Abstract
Colorectal cancer (CRC) is a malignant digestive tumor associated with high mortality rate. Currently, 5-Fu therapy is a frequently used treatment approach for CRC. Yet, acquirement of 5-Fu resistance ultimately leads to therapeutic failure in CRC patients. LncRNA NOP14-AS1 was upregulated in cancers, but its biological functions and mechanisms in 5-Fu resistant colorectal cancer remain elusive. We discovered that NOP14-AS1 was high-expressed in colorectal tumors and cancer cells. Silencing NOP14-AS1 sensitized CRC cells to 5-Fu. By creating a 5-Fu resistant CRC cell line (HT-29 5-Fu R) and observed that expression of NOP14-AS1 was remarkedly elevated in 5-Fu resistant cells compared to parental cells. Additionally, we found that miRNA-30a-5p was a target of NOP14-AS1 and directly affected its function. miR-30a-5p overexpression sensitized CRC cells to 5-Fu treatment and targeted the glycolysis key enzyme, LDHA. Rescue experiments showed that restoring LDHA in CRC cells which were overexpressing miR-30c-5p successfully overridden 5-Fu resistance. Importantly, restoring miR-30a-5p in NOP14-AS1-overexpressing cells effectively restored 5-Fu sensitivity in HT-29 5-Fu R cells by targeting the LDHA-mediated glucose metabolism. In summary, our results revealed that lncRNA NOP14-AS1 promotes 5-Fu resistance by mediating the miR-30a-5p-LDHA axis.
Supplementary Information
The online version contains supplementary material available at 10.1007/s12672-025-02156-4.
Keywords: LncRNA-NOP14-AS1, 5-Fu resistance, MiR-30a-5p, Colorectal cancer, Glucose metabolism
Introduction
Colorectal cancer (CRC) is a commonly occurring malignancy, causing high mortality rates globally [1]. Despite advancements in administrative approaches improving outcomes for CRC patients overall, the prognosis and survival of colon cancer patients have not met expectations [2]. 5-Fluorouracil (5-Fu), a chemotherapy agent which has been used for over 40 years, disrupts nucleotide synthesis of cancer cells and is commonly used in cancer therapy [3]. However, a subset of cancer patients develop resistance to 5-Fu, significantly limiting its clinical applications [4, 5]. Investigating the molecular mechanisms behind 5-Fu resistance in colorectal patients is currently a critical task.
LncRNAs which have limited protein-coding capacities, are a newly identified class of RNAs which have relative larger nucleotides sizes [6]. LncRNAs are involved in various physiological and pathological processes in cancer cells [7, 8]. Furthermore, dysregulation of lncRNAs has been frequently noticed in CRC [9, 10]. LncRNA NOP14 antisense RNA 1 (NOP14-AS1) was positively associated with multiple cancers such as liver cancer and lung cancer [11]. Moreover, studies have revealed that expressions of NOP14-AS1 were significantly upregulated by cell death inducers including the traditional anticancer drug, cisplatin [11], suggesting NOP14-AS1 functions as an oncogenic molecule and is tightly associated with chemotherapy efficiency in cancer cells. Currently, the precise roles and molecular mechanisms of NOP14-AS1 in mediating 5-Fu sensitivity of CRC cells remain unclear and require further exploration.
Cancer cells display distinct metabolic characteristics, including elevated anaerobic glycolysis and impaired mitochondrial respiration [12, 13]. Metabolic reprogramming provides vital materials and intermediates for progressions of cancer cells [14]. Moreover, dysregulated cellular metabolism has been closely contributed to chemoresistance in cancer cells [14]. One enzyme involved in glucose metabolism, LDHA, catalyzes the conversion of pyruvate to lactate [15]. Studies revealed that LDHA was significantly upregulated in various cancers [16, 17] and acted as a critical biomarker, conferring selectively proliferative and chemoresistant advantages to tumor cells [18]. This study assessed the role and underlying mechanism of NOP14-AS1 in 5-Fu resistant CRC cells. Functional experiments were performed to exam the effects of NOP14-AS1 on chemoresistant CRC. Additionally, the molecular targets of NOP14-AS1 were validated. These findings will provide insights for developing effective therapeutic strategies to overcome 5-Fu resistance in CRC patients.
Materials and methods
Ethics approval and colon cancer tissue collection
Ethics was approved by the Ethics Committee of School of Medicine, Xuchang University [20]. Informed consents (Consent to Participate and Consent to Publish) were obtained from all participants or, if participants are under 18, from a parent and/or legal guardian. Adjacent normal colon tissues and CRC tissues were collected in 40 colorectal cancer patients in School of Medicine, Xuchang University (Table 1). Specimens were immediately frozen in liquid nitrogen, followed by transferring to −80 °C freezer.
Table 1.
Characteristics of CRC patients (n = 40)
Cell culture
CRC cells were obtained from Chinese academy of science and maintained in DMEM medium (Sigma-Aldrich, USA) containing FBS (10%) (Invitrogen, USA), 1 × penicillin and 1 × streptomycin (Invitrogen, USA) with 5% CO2 at 37 °C. HT-29 5-Fu resistant cells were established by stepwise exposing HT-29 parental cells to increased concentrations of 5-Fu for 3 months. Survival cells were pooled and cryopreserved for downstream experiments. Cells were used in the 2nd to 4th passages. Resistance was re-examined by 5-Fu treatment each three months to eliminate the potential cells which lost resistance.
Transfection
Transfection was conducted by Lipofectamine 3000 agent (Thermo Fisher, USA). SiRNA, miRNA and their controls were purchased from GenePharma (Shanghai, China). LDHA overexpression plasmid (#RC209378) was obtained from Origen.com. ORF of LDHA transcript 1 was amplified and cloned into pCMV6-Entry Mammalian Expression Vector using the Sgf I and Mlu I digestion sites. The insertion was sequenced and verified before used in experiments. Transfection was performed at 100 nM for 48 h.
Bioinformatics analysis
The analysis of the RNA-RNA interactions was predicted through starBase. Analysis of survival rates of CRC patients was performed by Kaplan–Meier method which analyzed the correlation between gene expression and survival rate in cancer patients. A steep slope suggests a higher death rate and a poorer prognosis, while a flatter slope indicates a lower death rate and a better prognosis. Gene expression correlations were analyzed by Pearson’s correlation coefficient.
RNA extraction and qRT-PCR
Total RNAs were extracted by Trizol reagent (Invitrogen, USA). Concentrations and purities of RNAs were checked using NanoDrop 1000 Spectrophotometer (Thermofisher, USA). cDNA was synthesized using the miScript Reverse Transcription Kit (Qiagen, USA). qPCR reactions were performed using the SYBR® Premix Ex Taq™ II (TaKaRa, China). The primer sequences used for PCR were: NOP14-AS1: Forward: 5’-CCATGCCCTCCTTGTTTACT-3’ and Reverse: 5’-GGGAAAGGGCTGTTATCATCTT-3’; LDHA: Forward: 5’-AGCCCGATTCCGTTACCT-3’ and Reverse: 5’-CACCAGCAACATTCATTCCA-3’; GLUT1: Forward: 5’-AGTTCTACAACCAGACATGGAGCCCGATTCCGTTACCT-3’ and Reverse: 5’-CAGGTTCATCATCAGCATTGCACCAGCAACATTCATTCCA-3’; PDK1: Forward: 5’-ATGATGTCATTCCCACAATG-3’ and Reverse: 5’-AAGAGTGCTGATTGAGTAAC-3’; β-actin: Forward: 5’-CTGAGAGGGAAATCGTGCGT-3’ and Reverse: 5’-CCACAGGATTCCATACCCAAGA-3’; miR-30a-5p: Forward: 5’-ACACTCCAGCTGGGTGTAAACATCCTCGACTG-3’ and Reverse: 5’-CTCAACTGGTGTCGTGGA-3’; U6: Forward: 5’-CTCGCTTCGGCAGCACA-3’ and Reverse: 5’-AACGCTTCACGAATTTGCGT-3’. Relative expressions were analyzed using the 2−ΔΔCt method.
Luciferase assay
Wild-type NOP14-AS1 (WT-NOP14-AS1) and miR-30a-5p binding site mutant NOP14-AS1 (Mut-NOP14-AS1) and 3’UTRs of wild-type LHDA (WT-LDHA) and miR-30a-5p binding site mutant LDHA (Mut-LDHA) were amplified and subcloned into pmiR-GLO vector (Promega, USA). The miR-30a-5p binding site mutant luciferase reporters (Mut-NOP14-AS1 and Mut-LDHA) were created based on WT-NOP14-AS1 and WT-LDHA as templates using the QuikChange® Site-Directed Mutagenesis kit (Agilent Technologies, USA.). The mutant sites were shown as bold: Mut-NOP14-AS1: 5'-ccUCCAUGCCCUCCUACAAAUGu-3' and Mut-LDHA: 5'-gagCCAGGUCCUACAAAUGc-3'. CRC cells were co-transfected with control miRNAs or microRNA-30a-5p and WT- or Mut- NOP14-AS1; WT- or Mut- LDHA 3’UTR. Luciferase activity was examined. The firefly luciferase enzyme activity was normalized to Renilla luciferase enzyme activity.
Cell viability assay
CRC cells were plated onto 96-well plate (5 × 103 per well) and cultured for 24 h. After treatments with 5-Fu, cells from each well were incubated with 100 µL (0.5 mg/ml) MTT (Sigma-Aldrich, USA) and incubated at 37 °C for 4 h. After incubation, DMSO (150 μL) was added. The absorbance at detected at 570 nm.
Cell apoptosis assay
The Annexin V-FITC Apoptosis Kit (Becton Dickinson, USA) was used. CRC cells (1 × 106) cells were resuspended in binding buffer (500 μL) followed by loading of Annexin V FITC (5 μL) and propidium iodide (5 μL) and cultured for 30 min without light. Late and early apoptotic rates of colon cancer cells were analyzed.
Measurement of glucose metabolism
Glucose uptake assay (ab136955, Abcam, USA) and lactate product assays (MAK329, Sigma-Aldrich, China) were applied based on protocols from kits to evaluate glucose metabolism rate. For detection of glucose uptake, cells were seeding at a density of 2 × 103 cells/well in 100 μL culture medium in a sterile 96-well plate for overnight. Cells were washed with PBS and starve in serum free medium overnight, followed by adding 10 μL of 10 mM 2-DG into medium and incubating from 20 min. After degrading endogenous NAD(P) by heating, 10 μL of Reaction Mix A from the kit were added into medium to incubate at 37 °C for 1 h. Extraction Buffer I/Extraction Buffer (90 μL) from the kit was added to each well and samples were heat at 90 °C for 40 min with sealing the microplate. After cooling to room temperature, Neutralization Buffer II/Neutralizing Buffer (12 μL) were added into well, followed by adding the prepared master mix of the Reaction Mix B (38 uL) containing Glutathione Reductase, DTNB/Substrate and Recycling Mix (38 μL). Samples were measured at OD 412 nm on a microplate reader at 37 °C protected from light. Relative glucose uptake was normalized by the ratio of the readings of the experimental group to those of the control group. For detection of lactate product, cells were seeding at a density of 2 × 103 cells/well in 100 μL culture medium in a sterile 96-well plate for 24 h. Cell culture medium of each sample (20 μL) was transferred into separate wells, followed by adding 80 μL of Reaction Mix from the kit per reaction well and mixing thoroughly. The initial absorbance was measured at OD565 nm on a microplate reader. After incubation for 20 min at room temperature, samples were measured at OD565 for the final absorbance. The relative lactate product was calculated by subtracting the initial OD565 from the final OD565 and then normalized by the ratio of the readings of the experimental group to those of the control group.
Western blot analysis
Proteins were extracted by RIPA buffer (Pierce, USA). Protein samples were separated onto 10% SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes. Membranes were incubated with primary antibodies overnight in a cold room. After completely washing off primary antibody residues, membranes were incubated with secondary antibody (anti-rabbit) for 1 h at room temperature. Proteins on membranes were examined using ECL Western Blotting Substrate (Beyotime, China).
Statistical analysis
Data were shown as the mean ± standard deviation (SD). Experiments were conducted in three biological replicates. Student’s t-test was used for comparing difference between two groups. One-way analysis of variance (One-way ANOVA) followed by Tukey’s post hoc test was applied to compare the differences among three or more groups. P < 0.05 was considered statistically significant.
Results
LncRNA NOP14-AS1 is high-expressed in CRC and promotes 5-Fu resistance
A previous study has reported that NOP14-AS1 was high-expressed in liver cancer and lung cancer [11], indicating that NOP14-AS1 plays oncogenic roles in colon cancer. In order to evaluate the roles of NOP14-AS1 in colon cancer, expression of NOP14-AS1 was examined in forty CRC tumor specimens as well as paired normal colon tissues using qRT-PCR. Expression of NOP14-AS1 was remarkedly upregulated in CRC specimens (Fig. 1A). Moreover, expressions of NOP14-AS1 were considerably higher in five CRC cell lines compared to normal cell line (Fig. 1B). To investigate the effects of NOP14-AS1 on the chemosensitivity of colon cancer cells, NOP14-AS1 was silenced in LoVo and DLD-1 cells (Fig. S1). Expectedly, it was observed that CRC cells with lower levels of NOP14-AS1 exhibited significantly elevated 5-Fu sensitivity compared to control cells (Fig. 1C–F, S2A, S2B). In summary, the above results indicate that NOP14-AS1 functions as a potential oncogene in colon cancer.
Fig. 1.
NOP14-AS1 is upregulated in colon cancer and promotes 5-Fu resistance. A Expressions of NOP14-AS1 were measured using qRT-PCR in forty human colon cancer tissues and their corresponding non-tumor tissues. Statistical analysis was performed using unpaired student’s t test. B Expressions of NOP14-AS1 were measured using qRT-PCR in a normal colon epidermal cell line and five colon cell lines. C HT-29 and (D) DLD-1 cells were transfected with siNOP14-AS1 or control siRNA. Cells were treated with indicated concentrations of 5-Fu for 48 h. Cell responses to 5-Fu treatment were examined using E cell viability assay and F apoptosis assay. *, p < 0.05; **, p < 0.01; *** p < 0.001
5-Fu resistant CRC cells display promoted NOP14-AS1 expression and glucose metabolism
A 5-Fu resistant CRC cell line (HT-29 5-Fu R) was established to explore the mechanisms of NOP14-AS1 in 5-Fu resistant CRC cells. Resistance was validated that HT-29 5-Fu R cells could withstand higher amount of 5-Fu treatment (Fig. 2A, B). The IC50 value of HT-29 cells was 7.23 µM, which was apparently lower than that of the HT-29 5-Fu R cells (22.31 µM). Additionally, the clonogenic assay demonstrated that 5-Fu resistant CRC cells had a better survival capacity compared to HT-29 parental cells under same 5-Fu treatment. Furthermore, the expression of NOP14-AS1 was elevated (p < 0.001) in HT-29 5-Fu R cells (Fig. 2C), suggesting that NOP14-AS1 facilitates 5-Fu resistance in colorectal cancer cells. Subsequently, we investigated which cellular characteristic was altered in 5-Fu resistant CRC cells. Previous studies have shown that glucose metabolism reprogramming of cancer cells was closely linked to chemoresistance [14]. As expected, we found that glucose uptake (Fig. 2D) and lactate production (Fig. 2E) were remarkedly increased in HT-29 5-Fu R cells. Furthermore, expressions of glucose metabolism key enzymes, HK2, GLUT1, and LDHA were increased in 5-Fu resistant CRC cells (Fig. 2F), implying that elevated glucose metabolism rate contributes to 5-Fu resistance of colon cancer cells. To assess whether NOP14-AS1 regulates glucose metabolism and 5-Fu resistance, NOP14-AS1 was knocked-down in HT-29 5-Fu R cells (Fig. 2G). Expected results demonstrated that silencing NOP14-AS1 effectively suppressed glucose uptake (Fig. 2H), lactate production (Fig. 2I) and expressions of glucose metabolism enzymes of HT-19 5-Fu R cells (Fig. 2J). Consistent results were obtained in HT-29 parental cells (Fig. S3A-S3C). Furthermore, silencing NOP14-AS1 remarkedly sensitized 5-Fu resistant HT-29 cells to 5-Fu treatment, as shown in Fig. 2K, L S4.
Fig. 2.
5-Fu resistant colorectal cancer cells show elevated NOP14-AS1 expression and glucose metabolism. A HT-29 parental and 5-Fu resistant cells were treated with 5-Fu. Cell viability was assessed using a cell viability assay and B clonogenic assay. C Expression levels of NOP14-AS1 were measured in HT-29 parental and 5-Fu resistant cells using qRT-PCR. D Glucose uptake, E lactate production and F glucose metabolism enzymes expressions were measured in HT-29 parental and 5-Fu resistant cells. G HT-29 5-Fu resistant cells were transfected with either control siRNA or NOP14-AS1 siRNA, and expression levels of NOP14-AS1 were measured. H Glucose uptake, I lactate production, and J expression levels of glucose metabolism enzymes were detected in the above transfected HT-29 5-Fu resistant cells. K Transfected cells were then exposed to 5-Fu for 48 h, and cell responses to 5-Fu were assessed using a cell viability assay and L apoptosis assay. *, p < 0.05; **, p < 0.01; *** p < 0.001
miR-30a-5p is sponged by NOP14-AS1 and negatively associated with colon cancer
We then investigated the mechanism of NOP14-AS1 in mediating 5-Fu resistance. The miRNA targets of NOP14-AS1 were predicted using the non-coding RNA service, starBase, as previous studies unveiled that lncRNAs acted as molecular sponges of miRNAs to influence downstream mRNA expressions [19]. Results illustrated that miR-30a-5p contained binding sites for NOP14-AS1 (Fig. 3A), indicating that NOP14-AS1 may downregulate miR-30a-5p by sponging it. We explored the roles of miR-30a-5p in colon cancer patients. As expected, a negative correlation (p < 0.001) was obtained between the expressions of NOP14-AS1 and miR-30a-5p in CRC specimens (Fig. 3B). Additionally, overexpression of NOP14-AS1 effectively downregulated miR-30a-5p in HT-29 and DLD-1 cells (Fig. 3C). Subsequently, luciferase assay was analyzed to confirm whether NOP14-AS1 directly interacts with miR-30a-5p in colon cancer cells. Co-transfection of miR-30a-5p with the Wild-Type NOP14-AS1 luciferase vector led to a decreased luciferase activity (p < 0.001) (Fig. 3D, E). Meanwhile, miR-30a-5p was unable to effectively suppress (p > 0.05) the luciferase activity of the mutant NOP14-AS1 (Fig. 3D, E). These results demonstrate that the lncRNA NOP14-AS1 inhibits miR-30a-5p expression through sponging it in colon cancer cells.
Fig. 3.
NOP14-AS1 sponges miR-30a-5p to block its expression in CRC cells. A The association between NOP14-AS1 and miR-30a-5p is predicted by StarBase. B Pearson’s correlation analysis shows a negative correlation between NOP14-AS1 and miR-30a-5p expressions in colon cancer tissues. C NOP14-AS1 is overexpressed in HT-29 and DLD-1 cells. D, E Luciferase assays are performed in HT-29 and DLD-1 cells by co-transfection with control miRNA or miR-30a-5p along with WT-NOP14-AS1 or Mut-NOP14-AS1. Luciferase activities are examined. **, p < 0.01; ***. p < 0.001
Subsequently, the biological roles of miR-30a-5p in colon cancer were examined. Results showed miR-30a-5p was significantly low-expressed in colon tumor specimens (Fig. 4A) and cancer cell lines (Fig. 4B), indicating it acts as a tumor suppressor in colon cancer. Consistently, overexpression of miR-30a-5p effectively suppressed glucose uptake and lactate production in CRC cells (Fig. 4C, D). Furthermore, CRC cells with high-level of miR-30a-5p displayed elevated 5- Fu sensitivity (Fig. 4E, F), suggesting that miR-30a-5p targets the glucose metabolism pathway to promote the 5-Fu sensitivity of CRC cells.
Fig. 4.
miR-30a-5p is negatively associated with colon cancer. A The expression of miR-30a-5p was examined in forty human colon cancer tissues and paired non-tumor specimens using qRT-PCR. Statistical analysis was performed using unpaired student’s t test. B The expression of miR-30a-5p was measured in a normal colon epidermal cell line and five colon cell lines using qRT-PCR. C HT-29 and DLD-1 cells were transfected with control miRNA or miR-30a-5p, and the glucose uptake was examined. D The lactate production in HT-29 and DLD-1 cells transfected with control miRNA or miR-30a-5p was examined. E, F The above cells were treated with 5-Fu for 48 h, and the cell viability was determined using a cell viability assay. *, p < 0.05; **, p < 0.01; ***, p < 0.001
miR-30a-5p directly targets LDHA to inhibit glucose metabolism of CRC cells
We have demonstrated an axis of NOP14-AS1-miR-30a-5p-glucose metabolism in HT-29 5-Fu R CRC cells. It is known that miRNAs block target transcripts via binding to the target mRNA 3’UTR [20]. We analyzed the mRNA targets of miR-30a-5p from starBase. Among the predicted candidates, we found LDHA, which is frequently upregulated in human cancers and converts pyruvate to lactate [15], contained miR-30a-5p binding sites in its 3’UTR (Fig. 5A). Expressions of LDHA and miR-30a-5p were negatively correlated in colon tumors from the TCGA cancer database (Fig. S5) and by Pearson’s correlation coefficient analysis (Fig. 5B). Additionally, expression of miR-30a-5p was negatively correlated with other glucose metabolism enzymes, GLUT1 (Fig. S6A) and PDK1 (Fig. S6B) in colon tumors. Consistently, LDHA was significantly high expressed in colon cancer tissues and cells (Fig. 5C, D). To assess whether the protein expression of LDHA was suppressed by miR-30a-5p, we overexpressed miR-30a-5p in DLD-1 and HT-29 cells. Western blot results showed that colon cancer cells with overexpression of miR-30a-5p displayed downregulated LDHA expressions (Fig. 5E). We then verified whether miR-30a-5p targeted the LDHA 3’UTR, results from luciferase assay showed that luciferase activity of CRC cells which were co-transfected with miR-30a-5p and WT-LDHA 3’UTR was significantly blocked (Fig. 5F, G). The luciferase activity of CRC cells which were co-transfected with Mut-LDHA 3’UTR and miR-30a-5p was not significantly affected (Fig. 5F, G). Our results consistently verified that miR-30a-5p bond to the LDHA 3’UTR in CRC cells.
Fig. 5.
LDHA is directly targeted by miR-30a-5p in CRC cells. A Targetscan.org predicted binding of miR-30a-5p on LDHA 3’UTR. B Pearson’s correlation analysis showed negative correlation between LDHA and miR-30a-5p expressions in colon cancer tissues. C LDHA expressions were measured by qRT-PCR in colon cancer tissues (n = 40) and normal colon tissues (n = 40). D LDHA expressions were compared in colon cancer cell lines and normal colon epithelial cells. E Protein expressions of LDHA were shown after transfecting colon cancer cells with control miRNA or miR-30a-5p. F Luciferase assays were performed in HT-29 and G DLD-1 cells that were co-transfected with control miRNA or miR-30a-5p along with WT-LDHA 3’UTR or Mut-LDHA 3’UTR. Luciferase activities were examined. **, p < 0.01; ***. p < 0.001
To validate whether miR-30a-5p suppressed glucose metabolism by targeting 3’UTR of LDHA, rescue experiments were conducted. HT-29 5-Fu R cells were transfected with controls, miR-30a-5p alone or plus LDHA overexpression plasmid. Results from Western blot validated that LDHA expression was rescued in HT-29 5-Fu R cells with co-transfection of miR-30a-5p plus LDHA (Fig. 6A). Expectedly, restoration of LDHA successfully restored glucose metabolism of HT-29 5-Fu R cells (Fig. 6B, C).
Fig. 6.
Restoring LDHA in miR-30a-5p-overexpressing CRC cells recovers glucose metabolism. A Protein expressions of LDHA were shown after transfecting HT-29 5-Fu resistant cells with control miRNA, miR-30a-5p alone, or miR-30a-5p plus LDHA overexpression plasmid. B Glucose uptake and C lactate production were examined in the above cells. *, p < 0.05
miR-30a-5p targets LDHA to re-sensitize 5-Fu resistant CRC cells
We subsequently asked whether inhibiting glucose metabolism could increase the sensitivity of 5-Fu. HT-29 5-Fu R cells were treated with PBS, glucose metabolism inhibitor (Oxamate), 5-Fu, or 5-Fu plus Oxamate. Cell viability assays showed that inhibiting glucose metabolism effectively improved the 5-Fu sensitivity of HT-29 5-Fu R cells (Fig. 7A). Furthermore, to validate whether miR-30a-5p increased 5-Fu sensitivity by blocking LDHA-mediated glucose metabolism, the transfected HT-29 5-Fu resistant cells mentioned above (Fig. 6A) were treated with 5-Fu. Consistently, cell viability and apoptosis assays supported that restoring LDHA in miR-30a-5p-overexpressing HT-29 5-Fu R cells successfully reversed the 5-Fu resistance (Fig. 7B, C, S7), confirming that miR-30a-5p targeted LDHA to sensitize 5-Fu R cells to 5-Fu.
Fig. 7.
Restoring LDHA in miR-30a-5p-overexpressing CRC cells recovers 5-Fu resistance. A HT-29 5-Fu resistant cells were treated with control, Oxamate alone, 5-Fu alone, or 5-Fu plus Oxamate. Cell viability was examined. B Control miRNA, miR-30a-5p alone, or miR-30a-5p plus LDHA overexpression plasmid was transfected into HT-29 5-Fu R cells, followed by treatment with control or 5-Fu. Cell responses to 5-Fu were determined by cell viability assay and C apoptosis assay. *, p < 0.05; **, p < 0.01
NOP14-AS1 promotes 5-Fu resistance by regulating the miR-30a-5p-LDHA axis
Given the above results, which have shown that NOP14-AS1 promotes 5-Fu resistance and inhibits miR-30a-5p, thus blocking glucose metabolism by targeting LDHA, we then asked whether the NOP14-AS1-mediated 5-Fu resistance is caused by the regulation of the miR-30a-5p-LDHA axis. Expression of NOP14-AS1 was positively correlated with expressions of LDHA, as well as other glucose metabolism enzymes in CRC tissues (Fig. 8A, S8A, S8B). HT-29 5-Fu R cells were transfected NOP14-AS1 overexpression vector alone, or NOP14-AS1 plus miR-30a-5p as well as control vector. Co-transfection of NOP14-AS1 with miR-30a-5p successfully restored miR-30a-5p and LDHA expressions compared to control and NOP14-AS1 transfection (Fig. 8B, C). As expected, HT-29 5-Fu R cells with NOP14-AS1 overexpression alone exhibited increased 5-Fu resistance (Fig. 8D, E). Meanwhile, HT-29 5-Fu resistant cells co-transfected with NOP14-AS1 and miR-30a-5p showed restored 5-Fu sensitivity (Fig. 8D, E, S9). In summary, these findings consistently demonstrate that NOP14-AS1 directly regulates the miR-30a-5p-LDHA axis, resulting in 5-Fu resistance in CRC cells.
Fig. 8.
Roles of the NOP14-AS1-miR-30a-5p-LDHA axis in 5-Fu resistant colon cancer cells. A Pearson’s correlation analysis showed positive correlation between NOP14-AS1 and LDHA expressions in colon cancer tissues. B HT-29 5-Fu R cells were transfected with control, NOP14-AS1 alone, or NOP14-AS1 plus miR-30a-5p. Expressions of miR-30a-5p and C LDHA were examined by qRT-PCR and Western blot. D The above cells were treated with 5-Fu. Cell responses to 5-Fu were determined by cell viability assay and E Annexin V apoptosis assay. *, p < 0.05; **, p < 0.01
Discussion
Colon cancer is a malignant disease which leads to high mortality rates worldwide [1]. The survival rate (5 years) of colon cancer patients remains low due to a significant number of newly diagnosed CRC patients being at an advanced or metastatic stage [2]. Adjuvant therapy, using either single-agent 5-Fu or a combination of 5-Fu with oxaliplatin, is widely used as an effective chemotherapy strategy for advanced CRC [3]. However, the development of 5-Fu resistance severely limits the effectiveness of these chemotherapy approaches [4, 5]. Currently, the molecular mechanisms of chemoresistant CRC are still being investigated. Investigating the precise mechanisms of 5-Fu resistance in CRC cells is a crucial task. Here, we explored the role and molecular mechanism of non-coding RNAs in modulating 5-Fu resistance of CRC cells. Expression of NOP14-AS1 was high-expressed in colorectal tumors and cell lines. Inhibiting NOP14-AS1 effectively sensitized CRC cells to 5-Fu, suggesting that blocking NOP14-AS1 could be a potential approach against chemoresistant CRC.
LncRNAs play critical roles in regulating progressions of various cancers through competitively binding with target microRNAs to form competing endogenous RNA (ceRNA) networks, ultimately blocking downstream signaling pathways of miRNAs [10]. Previous studies have shown that miR-30a-5p played tumor suppressive roles in the development and chemosensitivity of multiple cancers [21–25]. Here, we discovered that miR-30a-5p was low-expressed in CRC tumors and cell lines. Patients with lower miR-30a-5p expression had worse survival rates. Through bioinformatics analysis, we predicted that NOP14-AS1 contains potential binding sites for miR-30a-5p. This predicted association was further confirmed through luciferase and RNA pull-down assays. These consistent results indicate the existence of a NOP14-AS1-miR-30a-5p ceRNA network in 5-Fu resistant colon cancer cells.
Cancer cells show a distinct metabolic phenotype in which they use glucose for aerobic glycolysis instead of oxidative phosphorylation [26]. Furthermore, studies uncovered that targeting cellular metabolism of cancer cells can improve anti-cancer treatment [12, 14, 27]. Our findings demonstrated that glucose metabolism rate was significantly elevated in 5-Fu resistant colon cancer cells. Inhibiting glucose metabolism effectively restored 5-Fu sensitivity. Interestingly, NOP14-AS1 and miR-30a-5p played opposing roles in 5-Fu R CRC cells, as silencing NOP14-AS1 or overexpressing miR-30a-5p effectively suppressed glucose metabolism. We predicted the targets of miR-30a-5p through bioinformatics analysis. The binding of miR-30a-5p on LDHA 3’UTR was further confirmed by luciferase assay. Subsequent rescue experiments demonstrated that NOP14-AS1 facilitated 5-Fu resistance through the miR-30a-5p-LDHA pathway. Yet, the binding of miR-30a-5p and LDHA has been reported in breast cancer [28], our results are the first to integrate the role of NOP14-AS1 in promoting 5-Fu resistance and the miR-30a-5p-LDHA axis in chemoresistant CRC cells.
In summary, our study uncovered role and mechanism of NOP14-AS1 in 5-Fu resistant CRC cells. It introduces the NOP14-AS1-miR-30a-5p-LDHA axis as a novel biotarget for treating colon cancer cases that are resistant to 5-Fu.
Supplementary Information
Supplementary Material 1. Figure S1. NOP14-AS1 knockdown efficiency in CRC cells. HT-29 and DLD-1 cells were transfected with control siRNA or siNOP14-AS1, expressions of NOP14-AS1 were examined by qRT-PCR. **, p < 0.01; ***, p < 0.001.
Supplementary Material 2. Figure S2. Quantification of the Annexin V apoptosis results of Fig. 1Eand 1F. **, p < 0.01.
Supplementary Material 3. Figure S3. Effects of NOP14-AS1 knockdown on glucose metabolism of HT-29 parental cells.NOP14-AS1 was silenced in HT-29 cells, followed by measurements of glucose uptake,lactate production andexpressions of glucose metabolism enzymes. *, p < 0.05; **, p < 0.01.
Supplementary Material 4. Figure S4. Quantification of the Annexin V apoptosis results of Fig. 2L. **, p < 0.01.
Supplementary Material 5. Figure S5. Correlation between LDHA and miR-30a-5p expressions in colon cancer specimens analyzed from TCGA cancer database from starBase
Supplementary Material 6. Figure S6. Correlation between expressions of miR-30a-5p and glucose metabolism enzymes in colon tumors by Pearson’s correlation coefficient.Correlation between expressions of miR-30a-5p and GLUT1.Correlation between expressions of miR-30a-5p and PDK1
Supplementary Material 7. Figure S7. Quantification of the Annexin V apoptosis results of Fig. 7C. **, p < 0.01.
Supplementary Material 8. Figure S8. Correlation between expressions of NOP14-AS1 and glucose metabolism enzymes in colon tumors by Pearson’s correlation coefficient.Correlation between expressions of NOP14-AS1 and GLUT1.Correlation between expressions of NOP14-AS1 and PDK1
Supplementary Material 9. Figure S9. Quantification of the Annexin V apoptosis results of Fig. 8e. **, p < 0.01.
Acknowledgements
None.
Author contributions
Y.N.L. designed the study, performed the experiments, analyzed data and wrote the manuscript.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
This study was carried out in compliance with the Helsinki Declaration. Informed consents (Consent to Participate and Consent to Publish) were obtained from all participants or, if participants are under 18, from a parent and/or legal guardian.
Competing interests
The authors declare no competing interests.
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplementary Material 1. Figure S1. NOP14-AS1 knockdown efficiency in CRC cells. HT-29 and DLD-1 cells were transfected with control siRNA or siNOP14-AS1, expressions of NOP14-AS1 were examined by qRT-PCR. **, p < 0.01; ***, p < 0.001.
Supplementary Material 2. Figure S2. Quantification of the Annexin V apoptosis results of Fig. 1Eand 1F. **, p < 0.01.
Supplementary Material 3. Figure S3. Effects of NOP14-AS1 knockdown on glucose metabolism of HT-29 parental cells.NOP14-AS1 was silenced in HT-29 cells, followed by measurements of glucose uptake,lactate production andexpressions of glucose metabolism enzymes. *, p < 0.05; **, p < 0.01.
Supplementary Material 4. Figure S4. Quantification of the Annexin V apoptosis results of Fig. 2L. **, p < 0.01.
Supplementary Material 5. Figure S5. Correlation between LDHA and miR-30a-5p expressions in colon cancer specimens analyzed from TCGA cancer database from starBase
Supplementary Material 6. Figure S6. Correlation between expressions of miR-30a-5p and glucose metabolism enzymes in colon tumors by Pearson’s correlation coefficient.Correlation between expressions of miR-30a-5p and GLUT1.Correlation between expressions of miR-30a-5p and PDK1
Supplementary Material 7. Figure S7. Quantification of the Annexin V apoptosis results of Fig. 7C. **, p < 0.01.
Supplementary Material 8. Figure S8. Correlation between expressions of NOP14-AS1 and glucose metabolism enzymes in colon tumors by Pearson’s correlation coefficient.Correlation between expressions of NOP14-AS1 and GLUT1.Correlation between expressions of NOP14-AS1 and PDK1
Supplementary Material 9. Figure S9. Quantification of the Annexin V apoptosis results of Fig. 8e. **, p < 0.01.
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.