LncRNA ELFN1-AS1 promotes colon cancer occurrence and progression by regulating the miR-191-5p/ZBTB34 axis

Introduction

Colon cancer (CC) is the fourth most common cancer diagnosed and a major cause of cancer-related deaths worldwide. According to the 2020 Global Cancer Statistics, 1,148,511 new cases of CC were diagnosed (1). The mortality rate associated with CC accounts for about 5% of all cancer-related deaths and ranks after lung cancer, breast cancer, stomach cancer and liver cancer. Due to the asymptomatic nature of the disease, most CC patients are diagnosed at an advanced stage during their first treatment (2). CC cases in the early stage are generally treated with tumor resection, while cases of advanced CC are usually treated with cytotoxic drugs (oxaliplatin, 5-fluorouracil, capecitabine and irinotecan) and biological agents (panitinumab, bevacizumab and cetuximab) (3-5). Combined chemotherapy is initially effective for most patients. However, due to drug resistance, about 50% of CC patients develop recurrent disease, and the 5-year survival rate of late-stage patients decreases by more than 10% (6,7). In addition, about 25% of CC cases at initial diagnosis have liver metastases, and about 50% of patients develop liver metastases within 3 years after initial surgery (8). Therefore, further investigation into the molecular mechanisms involved in CC development is crucial to identify novel therapeutic targets and enhance patient survival.

Long non-coding RNAs (lncRNAs) are a subclass of transcripts exceeding 200 bp in length. It has been reported that lncRNAs promote various cellular functions, such as proliferation and migration. Abnormalities in these crucial cellular functions have been implicated in a variety of human diseases, including cancer (9,10). An increasing number of lncRNAs are found to play a role in CC progression. For example, LINC00662 regulates CLDN8 and IL22 expression and activates the extracellular signal-regulated kinase (ERK) signaling pathway by targeting miR-340-5p, promoting the occurrence and development of CC (11). LncRNA CYTOR is significantly overexpressed in CC and promotes epithelial-mesenchymal transition (EMT) and metastasis by interacting with β-Catenin (12). ELFN1-AS1 is a newly discovered lncRNA whose carcinogenic effects have been reported in various cancers, including CC (13,14). Our previous study also found that ELFN1-AS1 can drive CC cell proliferation and invasion by regulating the miR-191-5p/SATB1 axis (15). The further detailed mechanism of action of lncRNA in the progression of CC requires further refinement.

MicroRNA (miRNA) is a type of non-coding small RNA molecule of approximately 22 nucleotides in size, which inhibits RNA translation, promotes RNA degradation, and regulates transcription and splicing processes (16). Abnormal regulation of miRNA often promotes the formation and development of cancer. For instance, miR-378 acts as a tumor suppressor in CC by targeting SDAD1 to inhibit the proliferation, migration, and invasion of CC cells (16); conversely, miR-510 functions as a tumor promoter in CC by targeting SRCIN1 to promote the proliferation, migration, and invasion of CC cells (17).

The findings of the present study demonstrate that LncRNA ELFN1-AS1 plays a crucial role in CC both in vitro and in vivo. The expression levels of lncRNA ELFN1-AS1 were elevated in CC cells and tissues. The upregulation of ELFN1-AS1 promoted the growth, migration, and invasion of CC cells. More importantly, we identified ZBTB34 as a downstream gene regulated by miR-191-5p in this pathway. LncRNA ELFN1-AS1 participates in the development and progression of CC by regulating the miR-191-5p/ZBTB34 axis. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2792/rc).

Methods

Clinical sample

Thirty pairs of CC tumor and adjacent non-tumor tissue samples were collected from the Hospital of Chengdu University of Traditional Chinese Medicine. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All patients provided written informed consent, and the study was approved by the Hospital of Chengdu University of Traditional Chinese Medicine and its affiliated Ethics Committee.

Cell culture

Normal colon epithelial cell line NCM460 and CC cells (HCT-116, HT-29, LoVo and SW480) were acquired from Cell Bioscience Biotechnology Co., Ltd. (Shanghai, China). The human CC cell lines HCT-116, HT-29, LoVo, and SW480, as well as the normal colonic epithelial cell line NCM460, were obtained from iCell Bioscience Inc. (Shanghai, China). The key genetic mutation profiles of the cancer cell lines are as follows: HCT-116 harbors mutations in KRAS (G13D), PIK3CA (H1047R), and CTNNB1 (S45del) but is TP53 wild-type (WT); HT-29 carries BRAF (V600E), TP53 (R273H), SMAD4 (Q311*), and APC mutations; LoVo exhibits KRAS (G13D) and APC mutations and is microsatellite instability-high (MSI-H); SW480 has mutations in TP53 (R273H and P309S), KRAS (G12V), and APC. NCM460 is a non-tumorigenic cell line with no known cancer-associated driver mutations. Cells were cultured in a humidified atmosphere with 5% CO₂ at 37 °C, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA).

Fluorescent real-time quantitative polymerase chain reaction (RT-qPCR)

The tumor and non-tumor tissues of CC patients, as well as NCM460 cells and CC cell lines (HCT-116, HT-29, LoVo, and SW480), were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The extracted RNA was reverse-transcribed into complementary DNA (cDNA) using a Takara reverse transcription kit, and lncRNA ELFN1-AS1 (U6 as internal reference) was detected by RT-qPCR. The total RNA of HCT-116 and HT-29 cells in each group was isolated using TRIzol reagent (Invitrogen), and the extracted RNA was reverse-transcribed into cDNA using Takara reverse transcription kit. miR-191-5p (U6 as internal reference) and ZBTB34 (GAPDH as internal reference) were detected by RT-qPCR. The experiment was repeated three times, and the primer sequence is shown in Table 1.

Cell transfection

HT-29 and HCT-116 cells are inoculated into 6-well plates in appropriate quantities, and when the cell density reaches 60–70%, transfection is performed according to the instructions for the transfection reagent. Cells are transfected with pLKO.1-NC (sh-NC), pLKO.1-ELFN1-AS1 (sh-ELFN1-AS1), mimics NC, miR-191-5p mimics, inhibitor NC, and miR-191-5p inhibitor, pcDNA-3.1-NC, pcDNA-3.1-ZBTB34 into HT-29 and HCT-116 cells, respectively. Subsequently, RT-qPCR is used to detect the success of cell transfection.

Cell Counting Kit-8 (CCK-8) assay

Cell viability was evaluated using the CCK-8 kit (Solarbio, Beijing, China). Transfected HT29 and HCT116 cells were seeded at a density of 1.5×10⁴ cells/well in 96-well plates and incubated for the specified time intervals (0, 24, 48, 72 h). A volume of 10 µL of CCK-8 reagent was introduced into each well, following which it was placed in an incubator at 37 °C for 2 h. Optical density was measured at 450 nm using a microplate reader (DALB, Shanghai, China).

Wound healing assay

Approximately 5×10⁵ HT-29 and HCT-116 cells were seeded and, the following day, a vertical scratch was created on the cell monolayer using a pipette tip. The cells were washed thrice with phosphate-buffered saline (PBS) to remove the detached cells, and serum-free medium was added. Culturing was conducted at 37 °C in a 5% CO₂ incubator. Images were captured at 0 and 24 hours to assess cell migration.

Transwell assay

The invasiveness of HT-29 and HCT-116 cells in each group was detected using the Transwell assay. Briefly, the CC cells were implanted in 100 mL of melted matrix gel (Solarbio) in the Matrigel-coated upper chamber. Following a 24-hour incubation period, the cells were fixed in 4% paraformaldehyde for 20 min and stained with crystal violet. The number of invading cells was quantified by counting cells in five randomly selected microscopic fields and analyzed using ImageJ software (National Institutes of Health).

Targeting prediction of miR-191-5p to ELFN1-AS1 and ZBTB34

We utilized the StarBase database (https://starbase.sysu.edu.cn/) to predict the targeting of miR-191-5p and ELFN1-AS1. Furthermore, we integrated the top 20 predicted target genes from both miRDB and TargetScan database, and performed an intersection analysis to identify potential targets of miR-191-5p. Based on a review of existing literature and our findings, ZBTB34 was selected as a candidate downstream gene for further experimental validation.

Dual-luciferase reporter assay (DLR)

Following the amplification of the 3-untranslated region (UTR) of ZBTB34 and ELFN1-AS1, these fragments were independently cloned downstream of the firefly luciferase gene in the pGL3 vector of Promega, resulting in constructs designated as WT 3’-UTRs. Site-directed mutagenesis of the ZBTB343 -UTR at the miR-191-5p binding site was performed using the QuickChange Site-Directed Mutagenesis Kit from Stratagene (Cedar Creek, USA), generating a mutant (MUT) 3’-UTR. CC cells were transfected with either the WT-3’-UTR or MUT-3’-UTR, along with a miR-NC or miR-191-5p mimic. Luciferase activity was measured 48 hours post-transfection using a dual-luciferase assay system (MedChemExpress, USA). The experiment was repeated three times.

Western blot analysis

Western blot analysis of ZBTB34 protein expression in HT-29, HCT-116 cells, and mouse tumor tissues from various groups. To harvest total protein, we use radioimmunoprecipitation assay buffer (RIPA) lysis buffer. Protein concentration was determined using the Pierce Bicinchoninic acid assay (BCA) Protein Assay Kit. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes, which were then blocked with 5% skim milk. The membranes were subsequently incubated with primary antibodies overnight at 4 °C. Following this, they were incubated with anti-rabbit or anti-mouse secondary antibodies for 1 h at room temperature. Finally, an enhanced chemiluminescence (ECL) substrate was added, and the blots were visualized and imaged using a Tanon 5200 chemiluminescence imaging system.

Nude mouse xenograft experiment

All animal experiments were performed in accordance with the Regulations for the Administration of Laboratory Animals (State Science and Technology Commission of the People’s Republic of China, 1988, revised 2017) and the Guideline on Treating Laboratory Animals with Care (Ministry of Science and Technology of China, 2006). Animal experiments were approved by the Animal Ethics Committee of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine (No. 2023DL-039). Nine BALB/c male nude mice (weighing 18–20 g, aged 5 weeks) were completely randomly selected and divided into three groups: lentiviral vector (LV)-negative control (NC) group, LV-ELFN1-AS1, and LV-ELFN1-AS1 + anti-miR191-5p.

HT29 cells were subcutaneously injected into each group of nude mice as follows: the control group received a cell suspension (2×10⁶ cells), the lncRNA ELFN1-AS1 knockdown group received a cell suspension (2×10⁶ cells), and the LV-ELFN1-AS1 + anti-miR191-5p group received a cell suspension (2×10⁶ cells), thereby constructing a mouse CC animal model. During the research, tumor size was assessed weekly by monitoring the length (L) and width (W) of the tumors using Vernier calipers. The formula used to calculate tumor volume was as follows: V (mm³) = (L × W²)/2. After four weeks, the mice were euthanized with 100 mg/kg sodium pentobarbital, and the xenografted tumor tissues were removed. Tumor weights were recorded, and tumor tissues were processed for Western blot analysis and hematoxylin and eosin (H&E) staining.

Histopathological analysis of tumor tissues

Tumor tissues from three groups of mice (LV-NC group, LV-ELFN1-AS1, LV-ELFN1-AS1 + anti-miR191-5p) were collected and fixed in 4% paraformaldehyde, followed by embedding in paraffin. Serial tissue sections (with a thickness of 3 µm) were prepared and subjected to H&E staining. Subsequently, the sections were examined under an optical microscope and photographed, revealing blue nuclei and red cytoplasm in the resulting images.

Statistical analysis

All data were processed using GraphPad Prism 8.0 statistical software, with mean ± standard deviation notation used for presentation. Comparisons between two groups were performed using t-tests, while differences among multiple groups were analyzed by one-way analysis of variance (ANOVA). Post-hoc comparisons following a significant one-way ANOVA were conducted using Tukey’s multiple comparison test. Statistical significance was set at P<0.05.

Results

Upregulated expression of lncRNA ELFN1-AS1 in CC tissues and cells

In this research, we use RT-qPCR to quantify the expression levels of LncRNA ELFN1-AS1 in tumor and non-tumor tissues of CC patients. The results indicated a markedly higher expression of ELFN1-AS1 in CC tissues compared to non-tumor tissues (Figure 1A). An analysis of the correlation between lncRNA ELFN1-AS1 expression and CC survival rates, conducted on the GEPIA website (http://gepia2.cancer-pku.cn/#index), revealed that patients with high lncRNA ELFN1-AS1 expression exhibited a significantly reduced overall survival rate (Figure 1B). RT-qPCR results further demonstrated that ELFN1-AS1 expression was upregulated in a panel of CC cell lines (HCT-116, HT-29, LoVo, SW480), with the highest observed in HCT-29 cells followed by HT-116 cells (Figure 1C). Therefore, we selected HCT-116 and HT-29 cells as representative models to investigate the role of ELFN1-AS1 in CC. Furthermore, cytoplasm/nucleus fractionation experiments confirmed that ELFN1-AS1 is primarily enriched in the cytoplasm of HT-29 and HCT-116 cells (Figure 1D), suggesting its potential involvement in cytoplasmic functions, such as acting as a miRNA sponge. Based on the Kaplan-Meier survival analysis (Figure 2A), the prognostic significance of ELFN1-AS1 expression was evaluated by comparing survival outcomes between high and low expression groups. The results demonstrate a statistically significant difference in survival probability over time (log-rank P=0.009). Specifically, patients with high ELFN1-AS1 expression exhibited markedly poorer overall survival compared to those in the low expression group. The accompanying risk table, which tracks the number of patients at risk at successive time points, confirms a consistent decline in survival among the high-expression cohort and accounts for censoring during follow-up. These findings indicate that elevated ELFN1-AS1 expression serves as a robust marker of adverse prognosis in the studied cancer cohort. Our systematic pan-cancer analysis (Figure 2B) reveals that the lncRNA ELFN1-AS1 is not a significant prognostic factor in the majority of cancer types. This finding does not conflict with its previously reported oncogenic roles but, instead, collectively underscores its strongly context-dependent functionality. While existing literature has primarily focused on elucidating its tumor-promoting role in specific settings, our work delineates the boundaries of its influence, demonstrating that ELFN1-AS1 is not a universal driver gene. This cautions against the over-extrapolation of the prognostic value of any single lncRNA and emphasizes that its assessment must be strictly confined to its specific biological context.

Figure 1 The expression of lncRNA ELFN1-AS1 is up-regulated in colon cancer tissues and cells. (A) The expression of ELFN1-AS1 is significantly elevated in colon cancer tissues. (B) GEPIA survival analysis of the correlation between ELFN1-AS1 and survival rate in colon cancer. (C) The expression of ELFN1-AS1 in colon cancer cells is higher than that in normal colon epithelial cell lines. (D) RT-qPCR analysis of nuclear and cytoplasmic ELFN1-AS1 in HT-29 and HCT-116 cells. **, P<0.01; ***, P<0.001. ELFN1-AS1, ELFN1 antisense RNA 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GEPIA, Gene Expression Profiling Interactive Analysis; HR, hazard ratio; lncRNA, long non-coding RNA; RT-qPCR, real-time quantitative polymerase chain reaction; TPM, transcript per million.

Figure 2 Survival analysis of ELFN1-AS1 in colon cancer. (A) Survival analysis of ELFN1-AS1 in colon cancer. (B) Survival analysis of ELFN1-AS1 in pancancer. *, P<0.05; **, P<0.01; ***, P<0.001 compared with normal/NCM460. CI, confidence interval; ELFN1-AS1, ELFN1 antisense RNA 1; HR, hazard ratio.

Concurrently, the tumor, node, metastasis (TNM) staging system is used to describe the extent of CC. It is based on the size and invasiveness of the primary tumor (T), the involvement of regional lymph nodes (N), and the presence of distant metastasis (M). Higher T, N, and M values indicate more advanced disease. CC is staged from I to IV based on the TNM scores, with higher stages representing more severe disease and poorer prognosis. In essence, a higher TNM stage reflects larger tumor size, greater lymph node involvement, and more distant metastasis. By integrating transcriptome data and clinical information from TCGA database, we analyzed the correlation between lncRNA ELFN1-AS1 expression and the staging and grading of CC. Our findings revealed a significant positive correlation between lncRNA ELFN1-AS1 expression and T staging (primary tumor extent), N staging (regional lymph node involvement), and M staging (distant metastasis) (Figure 3A-3C), as well as a significant positive correlation with CC progression (Figure 3D).

Figure 3 According to different clinical classifications, the expression of lncRNA ELFN1-AS1 is up-regulated in colon cancer tissues and cells. (A) ELFN1-AS1 is significantly elevated in T2–4 (where the tumor has invaded surrounding tissues and organs, including blood vessels, etc.); (B) there is no significant difference in the expression of ELFN1-AS1 between N0 (no regional lymph node involvement) and N1–2; (C) the expression of ELFN1-AS1 is significantly higher in M0 (no distant metastasis) than in M1; (D) the expression of ELFN1-AS1 is significantly higher in stage 2–4 (where the tumor has invaded beyond the colon wall and may have lymph node and distant metastasis) than in stage 1 (the early stage, where the tumor is confined to the colon wall without invasion into surrounding tissues, lymph node involvement, or distant metastasis). ELFN1-AS1, ELFN1 antisense RNA 1; lncRNA, long non-coding RNA; M, metastasis; N, node; T, tumor.

ELFN1-AS1 silencing inhibits proliferation, migration, and invasion of CC cells

To further elucidate the role of lncRNA ELFN1-AS1 in CC progression, we experimentally verified the effects of ELFN1-AS1 modulation on CC cell proliferation, migration, and invasion. Initially, we employed cell transfection to downregulate ELFN1-AS1 expression in HCT-116 and HT-29 cells. Concurrently, we detected the efficiency of ELFN1-AS1 knockdown in these cells via RT-qPCR (Figure 4A). Subsequently, CCK-8 assays showed that ELFN1-AS1 downregulation significantly impaired the proliferative capacity of CC cells (Figure 4B). Furthermore, wound healing and Transwell migration assays indicated that reduced ELFN1-AS1 expression significantly inhibited the migratory and invasive capabilities of CC cells (Figure 4C,4D).

Figure 4 Down-regulation of lncRNA ELFN1-AS1 inhibits the proliferation, migration and invasion of colon cancer cells. (A) Expression of ELFN1-AS1 in HCT-116 and HT-29 cells treated with sh-NC or sh-ELFN1-AS1; (B) CCK-8 assay; (C) scratch assay for cell migration detection; (D) transwell assay for cell invasion detection. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Non-invading cells on the upper membrane were then removed with a cotton swab. Invading cells on the lower membrane were stained with 0.1% crystal violet solution for 20 minutes at room temperature. After washing with distilled water and air-drying, stained cells were counted in five randomly selected fields per well under a light microscope. ***, P<0.001 vs. sh-NC. CCK-8, Cell Counting Kit-8; ELFN1-AS1, ELFN1 antisense RNA 1; lncRNA, long non-coding RNA; OD, optical density; sh-NC, short hairpin negative control.

miR-191-5p is the target of lncRNA ELFN1-AS1

ELFN1-AS1 is predominantly localized in the cytoplasm, suggesting a potential role as a competing endogenous RNA (ceRNA) in the regulation of downstream targets. A review identified miR-191-5p as a well-characterized tumor suppressor that inhibits tumor cell proliferation and invasion. Consequently, we utilized the StarBase database (https://starbase.sysu.edu.cn/) to predict potential target interactions. Knockdown of ELFN1-AS1 significantly increased miR-191-5p levels in HCT-116 and HT-29 cells (Figure 5A). Consistent with this inverse relationship, miR-191-5p expression was significantly lower in colon cancer tissues than in non-tumor colon tissues (Figure 5B). Furthermore, the putative binding sites between miR-191-5p and ELFN1-AS1 are depicted in Figure 5C. Subsequently, the predicted miR-191-5p binding site of ELFN1-AS1 (ELFN1-AS1-WT) and the mutated miR-191-5p binding site of ELFN1-AS1 (ELFN1-AS1-MUT) were cloned into luciferase reporter plasmids. We observed that co-transfection with miR-191-5p and ELFN1-AS1-WT significantly reduced luciferase activity, whereas co-transfection with miR-191-5p and ELFN1-AS1-MUT had no effect on luciferase activity, indicating that miR-191-5p can directly bind to ELFN1-AS1 (Figure 5D).

Figure 5 miR-191-5p is the target gene of lncRNA ELFN1-AS1. (A) Expression of miR-191-5p in different treatment groups; (B) RT-qPCR assay demonstrated the level of miR-191-5p in colon cancer tissues; (C) StarBase predicted the targeting relationship between the two; (D) DLR confirmed the targeting relationship. ***, P<0.001 vs. miR-NC. DLR, dual-luciferase reporter assay; ELFN1-AS1, ELFN1 antisense RNA 1; lncRNA, long non-coding RNA; RT-qPCR, real-time quantitative polymerase chain reaction; sh-NC, short hairpin negative control; WT, wild type.

Effect of miR-191-5p on the proliferation, migration and invasion of CC cells

In vitro experiments verified the effects of miR-191-5p on CC cell proliferation, migration and invasion. CCK-8 assays revealed that the overexpression of miR-191-5p significantly inhibited the proliferative capacity of CC cells (Figure 6A). Subsequently, wound healing assays and Transwell assays (Figure 6B,6C) revealed that the overexpression of miR-191-5p significantly reduced the migratory and invasive capabilities of CC cells. Collectively, these findings indicate that miR-191-5p is involved in the development and progression of CC.

Figure 6 Effects of miR-191-5p on proliferation, migration and invasion of colon cancer cells. (A) CCK-8 assay for the proliferation of colon cancer HT-29 and HCT-116 cells; (B) scratch assay; (C) transwell assay. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Non-invading cells on the upper membrane were then removed with a cotton swab. Invading cells on the lower membrane were stained with 0.1% crystal violet solution for 20 minutes at room temperature. After washing with distilled water and air-drying, stained cells were counted in five randomly selected fields per well under a light microscope. *, P<0.05; ***, P<0.001 vs. miR-NC. CCK-8, Cell Counting Kit-8; OD, optical density.

MiR-191-5p regulates ZBTB34 expression and CC cell behavior

MiRNAs primarily function by regulating their downstream target genes. Using the miRDB and TargetScan databases to predict the target genes of miR-191-5p, we identified four potential targets: TAF5, TMOD2, ZBTB34, and SATB1 (Figure 7A). Considering the existing literature and the novelty of our study, we chose ZBTB34 for further validation. Through StarBase prediction, we determined the binding sites of miR-191-5p on the ZBTB34 mRNA (Figure 7B). Luciferase reporter assays demonstrated that in HCT-116 and HT-29 cells, co-transfection with miR-191-5p mimic and WT-ZBTB34-3’-UTR resulted in a significant reduction in luciferase activity (Figure 7C), suggesting that miR-191-5p may regulate ZBTB34 expression in CC cells. Additionally, RT-qPCR analysis revealed a significant upregulation of ZBTB34 expression in CC tissues compared to adjacent non-tumor tissues (Figure 7D). Western blot analysis further confirmed that overexpression of miR-191-5p in HCT-116 and HT-29 cells inhibited ZBTB34 protein levels (Figure 7E). Therefore, these data collectively demonstrate that ZBTB34 is a downstream gene regulated by miR-191-5p in this pathway.

Figure 7 miR-191-5p targeted ZBTB34. (A) Intersection of downstream target genes predicted by miRDB database and TargetScan; (B) prediction of the targeting relationship between the two by starbase; (C) confirmation of the targeting relationship by DLR; (D) detection of ZBTB34 expression in tumor and non-tumor tissues by RT-qPCR; (E) detection of ZBTB34 protein expression in different treatment groups by Western blot. ***, P<0.001 vs. miR-NC. DLR, dual-luciferase reporter assay; RT-qPCR, real-time quantitative polymerase chain reaction; WT, wild type.

To further elucidate the function of ZBTB34 within cells, we conducted a co-expression analysis of ZBTB34 (Figure 8A). The top 20 genes, based on correlation coefficients with ZBTB34, were subjected to protein-protein interaction network analysis using the STRING database (https://string-db.org/) (Figure 8B). The results indicated significant enrichment of these genes in mitochondrial-related functions, including TUFM, NDUFS8, MRPS7, MRPL4, UQCRC1, ATP5F1D, and others. These genes mainly encode mitochondrial ribosomal proteins, subunits of oxidative phosphorylation (OXPHOS) complexes, and proteins involved in adenosine triphosphate (ATP) synthesis.

Figure 8 ZBTB34 co-expression analysis. (A) Volcano plot of correlation analysis for the top 20 genes with the highest correlation coefficients of ZBTB34; (B) core targets of ZBTB34.

These findings suggest that ZBTB34 may be involved in regulating mitochondrial function and cellular energy metabolism. Mitochondrial dysfunction is known to be closely associated with malignant behaviors such as tumor cell proliferation, apoptosis evasion, and metabolic reprogramming. The high expression of ZBTB34 may promote tumor progression by affecting the balance of energy metabolism or oxidative stress responses in CC cells. In summary, ZBTB34 may promote the proliferation, survival, and invasive ability of CC cells by regulating processes such as mitochondrial respiratory chain complex assembly, ATP production, or reactive oxygen species (ROS) metabolism.

LncRNA ELFN1-AS1/miR-191-5p/ZBTB34 axis regulates CC cell proliferation, migration, and invasion

To verify the function of ELFN1-AS1/miR-191-5p/ZBTB34 in CC, rescue experiments were conducted on HCT-116 and HT-29 cells. HCT-116 (sh-ELFN1-AS1) and HT-29 (sh-ELFN1-AS1) cells stably transfected with sh-ELFN1-AS1 were cultured and divided into three groups, specifically transfected with NC, anti-miR-191-5p, and pcDNA-ZBTB34. The CCK-8 assay results revealed that the proliferative capacity of the anti-miR-191-5p group and pcDNA-ZBTB34 group was significantly enhanced (Figure 9A). Furthermore, anti-miR-191-5p and pcDNA-ZBTB34 promoted the migration and invasion of HCT-116 (sh-ELFN1-AS1) and HT-29 (sh-ELFN1-AS1) cells (Figure 9B,9C).

Figure 9 Effects of ELFN1-AS1, miR-191-5p and ZBTB34 on proliferation, migration and invasion of colon cancer cells. (A) CCK-8 experiment; (B) the scratch test is used to detect cell migration; (C) transwell assay is used to detect cell invasion. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Non-invading cells on the upper membrane were then removed with a cotton swab. Invading cells on the lower membrane were stained with 0.1% crystal violet solution for 20 minutes at room temperature. After washing with distilled water and air-drying, stained cells were counted in five randomly selected fields per well under a light microscope. ***, P<0.001 vs. sh-NC; ^###, P<0.001: sh-ELFN1-AS1 + anti-miR-191-5p vs. sh-ELFN1-AS1; ^&&&, P<0.001: sh-ELFN1-AS1 + pcDNA-ZBTB34 vs. sh-ELFN1-AS1. CCK-8, Cell Counting Kit-8; ELFN1-AS1, ELFN1 antisense RNA 1; OD, optical density; sh-NC, short hairpin negative control.

Tumor growth and ZBTB34 expression

The nude mouse tumor experiment confirmed that ELFN1-AS1 knockdown inhibits colon cancer progression. As shown in Figure 10A,10B, tumor growth was significantly suppressed in the LV-ELFN1-AS1 group compared to the LV-NC group, while the LV-ELFN1-AS1 + anti-miR-191-5p group reversed this effect. ZBTB34 protein expression in tumor tissues was detected (Figure 10C). The expression level was significantly decreased in the LV-ELFN1-AS1 group versus the LV-NC group, and significantly increased in the LV-ELFN1-AS1 + anti-miR-191-5p group versus the LV-ELFN1-AS1 group.

Figure 10 Down-regulation of lncRNA ELFN1-AS1 inhibits the progression of colon cancer in vivo. (A) Subcutaneous tumors in mice; (B) tumor mass and volume; (C) Western blot was used to detect the expression of ZBTB34 in tumor tissues. **, P<0.01 vs. LV-NC; ^##, P<0.01 vs. LV-ELFN1-AS1. ELFN1-AS1, ELFN1 antisense RNA 1; lncRNA, long non-coding RNA; LV, lentiviral vector; NC, negative control.

Histological analysis

HE staining of tumor tissues showed that, compared to the LV-NC group, the LV-ELFN1-AS1 group exhibited looser cell arrangement and lower malignancy; conversely, the LV-ELFN1-AS1 + anti-miR-191-5p group reversed this inhibitory effect (Figure 11).

Figure 11 Tumor tissues were fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and sectioned at 4 µm thickness. Sections were deparaffinized, rehydrated, stained with hematoxylin for 5 minutes, differentiated in 1% acid alcohol for 2–3 seconds, blued in running tap water for 5 minutes, and then counterstained with eosin for 2 minutes. After dehydration and clearing, sections were mounted and observed under a light microscope (H&E staining, ×400). ELFN1-AS1, ELFN1 antisense RNA 1; H&E, hematoxylin and eosin; LV, lentiviral vector; NC, negative control.

Discussion

CC is a prevalent malignant tumor of the digestive tract, characterized by a low survival rate and high mortality rate. With shifts in diet and lifestyle, the incidence of CC has been increasing annually, and there is a trend towards younger onset (<55 years) (18). CC is a heterogeneous multifactorial disease exhibiting significant variability in prognosis and treatment responses among patients. The biological behaviors of tumor cells, including proliferation, migration, invasion, and apoptosis, are intimately linked to tumor progression. Research indicates that the cellular gene regulatory network in CC may provide novel insights for improving prognosis and identifying effective therapeutic strategies (19). Furthermore, tumor-related transcription factors can modulate these biological behaviors. LncRNAs are eukaryotic transcripts exceeding 200 nucleotides in length, capable of participating in the regulation of related gene transcription through chromatin modification and transcriptional control. Numerous studies have demonstrated that certain transcription factors can bind to lncRNAs, thereby regulating their transcription, such as FOXA1, MYC, YY1, etc.

In prior studies, ELFN1-AS1 has been established as an lncRNA involved in various malignancies. In ovarian cancer, experiments have demonstrated that ELFN1-AS1 regulates CLDN4 expression by sponging miR-497-3p, thereby promoting the proliferation, migration, and invasion of ovarian cancer cells (20). For esophageal cancer, ELFN1-AS1 is highly expressed and serves as a prognostic indicator, promoting esophageal cancer progression through the regulation of GFPT1 expression (21). In addition, ELFN1-AS1 has been found to be significantly upregulated in non-small cell lung cancer tissues, correlating with TNM staging, lymph node metastasis and overall survival, and promotes carcinogenesis by enhancing CCNE1 expression (22). Collectively, these findings indicate that ELFN1-AS1 plays an oncogenic role in various cancer types. Its role in CC, however, remains to be elucidated. In our study, we observed that ELFN1-AS1 is significantly upregulated in CC tissues. We further demonstrated that inhibiting ELFN1-AS1 expression suppresses CC development both in vivo and in vitro, particularly by impairing the migration and invasion capabilities of CC cells. These findings provide theoretical evidence for the potential of ELFN1-AS1 as a diagnostic and therapeutic target in CC. ELFN1-AS1 may potentially serve as a biomarker for colon cancer prognosis in the future.

miRNAs are non-coding single-stranded RNA molecules encoded by endogenous genes, typically ~22 nucleotides in length. They bind to the 3’-UTR of downstream mRNAs and cause mRNA degradation or translation inhibition (22). LncRNA Sponges adsorb miRNA to regulate mRNA transcription (23,24). Therefore, exploring the interaction between miRNAs and lncRNAs in CC progression and elucidating their molecular mechanisms provides a theoretical basis for the CC treatment. Previous reports have indicated a role for miR-191-5p in renal cell carcinoma (25). Our study confirmed that miR-191-5p is downregulated in CC and overexpression of miR-191-5p inhibits CC progression in CC cells. Furthermore, through bioinformatics analysis combined with dual-fluorescent reporter gene experiments, we demonstrated that miR-191-5p is negatively regulated by ELFN1-AS1 in CC.

ZBTB protein is an important member of the C2H2-type zinc finger protein family, which regulates the transcriptional activity of downstream genes as a transcription factor. It is widely involved in the transcriptional regulation, cell proliferation, differentiation, apoptosis and other cellular processes (26). In previous studies, ZBTB16 (27), ZBTB20 (28), and BCL6 [ZBTB27 (29)] from the ZBTB protein family have been shown to be associated with the development and prognosis of a variety of tumors. Zinc finger and BTB domain containing 34 (ZBTB34) was identified by Qi et al. in 2006 (26) as a novel zinc finger protein. They found that ZBTB34 is widely expressed in most adult tissues and is primarily localized to the cell nucleus. In a previous study, it was discovered that ROS-induced anticancer drugs can kill cancer cells by downregulating miR-27a or miR-17/miR-20a (30). These miR-27a can regulate the expression of ZBTB34, thereby inhibiting the expression of specific protein (SP) transcription factors. A recent study found that ZBTB34 and lncRNA CPLC are closely related in the progression of CC, indicating that the lncRNA CPLC/miR-4319/ZBTB34 signaling axis plays a role in regulating the development of CC. Our study delineates a novel oncogenic signaling axis in CC, wherein the long non-coding RNA ELFN1-AS1, predominantly localized in the cytoplasm, functions as a ceRNA to sequester miR-191-5p. This molecular sponge effect was confirmed by DLR and the observed inverse correlation in expression levels. The sequestration of miR-191-5p leads to the de-repression of its downstream target, the transcription factor ZBTB34, a direct interaction also validated by luciferase assays and Western blot analysis.

The functional significance of this axis is underscored by our comprehensive rescue experiments. The suppressive effects of ELFN1-AS1 knockdown on CC cell proliferation, migration, and invasion in vitro were effectively reversed by either inhibiting miR-191-5p or exogenously overexpressing ZBTB34. Consistent with these findings, our in vivo xenograft model confirmed that ELFN1-AS1 silencing suppressed tumor growth and ZBTB34 expression, and this suppression was again rescued by co-delivery of a miR-191-5p inhibitor.

Our discovery that ELFN1-AS1 drives proliferative signaling directly aligns with the foundational cancer hallmark of “evading growth suppressors”, as it incapacitates the tumor-suppressive function of miR-191-5p (31). This places our lncRNA of interest within the conceptual framework of core cancer-maintaining mechanisms. More importantly, the dynamic and reversible phenotype controlled by the ELFN1-AS1/miR-191-5p/ZBTB34 axis provides a vivid functional example of the “dual roles” of regulatory networks in cell fate decisions, a concept recently emphasized in the context of cellular senescence and its regulators (32). Just as senescent cells can exert both tumor-promoting and tumor-suppressive effects depending on context (32), our work demonstrates that the same molecular axis can be manipulated to switch CC cells between a highly proliferative and a suppressed state. This reinforces the notion that understanding the context of such networks is critical for therapeutic targeting.

Ultimately, we concluded that increased expression of ELFN1-AS1 in CC promotes tumor progression by regulating the miR-191-5p/ZBTB34 axis. Therefore, downregulating ELFN1-AS1 can inhibit ZBTB34 expression by targeting miR-191-5p, thereby suppressing the development of CC. Moreover, the ELFN1-AS1/miR-191-5p/ZBTB34 axis serves as a compelling illustration of a broader principle in cancer biology: that cell fate is governed by malleable regulatory circuits, the manipulation of which holds significant therapeutic promise. In summary, this axis has significant potential in targeted therapy for CC. This study thoroughly explores the pathogenesis of CC, providing diagnostic and therapeutic implications for patients, and offering a new treatment option and research direction.

Conclusions

In conclusion, this study demonstrates that ELFN1-AS1 promotes colon cancer occurrence and progression by regulating the miR-191-5p/ZBTB34 axis. ELFN1-AS1 functions as a ceRNA by sponging miR-191-5p, thereby upregulating ZBTB34 expression. Our findings suggest that ELFN1-AS1 may serve as a potential prognostic biomarker and therapeutic target for colon cancer. Further in vivo and clinical studies are warranted to validate its translational potential.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MADR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2792/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2792/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2792/prf

Funding: This study was supported by the Natural Science Foundation of Sichuan Province (No. 2022NSFSC0812).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2792/coif). All authors report that this study was supported by the Natural Science Foundation of Sichuan Province (No. 2022NSFSC0812). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Hospital of Chengdu University of Traditional Chinese Medicine. Written informed consent was obtained from all the participants prior to the enrollment of this study. Animal experiments were performed under a project license (No. 2023DL-039) granted by the Animal Ethics Committee of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, in compliance with the Regulations for the Administration of Laboratory Animals (State Science and Technology Commission of the People’s Republic of China, 1988, revised 2017) and the Guideline on Treating Laboratory Animals with Care (Ministry of Science and Technology of China, 2006), and associated guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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