogenesis factors and metabolic enzymes may provide promising avenues for improving CRC prognosis and developing effective therapeutic strategies.
As a critical metabolic feature of cancer cells, lipid metabolism has been widely reported to participate in cancer initiation, progression and drug resistance [
8]. Fatty acid synthase (FASN) is a central enzyme of lipid metabolism that can catalyse de novo fatty acid (FA) biosynthesis to promote cell growth and survival [
9]. For instance, loss of FBXW7β function facilitates FASN protein stability and promotes lipogenesis and growth in CRC [
10]. However, the mechanisms underlying the dysregulation of lipogenesis and aberrant FASN expression in CRC still need to be clarified.
Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides in length that have limited or no ability to encode proteins, but they can regulate protein-coding gene expression by modulating mRNA transcription, translation, and post- translational modification [
11,
12]. Over the past decade, lncRNAs have been reported to play vital roles in various cellular functions in CRC, including proliferation, apoptosis, migration and invasion [
13]. Previously, we reported that lncRNAs played important roles in CRC tumorigenesis and progression [
14‐
16]. Furthermore, several lncRNAs were reported to be critical players in lipid metabolism modulation by CRC cells. For example, ZFAS1 binds with PABP2 to stabilize SREBP1 mRNA, thus facilitating lipid accumulation in CRC cells [
17]. SNHG16 acts as a “sponge” for miRNAs to promote lipid metabolism by upregulating SCD [
18]. However, additional crosstalk between lncRNAs and metabolic enzymes or processes still needs to be characterized.
In the present study, we identified a novel lncRNA, POU6F2-AS1, whose expression is significantly upregulated in CRC; this upregulation phenotype is positively associated with poor clinical outcomes in CRC patients. Moreover, we found that POU6F2-AS1 drives the growth and lipogenesis of CRC cells in vitro and in vivo, and these effects partially dependent on YBX1-mediated transcriptional activation of FASN. We also demonstrated that METTL3-induced m6A modification maintains POU6F2-AS1 stability and increases its expression. Taken together, these findings revealed that POU6F2-AS1 may serve as a promising biomarker and therapeutic target for CRC.
Materials and methods
Clinical specimens
This study was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University (XYFY2022-KL473-01), and consent was obtained from all participants. A cohort comprising 84 pairs of CRC tissues and corresponding adjacent normal tissues (ANTs) was collected from CRC patients who underwent radical resection or palliative resection at the Department of Gastrointestinal Surgery of the Affiliated Hospital of Xuzhou Medical University between January 2023 and April 2023 and was used for extracting RNA to detect the expression level of POU6F2-AS1. In addition, a cohort of 60 pairs consisting of paraffin-embedded CRC tissues and corresponding ANTs was collected for tissue microarray (TMA) analysis. All CRC patients had a pathologically confirmed diagnosis and did not receive preoperative radiotherapy or chemotherapy. Clinicopathological characteristics, such as patient age and sex and tumour size and depth of invasion, etc., were also obtained.
Cell lines and culture
The human normal colorectal epithelial cell line FHC was obtained from the American Type Culture Collection (Manassas, VA, USA), while several human CRC cell lines (HCT116, DLD1, LoVo, SW620, HT29 and SW480) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCT116 cells were cultured in McCoy´s 5 A medium supplemented with 10% foetal bovine serum (FBS) (Gibco, MA, USA), while SW620 cells were cultured in L-15 medium (Gibco, MA, USA). DLD1, HT-29, and LoVo cells were cultured in RPMI 1640 medium (Gibco, MA, USA), and FHC and SW480 cells were cultured in DMEM/high-glucose medium (Gibco, MA, USA). All the cells were cultured in a constant temperature incubator at 37 °C containing 5% CO2 and subjected to mycoplasma testing to confirm that they were free of contamination before use.
BODIPY staining
The lipophilic fluorescence dye BODIPY (Molecular Probes, MA, USA) was used to monitor the content of neutral lipids in CRC cells. The transfected CRC cells were inoculated into 24-well plates containing coverslips and fixed with 4% paraformaldehyde for 30 min after 24 h. The cells were subsequently incubated with 2 µM BODIPY working solution in the dark for 30 min at 37 °C. The staining procedure was stopped by washing the cells with PBS, after which the nuclei were counterstained with Hoechst 33,342 (Beyotime, Shanghai, China) for 10 min. Representative images were taken with a confocal laser scanning microscope (Carl Zeiss, Germany).
Oil Red O staining
Oil Red O staining was performed using Oil Red O Saturated Solution (Solarbio Life Sciences, Beijing, China) according to the manufacturer’s instructions. Briefly, an Oil Red O working solution was prepared by mixing 3 parts of Oil Red O saturated solution with 2 parts deionized water. The transfected CRC cells were inoculated on 14 mm round coverslips in 24-well plates, washed twice with PBS after 24 h, and fixed in 4% paraformaldehyde for 30 min before staining. For frozen tissue sectioning, optimal cutting temperature (OCT)-embedded tissue was cut into 8 μm sections, which were fixed in 4% paraformaldehyde for 30 min before staining. The cells or tissue sections were incubated in 60% isopropanol for 30 s and then in Oil Red O working solution for 20 min at room temperature. Then sections were subsequently washed with 60% isopropanol and deionized water again, counterstained with haematoxylin (Solarbio Life Sciences, Beijing, China), and photographed by an Olympus microscope (Tokyo, Japan). The Oil Red O staining area was calculated as Oil Red O+ area divided by the area of hematoxylin staining and normalized to the average of the control.
Triglyceride (TAG) assay
For the quantitative estimation of triglycerides in cells, a Triglyceride Assay Kit (Promega, WI, USA) was used in accordance with the manufacturer’s protocols. Transfected cells were inoculated into 96-well plates and subjected to a triglyceride assay after 24 h. The cells were washed twice with PBS, and glycerol detection reagent was added, followed by incubation at room temperature for 1 h to detect luminescence. The luminescence signal was measured using a Tecan Spark zymography (Zurich, CH).
Biotinylated RNA pull-down assay
In brief, T7 promoter-containing DNA was obtained by PCR amplification and purified using the TIANgel Purification Kit (TIANGEN BIOTECH, Beijing, China). The purified T7 promoter-containing DNA was incubated with a biotin RNA labelling mixture (Promega Corporation, MA, USA), T7 RNA polymerase (Thermo Fisher Scientific, MA, USA) and an RNase inhibitor for 2 h at a constant temperature of 37 °C in a PCR instrument and purified using an RNAclean Kit (TIANGEN BIOTECH, Beijing, China) to obtain the biotin-labelled RNA probe. Biotinylated RNAs were incubated with protein extracted from CRC cells and mixed with precleared streptavidin agarose resin (Thermo Fisher Scientific, MA, USA). The RNA-protein complexes were obtained by collecting the agarose resin by centrifugation, and the proteins bound therein were eluted and denatured, followed by electrophoresis on SDS-PAGE gels. Afterwards, the proteins were silver stained, and the differential bands were excised for mass spectrometry analysis (Shanghai Applied Protein Technology Co. Ltd., China). To identify which specific region of POU6F2-AS1 interacts with YBX1, we truncated POU6F2-AS1.
Luciferase reporter assay
The luciferase plasmid was constructed by Gene Create Biologicals (Wuhan, China). To investigate the transcriptional regulation of FASN, two different fragments of the FASN promoter region were PCR amplified and subsequently inserted into the KpnI/XhoI site upstream of firefly luciferase in the pGL3-Basic vector. Cells with or without knockdown or overexpression of YBX1/POU6F2-AS1 were transfected with the luciferase plasmid. Firefly luciferase activity was measured 48 h later using a Dual Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions, with Renilla luciferase serving as a transfection control.
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed using the BeyoChIP™ ChIP Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, 1 × 10
7 CRC cells were prepared and then cross-linked with 1% formaldehyde for 15 min at room temperature. The cross-linking process was quenched by adding 0.125 M glycine. Chromatin was isolated with the lysis buffer provided in the kit. Afterwards, sonication was performed to shear the DNA to 200–1000 bp. Immunoprecipitation of cross-linked protein/DNA was performed by incubating anti-YBX1 antibody (Proteintech, IL, USA) or normal rabbit IgG (Proteintech, IL, USA) with sheared chromatin overnight at 4 °C followed by incubation with Protein A/G Magnetic Beads for 2 h. Subsequently, cross-links of protein/DNA were released with 5 M NaCl, and the DNA was purified using a PCR Clean Up Kit (Beyotime, Shanghai, China). Finally, the purified DNA was subjected to qRT–PCR using the P1-P9 primer sequences listed in Additional file 1: Table
S4.
Patient-derived organoid (PDO) culture model
In this study, a PDO model was established using tumour tissues from CRC patients at the Department of Gastrointestinal Surgery of the Affiliated Hospital of Xuzhou Medical University. Briefly, freshly removed tumour tissues were cut into 1–3 mm3 pieces, washed several times with antibiotic-containing PBS, and digested with 200 U/ml collagenase (Sigma, MO, USA) and 100 U/ml hyaluronidase (Sigma, MO, USA) for 30 min at 37℃. The digested tissues were filtered through a 100 μm cell filter and then centrifuged to discard the supernatant, after which the cells were resuspended in Matrigel. Then, 50 µL of Matrigel resuspension solution was added to each well of a 48-well plate, and 450 µL of CRC organic medium (bioGenous, Suzhou, China) was added to each well after solidification. The medium was changed every 2–3 days, and the organoids were passaged every 10–15 days. Lentiviral transfection was performed 24 h after organoid passaging. Organoid images were obtained with an Olympus FSX100 microscope (Olympus, Tokyo, Japan).
Animal experiments
Female BALB/c nude mice ranging from 4 to 6 weeks old were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and housed under specific pathogen-free conditions. To construct a xenograft tumorigenesis model, transfected HCT116 cells were injected subcutaneously into the axillary region of mice (5 × 106 cells). Tumour growth was measured every 3 days, and the tumour volume (V) was calculated according to the following formula: V= (length × width2)/2. When the maximum tumour length diameter reached 15 mm, the mice were sacrificed, and the subcutaneous tumours were removed to calculate the mass and then subjected to HE and IHC staining. The Animal Care and Use Committee of Xuzhou Medical University provided a statement of ethical approval for all animal experiments in this study.
Statistical analysis
Statistical analyses were performed using SPSS 19.0 software (IBM, Armonk, NY, USA) and GraphPad Prism version 8.0 (La Jolla, CA, USA). All the data in the present study is presented as the means ± standard deviations (SD), and all the tests were two-sided. A P value < 0.05 was considered wo indicate statistical significance. Student’s t-test or one-way analysis of variance (ANOVA) was used to evaluate the significance of differences between groups. Correlations were detected by Spearman’s correlation coefficient. The relationship between POU6F2-AS1 expression and the clinicopathological parameters of CRC patients was calculated by the chi-square test or Fisher’s exact test. Overall survival (OS) was assessed by the Kaplan–Meier method and log-rank test. Univariate and multivariate Cox proportional hazard regression models were used to evaluate the effects of POU6F2-AS1 expression or other clinicopathological parameters on survival and the hazard ratio (HR).
Additional methods
Additional experiments, including cell transfection, RNA extraction, qRT–PCR, immunohistochemistry (IHC), RNA stability assays, fluorescence in situ hybridization (FISH) and immunofluorescence (IF) staining, Western blotting, Cell Counting Kit-8 (CCK–8) assays, 5-ethynyl-2’-deoxyuridine (EdU) assays, colony formation assays, methylated RNA Immunoprecipitation (MeRIP)-qPCR, RNA immunoprecipitation (RIP)-qPCR, RNA sequencing and bioinformatics analysis, application of palmitic acid (PA) and orlistat, and liquid chromatography mass spectrometry (LC-MS) based FA analysis are described in Additional file 2: Supplemental Materials and Methods.
Discussion
Transcriptome studies have demonstrated that protein-coding RNAs account for less than 2% of the transcribed human genome, and most of the remaining RNAs are noncoding RNAs (ncRNAs) [
40]. Currently, lncRNAs, which are functional ncRNAs, have attracted increasing amounts of attention in cancer research. Several phase I/II clinical trials targeting lncRNAs have been conducted to treat patients with cancer [
41]. The development of aberrantly expressed lncRNAs as therapeutic tools has yielded exciting prospects. Moreover, with the widespread use and improvement of high-throughput sequencing, additional opportunities have been provided for bioinformatics-based research and clinical validation of promising lncRNAs. In the present study, we identified the novel upregulated lncRNA POU6F2-AS1 in CRC via comprehensive analysis of publicly available datasets and validated the upregulation of POU6F2-AS1 in both CRC tissues and cell lines. Moreover, we found that high expression of POU6F2-AS1 was positively associated with advanced clinicopathological stage, large tumour size, and poor OS in patients with CRC, suggesting that POU6F2-AS1 might play an essential role in CRC tumorigenesis and progression. Previous study demonstrated that POU6F2-AS1 are upregulated in lung cancer and CRC and is an oncogenic factor [
42,
43]. However, the function and mechanism of POU6F2-AS1 in lipid metabolism has not been explored in CRC.
Gain- and loss-of-function experiments demonstrated that POU6F2-AS1 promotes the growth of CRC cells in vitro and in vivo. In addition, RNA sequencing analysis, BODIPY and Oil Red O staining and LC/MS data revealed that POU6F2-AS1 was able to affect lipid metabolism and FA metabolism. Reprogramming of lipid metabolism is a newly well-documented hallmark of cancer cells, and represents an attractive vulnerability that might be exploited as s therapeutic target [
6]. Proliferating cancer cells are likely to obtain energy via lipid reprogramming particularly through the synthesis of FAs; overall, FA metabolism involves FA uptake, de novo synthesis, and β‐oxidation to support unlimited cellular proliferation [
19,
20,
44]. In addition, cancer cells can utilize FAs to provide ingredients for synthesizing cellular organelles and membranes during rapid replication [
45].
Multiple metabolic pathways contribute to lipid accumulation in cancer cells, and most of these pathways are related to the rate-limiting lipogenic enzymes, whose upregulation induces FA synthesis [
46]. Accumulating evidence has shown that multiple lipogenic enzymes, such as FASN, ACC, SCD1, and ACLY, are aberrantly upregulated in a wide variety of cancers, driving FA synthesis and contributing to lipogenesis and the progression of cancer cells [
47‐
50]. Consistently, the expression of lipogenic enzymes is also markedly increased in CRC [
51]. Recently, lncRNAs were reported to participate in the regulation of lipogenic enzymes, thereby promoting the progression of cancers. For example, Zheng et al. revealed that the lncRNA TINCR could bind to ACLY and maintain its protein stability by protecting it from ubiquitin-mediated degradation, thereby facilitating de novo lipid biosynthesis and progression of nasopharyngeal carcinoma [
52]. Jia et al. reported that that the lncRNA NEAT1 facilitated FA metabolism in gastric cancer via the c‑Jun/c‑Fos/SREBP1 axis [
53]. Peng et al. demonstrated that the lncRNA FASRL could bind to ACACA and inhibit its phosphorylation, thus increasing FA synthesis in hepatocellular carcinoma [
54]. However, the crosstalk between lncRNAs and metabolic enzymes or processes in CRC is poorly understood. In the present study, our findings revealed that POU6F2-AS1 overexpression induces lipogenesis and proliferation in CRC cells by upregulating FASN, which is the key rate‐limiting lipogenic enzyme responsible for the terminal catalytic step in the synthesis of FAs. FASN is often upregulated in cancer cells and its depletion results in antitumour effects; thus, FASN has been defined as a promising therapeutic target [
55]. Consistent with this conclusion, we found that FASN knockdown could efficiently attenuated CRC cell lipogenesis and proliferation, while these processed were promoted by POU6F2-AS1 overexpression. These findings indicated cotreatment with POU6F2-AS1 siRNAs may improve the therapeutic efficacy of FASN inhibitors. However, the antitumour effects of combination therapies based on POU6F2-AS1 need to be further studied in the future.
LncRNAs typically exert their functions by acting as microRNA (miRNA) sponges and interacting with RBPs [
56,
57]. In the present study, by overlapping the MS analysis and two online RBP prediction results, we determined that POU6F2-AS1 specifically interacts with YBX1. Deletion-mapping analysis revealed that the 117–195 nt region of POU6F2-AS1 was responsible for the interaction between these two genes, which is consistent with the regions predicted by catRAPID. YBX1 has been shown to play vital roles in the tumorigenesis and progression of malignancies, including CRC. Accumulating evidence has indicated that YBX1 is a vital functional partner of multiple lncRNAs in cancer. For instance, YBX1 is an interactive partner of lincNMR that regulates the expression of downstream RRM2, TYMS and TK1 by binding to their promoters, governing nucleotide metabolism and cell proliferation in cancer [
58]. The lncRNA MILIP bound with YBX1 to increase the translation of Snail, leading to the enhanced metastasis of clear cell renal cell carcinoma cells [
59]. LncRNA RMRP recruits YBX1 to upregulate the transcription of TGFBR1, thereby promoting the proliferation and progression of non-small cell lung cancer [
60]. Previous studies demonstrated that YBX1, a well-established transcription factor, may bind to the promoters of certain genes, thereby initiating their transcriptional activation. For example, NOTCH3 [
61], CBX3 [
62], and ANXA8 [
63] are regulated by YBX1 via transcriptional activation. In this study, we found that POU6F2-AS1 tethers YBX1 to the promoter of FASN to promote FASN activation. Furthermore, ChIP assays revealed that the region spanning from − 583 to -10 nt in the FASN promoter contains the binding sites for YBX1. Coincidently, YBX1 binding site-2 (YBX-2), YBX-3, YBX-5, and YBX-6, which were predicted by the JASPAR website, were also located in this region. These findings are consistent with conclusions reported in the literature that transcriptional regulation is one of the most important mechanisms of FASN overexpression in cancer cells [
36]. However, we did not detect whether histone proteins or other transcription factors in this region cooperatively interact with YBX1 to initiate FASN transcription. Additionally, as a multifunctional DNA/RNA binding protein RNA, YBX1 also participates in the regulation of multiple DNA/RNA-dependent events, including pre-mRNA splicing, mRNA packaging, translation and stabilization, etc [
29,
64]. However, whether YBX1 cooperates with POU6F2-AS1 to regulate FASN via these mechanisms needs to be explored in the future.
In addition, our work addressed the mechanism of POU6F2-AS1 upregulation in CRC. The m
6A modification, a type of reversible epigenetic regulatory mechanism, has been widely reported to be involved in the stabilization and expression of RNAs, including lncRNAs, in cancer [
65,
66]. For example, METTL3-mediated m
6A modification increases the stabilization and expression of THAP7-AS1 in an IGF2BP1-dependent manner [
37]. METTL3 catalyzes m
6A modification, and YTHDC1 recognizes and stabilizes m
6A to increase expression of TERRA [
67]. Since the bioinformatics analysis predicted high-confidence m
6A sites in the sequence of POU6F2-AS1, we further assessed whether POU6F2-AS1 was an m
6A-incucible lncRNA. Coincidently, we observed that knockdown of METTL3 suppressed the expression and stability of POU6F2-AS1 in CRC cells and decreased m
6A level of POU6F2-AS1. IGF2BP2 has been shown to mediate the stability of m
6A-modified RNAs, such as CREB1 [
68], HMGA1 [
69], MSX1 and JARID2 [
70] in CRC. Here, we also found that IGF2BP2 could recognize m
6A sites and maintain the stability of POU6F2-AS1. Overall, these results suggested that METTL3-catalyzed and IGF2BP2- stabilized m
6A modification may be involved in the upregulation of POU6F2-AS1 in CRC.
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