NONHSAG028908.3 sponges miR‑34a‑5p to promote growth of colorectal cancer via targeting ALDOA
- Authors:
- Published online on: March 15, 2023 https://doi.org/10.3892/or.2023.8526
- Article Number: 89
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Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Colorectal cancer (CRC), the third most common malignant cancer, exhibits high mortality worldwide (1). It was revealed that a total of 53,200 individuals succumbed to CRC in the United States, in 2020 (2). Although the diagnosis and treatment of CRC have improved, patients particularly those with advanced CRC still have a poor prognosis (3). Therefore, it is worth clarifying the molecular mechanism of CRC for effective treatment.
Recently, long non-coding RNAs (lncRNAs) have been demonstrated as the pivotal regulators of various tumorigenic processes such as cell proliferation, migration, and apoptosis (4–6). Research has revealed that several lncRNAs, such as lncRNA GASS, ADIPOQ, FEZF1-AS1 and GLCC1, are aberrantly expressed in CRC and play vital roles in the development of CRC (7–10). Among lncRNAs, NONHSAG028908.3 also known as LINC01123, has been demonstrated to promote cell proliferation, migration and invasion in different types of cancer, including non-small cell lung cancer, osteosarcoma and hepatocellular carcinoma (11–13), however the role of NONHSAG028908.3 in CRC progression is poorly understood. Data obtained from The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/) database reveals that the expression of NONHSAG028908.3 is significantly upregulated in CRC tissues compared with normal tissues (P<0.001). In addition, patients with high expression of NONHSAG028908.3 may have low survival probability. Thus, it is hypothesized that NONHSAG028908.3 contributes to the progression of CRC.
In addition, lncRNAs act as a sponge to bind with microRNAs (miRNAs or miRs), mRNA or proteins to perform biological functions (14). Studies indicate that the aberrant expression of miRNAs participates in regulating tumorigenic processes in CRC (15). For instance, Huang et al reported that reduced expression of miR-4319 was correlated with poor prognosis in patients with CRC, and miR-4319 suppressed CRC progression by targeting ABTB1 (16). Although some studies have revealed that miR-34a-5p is usually downregulated in CRC (17–20), relevant molecular mechanisms of miR-34a-5p in CRC have not been fully elucidated. The regulatory association between NONHSAG028908.3 and miR-34a-5p in CRC remains unclear.
Aberrant metabolism, particularly abnormal activation of the glycolytic pathway, is a general feature of cancer. Cancer cells usually exhibit increased glycolysis to produce more energy (21). Moreover, a high rate of glycolysis has been demonstrated to be associated with a poor prognosis in patients with cancer (22). As a glycolytic enzyme, aldolase, fructose-bisphosphate A (ALDOA), has been demonstrated to be upregulated in various types of cancer (23–25). Nevertheless, the role of ALDOA in CRC is controversial. Li et al reported that downregulation of ALDOA promoted the migration of CRC cells (SW480 and SW620) (26). By contrast, Dai et al revealed that ALDOA was highly expressed in CRC tissues and was associated with a poor prognosis of CRC (27). Therefore, it is necessary to reveal the function of ALDOA in CRC. Moreover, whether NONHSAG028908.3 targets miR-34a-5p/ALDOA to regulate tumorigenic processes in CRC warrants investigation.
The present study explored the function of NONHSAG028908.3 in CRC, and further clarified its regulatory effect on the miR-34a-5p/ALDOA axis.
Materials and methods
Cell culture
The normal human colon mucosal epithelial cell line (NCM460; cat. no. CP-H040) and human CRC cell lines (LoVo, cat. no. CL-0144; HCT116, cat. no. CL-0096; and Caco-2, cat. no. CL-0050) were purchased from Procell Life Science & Technology Co., Ltd. HT-29 (cat. no. ZQ0057), a CRC cell line, was obtained from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. The cell lines used have been authenticated using STR profiling. NCM460 cells were incubated in human colonic mucosal epithelial cell complete medium (cat. no. CM-H040; Procell Life Science & Technology Co., Ltd.). HT-29 and Caco-2 cells were incubated in DMEM (cat. no. SH30022; Hyclone; Cytiva) and MEM (cat. no. 41500-067; Gibco; Thermo Fisher Scientific, Inc.) medium supplemented with 10% fetal bovine serum (FBS; cat. no. 04-011-1A, Biological Industries; Sartorius AG), respectively. The LoVo cells were incubated in Ham's F-12K (cat. no. PM150910; Procell Life Science & Technology Co., Ltd.) medium which contained 10% FBS. McCoy's 5A (cat. no. PM150710; Procell Life Science & Technology Co., Ltd.) medium including 10% FBS was used to culture HCT116 cells. Penicillin (100 U/ml) and streptomycin (0.1 mg/ml) were added into all the aforementioned media. The cells were maintained in a humidified incubator at a temperature of 37°C and a CO2 ratio of 5%. Cells growing in the logarithmic phase, were used in the subsequent experiments.
Cell transfection
NONHSAG028908.3 siRNAs [si-NONHSAG028908.3-1 forward (F), 5′-GCUAGAUUGCUAUAGUCUATT-3′ and reverse (R), 5′-UAGACUAUAGCAAUCUAGCTT-3′; and si-NONHSAG028908.3-2 F, 5′-GAGAAGUUCUGCAGAUGUATT-3′ and R, 5′-UACAUCUGCAGAACUUCUCTT-3′], ALDOA siRNA (si-ALDOA F, 5′-GGAGGAGUAUGUCAAGCGAGCTT-3′ and R, 5′-UCGCUUGACAUACUCCUCCUGTT-3′); negative control (si-NC F, 5′-UUCUCCGAACGUGUCACGUTT-3′ and R, 5′-ACGUGACACGUUCGGAGAATT-3′); miR-34a-5p mimics (F, 5′-UGGCAGUGUCUUAGCUGGUUGU-3′ and R, 5′-AACCAGCUAAGACACUGCCAUU-3′); NC mimics (F, 5′-UUCUCCGAACGUGUCACGUTT-3′ and R, 5′-ACGUGACACGUUCGGAGAATT-3′); miR-34a-5p inhibitor (F, 5′-ACAACCAGCUAAGACACUGCCA-3′); and NC inhibitor (F, 5′-UUGUACUACACAAAAGUACUG-3′) were obtained from JTS scientific; http://www.jtsbio.com/. The NONHSAG028908.3-overexpressing plasmid and corresponding empty vector were prepared by General Bio Co., Ltd. Lipofectamine 2000 reagent (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.) was used for transfection following the manufacturer's protocols.
When the degree of cell confluence reached 70%, cells in 6-well plates were cultured with serum-free medium for 1 h. Subsequently, 100 µl Opti-MEM was used to dilute 8 µl Lipofectamine 2000 reagent, which was incubated at room temperature for 5 min. A total of 100 pmol of each construct was diluted with 100 µl Opti-MEM following incubation for 5 min, and then was transfected into cells. All dilutions were performed at room temperature for 10 min. Following the addition of the dilutions to the cells for 4 h, the medium was replaced with complete medium and further incubated for 48 h.
Reverse transcription-quantitative PCR (RT-qPCR)
The total RNA from cultured cells was extracted via RNzol-Total RNA isolation Kit (cat. no. RP1001; BioTeke Corporation), and the concentration of RNA was detected using an ultraviolet spectrophotometer NANO 2000 (Thermo Fisher Scientific, Inc.). MiR-34a-5p was extended with a specific loop primer, 5′-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACACAACC-3′. The total RNA was reverse transcribed into complementary DNA (cDNA) using Super M-MLV reverse transcriptase (cat. no. PR6502; BioTeke Corporation). Real-time PCR was performed using SYBR Green (cat. no. S9430; MilliporeSigma). The signals were measured via Exicycler™ 96 PCR instrument (Bioneer Corporation) and the thermocycling program consisted of holding at 94°C for 5 min, followed by 40 cycles of 15 sec at 94°C, 25 sec at 60°C, and 30 sec at 72°C. Gene expression was normalized to β-actin or U6. The relative mRNA expression was calculated via the 2−ΔΔCq method (28). All primer sequences are presented in Table I.
MTT assay
The LoVo, HCT116, and NCM460 cells (3×103 cells/well) were seeded into 96-well plates. Cells were transfected with corresponding si-NC, si-NONHSAG028908.3-1, si-NONHSAG028908.3-2, vector, NONHSAG028908.3, NC inhibitor, miR-34a-5p inhibitor, NC mimics, miR-34a-5p mimics or si-ALODA after cell adherence. The MTT reagent (0.5 mg/ml; cat. no. M-2128; MilliporeSigma) was added to the plates according to the protocol at 0, 24, 48 and 72 h, respectively. After incubation at 37°C for 4 h, the formazan crystals were dissolved using DMSO. Subsequently, the OD values were determined using a microplate reader (ELX-800; BioTek Instruments, Inc.) at 570 nm.
BrdU analysis
The LoVo and HCT116 cells were treated with 10 µM BrdU solution (cat. no. B110731; Aladdin Biochemical Technology Co., Ltd) for 1 h at post transfection. Cells were fixed using 4% paraformaldehyde for 15 min at room temperature, and then 0.1% Triton X-100 (cat. no. ST795; Beyotime Insitute of Biotechnology) was added for 30 min. Subsequently, 10% goat serum (cat. no. SL038; Beijing Solarbio Science & Technology Co., Ltd.) was used for blocking at room temperature for 15 min. Accordingly, the cells were then incubated at 4°C overnight, with anti-BrdU antibody (1:200; cat. no. 66241-1; ProteinTech Group, Inc.). Following incubation with a fluorescence secondary antibody (1:200; cat. no. A0521; Beyotime Institute of Biotechnology), the cells were stained using DAPI (cat. no. C1002; Beyotime Institute of Biotechnology). The images were captured via fluorescence microscope at a magnification of ×400.
Flow cytometry
Following transfection for 48 h, 2×104 cells (LoVo and HCT116) were collected and washed with PBS. Pre-cooled ethanol (concentration, 70%) was used for fixing cells at 4°C for 12 h. Following centrifugation at 1,000 × g at 4°C for 5 min, cells were resuspended in 500 µl cell staining buffer (cat. no. C1052-1; Beyotime Institute of Biotechnology). Subsequently, the cells were stained with propidium iodide (25 µl; cat. no. C1052-2; Beyotime Institute of Biotechnology) and RNase A (10 µl; cat. no. C1052-3; Beyotime Institute of Biotechnology) at 37°C for 30 min in the dark according to the manufacturer's instructions. The cell cycle was then assessed via flow cytometry (NovoCyte; Agilent Technologies, Inc.) using NovoExpress software (version 1.4.1; Agilent Technologies, Inc.).
Western blotting
Proteins from cells were extracted using RIPA lysis buffer with 1% PMSF (cat. nos. P0013B and ST506, respectively; Beyotime Institute of Biotechnology). A BCA kit (cat. no. P0009; Beyotime Institute of Biotechnology) was used to assess the protein concentration. Total protein (30 µg per lane) was separated using 10–12% SDS-PAGE (cat. no. P0015; Beyotime Institute of Biotechnology) and accordingly transferred to a PVDF membrane (cat. no. LC2005; Thermo Fisher Scientific, Inc.). Subsequently, the membrane was placed in Tris-buffered saline with 1.5% Tween-20 (TBST) for 5 min, and then incubated with primary antibody anti-cyclin D1 (1:1,000; cat. no. A19038; ABclonal Biotech Co., Ltd.), anti-CDK4 (1:1,000; cat. no. A0366; ABclonal Biotech Co., Ltd.), anti-mature matrix metalloproteinase (MMP)-2 (1:1,000; 10373–2-AP; ProteinTech Group, Inc.), anti-mature MMP-9 (1:500; cat. no. 10375-2-AP; Proteintech Group, Inc.) and anti-ALDOA (1:1,000; cat. no. A1142; ABclonal Biotech Co., Ltd.) overnight at 4°C. The membrane was washed using TBST following treatment with HRP-labeled secondary antibody (1:10,000; cat. no. SA00001-2; Proteintech Group, Inc.) at 37°C for 40 min. ECL (cat. no. E003; 7sea Technology Co., Ltd.) was then added into membranes to visualize the protein bands. The relative density was evaluated via Gel-Pro-Analyzer software (version 4.0; Beijing Liuyi Biotechnology Co., Ltd.).
Wound healing assay
When LoVo and HCT116 cells reached 100% confluency, the medium was replaced with serum-free medium including 1 µg/ml mitomycin C (cat. no. M0503; MilliporeSigma) for 1 h. A 200-µl pipette tip was used to a scratch the layer of the cells at 0 h, and the images were captured with microscope at a magnification of ×100. The cells were then incubated at 37°C for 24 h. Subsequently, the images were captured.
Transwell assay
The invasion capabilities of LoVo and HCT116 cells were determined via 24-well Transwell chamber assays (cat. no. 3422, Corning, Inc.). LoVo and HCT116 cells were collected at 48 h post transfection. A cell suspension (2×104 cells; 200 µl without FBS) was added to the upper chamber, which was precoated with 40 µl Matrigel for 2 h at 37°C, while the lower chamber contained 30% FBS. After the cells were incubated for 24 h, they were fixed with 4% paraformaldehyde at room temperature for 15 min. Crystal violet (0.4%; cat. no. 0528; Amresco, LLC) was used to stain the cells at room temperature for 5 min, and then the number of cells passing through the Matrigel was determined. Five fields were randomly selected for cell counting, and the experiment was repeated three times.
Dual-luciferase reporter assay
The sequences of wild-type (wt) NONHSAG028908.3 (wt-NONHSAG028908.3), mutant (mut) NONHSAG028908.3 (mut-NONHSAG028908.3), wt-ALDOA or mut-ALDOA were subcloned into pmirGLO vectors (GenScript Biotechnology Co., Ltd.; http://www.genscript.com.cn/). The molecular sequences were as follows: wt-NONHSAG028908.3, 5′-CCCAUCAGCAGCCACUGCCC-3′; mut-NONHSAG028908.3, 5′-CCCAUGUCGUCCGUGACGGC-3′; wt-ALDOA, 5′-CACCCUUUCCGGCACACUGCCA-3′; mut-ALDOA, 5′-CACCCUUUCCGGCUGUGACGGA-3′. Accordingly, these reporter vectors with miR-34a-5p mimics or NC mimics were co-transfected into 293T cells (cat. no. ZQ0033; Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.) using Lipofectamine 2000 (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, the transfected cells were harvested, and the luciferase activities were measured using the dual-luciferase reporter kit (cat. no. KGAF040; Nanjing KeyGen Biotech Co., Ltd.) normalized to Renilla luciferase activity.
Bioinformatics analysis
The expression pattern of NONHSAG028908.3 in CRC tissues and normal tissues was predicted using the online database, TCGA. The data of the correlation between NONHSAG028908.3 and miR-34a-5p and/or ALDOA expression levels in CRC cells or tissues were obtained from the Cancer Cell Line Encyclopedia (CCLE; http://sites.broadinstitute.org/ccle) and TCGA datasets.
Statistical analysis
Results were conducted using GraphPad 8.0 software (GraphPad software Inc.). The unpaired Student's t-test was performed to analyze the differences between two groups. One-way ANOVA followed by Bonferroni's post hoc test was used to analyze multiple comparisons. Pearson's correlation coefficient analysis was used to analyze correlations. Data is presented as the mean ± standard deviation (SD). P<0.05 was considered to indicate a statistically significant difference.
Results
NONHSAG028908.3 is upregulated in CRC tissues and cells
To determine the expression level of NONHSAG028908.3 (www.noncode.org) in CRC tissues and cells, TCGA database was employed and it was revealed that NONHSAG028908.3 was markedly upregulated in CRC tissues compared with that in normal tissues (Fig. 1A; P<0.001). In addition, normal colorectal cell line (NCM460) and four types of CRC cell lines were selected to determine the expression of NONHSAG028908.3 using RT-qPCR. The results revealed that the expression of NONHSAG028908.3 was also increased in HT-29, LoVo, HCT116 and Caco-2 cells compared with NCM460 cells (Fig. 1B; P<0.01). Among these cell lines, two NONHSAG028908.3 high-expressing cell lines, LoVo and HCT116, were selected to generate NONHSAG028908.3-silencing cell lines. RT-qPCR assay indicated that the expression of NONHSAG028908.3 was downregulated both in LoVo and HCT116 cells transfected with si-NONHSAG028908.3-1/2 (Fig. 1C and D; P<0.001).
Silencing of NONHSAG028908.3 suppresses the proliferation of CRC cells
To reveal the function of NONHSAG028908.3 on cell proliferation, an MTT assay was carried out on LoVo, HCT116 and NCM460 cells after transfection. The results of the MTT assay demonstrated that proliferation abilities of CRC cells (LoVo and HCT116) were decreased once transfected with the si-NONHSAG028908.3-1/2 (Fig. 2A and B; P<0.01). Overexpression of NONHSAG028908.3 promoted cell growth in NCM460 cells, a normal colorectal cell line (Fig. S1). In addition, it was found that the number of stained BrdU-positive cells was observably reduced under NONHSAG028908.3 silencing (Fig. 2C and D). In addition, the results of the flow cytometric assays demonstrated that knockdown of NONHSAG028908.3 blocked the cell cycle in the G1 phase (Fig. 3A and B; P<0.05). When the expression of NONHSAG028908.3 was inhibited, the levels of cell cycle-related proteins, cyclin D1 and CDK4, were downregulated in LoVo and HCT116 cells (Fig. 3C and D; P<0.001).
Knockdown of NONHSAG028908.3 restrains CRC cell migration and invasion
To further characterize the role of NONHSAG028908.3 in CRC, wound healing and Transwell assays were performed for monitoring cell migration and cell invasion, respectively. The findings of the wound healing assays revealed that the migration rate of CRC cells was significantly suppressed after inhibition of NONHSAG028908.3 (Fig. 4A; P<0.01). Similarly, the results of the Transwell invasion assays revealed that the number of cells passing through the membranes was markedly diminished when CRC cells were transfected with si-NONHSAG028908.3 (Fig. 4B; P<0.01). Furthermore, the results of the western blot assays indicated that knockdown of NONHSAG028908.3 downregulated the levels of mature MMP-2 and mature MMP-9 (Fig. 4C; P<0.001).
NONHSAG028908.3 serves as a sponge to bind with miR-34a-5p
Previous research has revealed that lncRNAs can sponge miRNAs to regulate metastasis (29). In the present study, it was hypothesized that NONHSAG028908.3 served as a sponge to combine with miR-34a-5p, and their binding sites are presented in Fig. 5A. Accordingly, a luciferase reporter assay was carried out to verify the aforementioned hypothesis. The data demonstrated that miR-34a-5p mimics significantly diminished the luciferase activity of the wt-NONHSAG028908.3 reporter but did not suppress that of the mut-NONHSAG028908.3 reporter (Fig. 5A; P<0.01). Moreover, it was found that the knockdown of NONHSAG028908.3 significantly enhanced miR-34a-5p expression (Fig. 5B and C; P<0.01). These data indicated that NONHSAG028908.3 functioned as a sponge for miR-34a-5p. Additionally, the results of RT-qPCR confirmed the transfection efficiency of miR-34a-5p mimics both in LoVo and HCT116 cells (Fig. 5D and E; P<0.001). Overexpression of miR-34a-5p inhibited the CRC cell growth of LoVo and HCT116 cells according to the data of the MTT assays (Fig. 5F and G). The results of the wound healing assays demonstrated that the ability of cell migration was decreased in miR-34a-5p-overexpressing CRC cells compared with that in cells transfected with NC mimics (Fig. 5H-K).
MiR-34a-5p silencing abolishes the inhibitory effect of NONHSAG028908.3 knockdown on the malignant behavior of cells
To further explore the association between NONHSAG028908.3 and miR-34a-5p in CRC cells, rescue experiments were performed. Data from MTT assays demonstrated that the inhibitory effect of si-NONHSAG028908.3 on cell proliferation was reversed by silencing of miR-34a-5p in LoVo and HCT116 cells (Fig. 6A and B; P<0.05). Wound healing assay also confirmed this finding. In brief, NONHSAG028908.3 knockdown significantly suppressed cell proliferation and migration, whereas silencing of miR-34a-5p abolished the suppressive effect of NONHSAG028908.3 knockdown (Fig. 6C and D; P<0.05).
ALDOA directly targets miR-34a-5p and is positively correlated with NONHSAG028908.3
For the next experiment, pmirGLO luciferase reporter vectors including wt-ALDOA and mut-ALDOA were constructed. When the 293T cells were co-transfected with wt-ALDOA and miR-34a-5p mimics, the luciferase activity was significantly decreased (Fig. 7A; P<0.001). However, the luciferase activity of mut-ALDOA underwent no obvious change. Furthermore, the expression of ALDOA was assessed in miR-34a-5p-overexpressing cells. The results of RT-qPCR and western blot assays demonstrated that overexpression of miR-34a-5p negatively regulated the expression level of ALDOA in LoVo and HCT116 cells (Fig. 7B and C; P<0.001). Furthermore, knockdown of NONHSAG028908.3 significantly decreased the mRNA and protein levels of ALDOA, which were rescued via silencing of miR-34a-5p (Fig. 7D and E; P<0.05). The results obtained from TCGA and CCLE datasets revealed that the expression level of NONHSAG028908.3 was positively correlated with the expression level of ALDOA in CRC (Fig. S2; P=0.001). Collectively, these findings indicated that NONHSAG028908.3 may positively regulate ALDOA expression through sponging miR-34a-5p.
ALDOA silencing suppresses CRC cell growth and migration
Furthermore, ALDOA was successfully knocked down in LoVo and HCT116 CRC cells (Fig. 8A and B; P<0.001). The results of MTT assays demonstrated that silencing of ALDOA inhibited CRC cell proliferation (Fig. 8C and D). In addition, the migration rate of CRC cells transfected with si-ALDOA was markedly reduced compared with that in cells transfected with si-NC (Fig. 8E and H; P<0.01). Thus, the data indicated that knockdown of ALDOA suppressed CRC cell proliferation and migration.
Discussion
Although treatment of CRC has greatly improved, CRC often results in an unfavorable outcome due to the malignant metastasis of CRC cells. In consequence, researchers are urgently seeking new biomarkers for the diagnosis and prognosis of CRC. Numerous studies have demonstrated that tumor-associated genes are frequently aberrantly expressed. A number of genomes are transcribed as noncoding RNAs, of which lncRNAs and miRNAs make up the majority of noncoding RNAs (30,31). Research has revealed that lncRNAs participate in a metastatic cascade and a high abundance of cancer-related lncRNAs regulate CRC pathogenesis (32,33).
To the best of our knowledge, the function of NONHSAG028908.3 has not been fully clarified in human types of cancer. In the present study, data from TCGA database was retrieved, and the results revealed that NONHSAG028908.3 was upregulated in CRC tissues. In addition, a high level of NONHSAG028908.3 was also detected in CRC cells. Furthermore, the promoting effect of NONHSAG028908.3 on cell proliferation and migration was revealed. The aggregation of cyclin D1 and CDK4 has also been demonstrated to be of great importance in the cell cycle (34). Upregulation of cyclin D1 has been revealed to cause dysregulated CDK activity, rapid cell growth under conditions of restricted mitogenic signaling, and finally, neoplastic growth (34). In the present study it was revealed that NONHSAG028908.3 silencing led to G1 cell cycle arrest involving the alleviation of cyclin D1 and CDK4. Moreover, previous studies indicated that MMP-2 and MMP-9 accelerated cell invasion via participating in extracellular matrix degradation (35). Similarly, the data of the present revealed that inhibition of NONHSAG028908.3 suppressed cell invasion, partly by a mechanism associated to the decrease of mature MMP-2 and MMP-9. All aforementioned findings demonstrated the oncogenic property of NONHSAG028908.3 in CRC cell growth.
Mechanistic insights using a luciferase reporter assay confirmed that NONHSAG028908.3 exhibited a high affinity to interact with miR-34a-5p. It has been established that miR-34a-5p is often downregulated and plays a tumor suppressive role in multiple cancers, including CRC. Moreover, a previous study demonstrated that miR-34a-5p was downregulated in CRC tissues, and miR-34a-5p expression was positively related with disease-free survival (17). The data of the present study also confirmed that overexpression of miR-34a-5p suppressed CRC cell proliferation and migration. In addition, the findings of other studies are consistent with the findings of the present study. For instance, Li et al demonstrated that miR-34a-5p was negatively associated with lncARSR expression, and inhibited CRC cell invasion and metastasis (18). Gao et al revealed that miR-34a-5p restrained recurrence of CRC by facilitating cell apoptosis in a p53-dependent manner (17). Bao et al reported that inhibiting miR-34a expression aggravated CRC cell invasion via promoting epithelial-mesenchymal transition (36). Additionally, the results of the present study also revealed that depletion of NONHSAG028908.3 restrained cell proliferation and migration, which was abrogated via miR-34a-5p inhibition. This indicated that NONHSAG028908.3 served as a sponge to bind to miR-34a-5p, thus facilitating cell proliferation and migration.
Previous research demonstrated that increased glycolysis promotes cancer cell growth and survival (37). Thus, targeting glycolysis to inhibit cancer appears to be an effective method. In the present study, it was demonstrated that miR-34a-5p directly targeted ALDOA, which was regulated by NONHSAG028908.3. ALDOA, a pivotal glycolytic enzyme, acts as a catalyst in a conversion reaction of fructose-1, 6-bisphosphate (38). As aforementioned, the function of ALDOA in CRC has not been fully elucidated. Dai et al revealed that the expression of ALDOA was increased in CRC tissues and liver metastatic CRC tissues, and high expression of ALDOA was correlated with poor prognosis of CRC (27). Kawai et al demonstrated that ALDOA was positively associated with CRC cell proliferation and invasion (39). Similar to these previous studies, it was observed in the present study that ALDOA was upregulated in CRC cells (LoVo and HCT116). Knockdown of ALDOA inhibited CRC cell growth and migration. In addition, silencing of NONHSAG028908.3 attenuated the expression level of ALDOA, which was reversed by suppressing miR-34a-5p. However, Li et al reported that downregulation of ALDOA promoted the migration of CRC cells, SW480 and SW620 (26). Since the aforementioned study used different cells from the ones used in the present study, it is hypothesized that the different results may be caused by cell differences. In addition to ALDOA, other targets of miR-34a-5p, including TNFAIP8, flotillin-2, and hexokinase-1 have been revealed (18,40,41). Among them, Li et al reported that hexokinase-1 is also the key enzyme of glycolysis and participates in CRC progression (18). However, it is not yet clear whether increased ALDOA expression is related to changes in other miR-34a-5p targets. Therefore, in future experiments, this will be investigated. In addition, lack of an animal study is a limitation of the present study. In a future study, an animal model will be established to completely confirm the function of NONHSAG028908.3 on tumor growth and metastasis in vivo. In addition, a sufficient number of clinical samples will be collected to further detect the expression levels of NONHSAG028908.3, miR-34a-5p, and ALDOA in CRC tissues and adjacent normal tissues, and analyze their correlation.
Collectively, the data of the present study revealed the importance of the NONHSAG028908.3/miR-34a-5p/ALDOA axis in modulating CRC progression. Upregulation of NONHSAG028908.3 was associated with the aggressive phenotypes of CRC. NONHSAG028908.3 served as a sponge to combine with miR-34a-5p and formed a positive feedback loop to regulate ALDOA. The present study provides insights into and a new mechanism for CRC treatment.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
The present study was supported by the National Natural Science Foundation of China (grant nos. 81470086 and 81871465).
Availability of data and materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
CW performed experiments, analyzed the data and wrote the manuscript. HX and GY performed experiments and analyzed the data. ZL designed the experiments and revised the manuscript. CW and ZL confirm the authenticity of all the raw data. All authors read and approved the final manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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