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World Journal of Surgical Oncology logoLink to World Journal of Surgical Oncology
. 2026 Jun 29;24:373. doi: 10.1186/s12957-026-04474-5

Silencing of ELFN1-AS1 induces prostate cancer cell apoptosis and autophagy by regulating miR-28-5p/BCAM axis

Shuai Luo 1,2, Dingwen Gui 1,2, Yankuang Guo 1,2, Zuwei Xu 1,2, Zheng Fang 3, Geng Huang 1,2,✉, Wenbing Wu 1,2,✉
PMCID: PMC13579999  PMID: 42374482

Abstract

Background

Prostate cancer (PCa) is the most frequently diagnosed non-cutaneous malignancy in men worldwide and constitutes a leading contributor to cancer-related mortality globally. This study aimed to investigate the biological functions and molecular mechanisms of long noncoding RNA ELFN1-AS1 in regulating PCa cell phenotypes.

Methods

ELFN1-AS1 expression in PCa cells was quantified via RT-qPCR analysis. CCK-8, Transwell, and flow cytometry analysis were carried out to measure PCa cell viability, invasion, and apoptosis. Monodansylcadaverine staining was used to visualize and assess autophagosome formation in PCa cells. Western blot analysis was performed to measure protein levels of apoptotic markers, autophagic markers, and the downstream factor basal cell adhesion molecule (BCAM). The binding relationship among ELFN1-AS1, miR-28-5p, and BCAM were validated by luciferase reporter assay and RNA immunoprecipitation assays.

Results

The results demonstrated that ELFN1-AS1 and BCAM were highly expressed in PCa cells, while miR-28-5p expression was downregulated compared with normal prostate epithelial cells. Silencing of ELFN1-AS1 effectively reduced optical density values and numbers of invaded cells while concurrently elevating apoptotic proportion. Moreover, ELFN1-AS1 knockdown promoted the formation of autophagosomes and increased LC3II/LC3I ratio. Importantly, ELFN1-AS1 acted as a molecular sponge for miR-28-5p to regulate BCAM expression. Additionally, the inhibitory effects of ELFN1-AS1 depletion on the malignant biological behaviors of PCa cells were notably reversed by BCAM overexpression.

Conclusion

Our findings indicate that the silencing of ELFN1-AS1 promotes PCa cell apoptosis and autophagy by interacting with miR-28-5p to regulate BCAM expression.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12957-026-04474-5.

Keywords: BCAM, Competing endogenous RNA, ELFN1-AS1, miR-28-5p, Prostate cancer

Introduction

Prostate cancer (PCa) is the most common malignancy in the male urinary system, seriously threatening the health and well-being of men across the globe [1]. The pathogenesis of the disease is multifactorial, with known risk factors including smoking, dietary habits, occupational exposure, and specific medications [2]. In recent years, the incidence of PCa has displayed an upward tendency [3, 4]. Epidemiological estimates indicate that 5% of patients with newly diagnosed PCa present with distant metastatic disease at the time of initial diagnosis [5]. Over the past decades, remarkable advancements have been made in the therapeutic strategies for PCa, encompassing radical prostatectomy, brachytherapy, external beam radiotherapy, and other targeted interventions [6–8]. Despite these progressive developments, suboptimal clinical outcomes as well as delayed or missed diagnosis remain formidable clinical challenges associated with this malignancy [9, 10]. Accordingly, it is imperative to identify reliable molecular markers for early detection and risk stratification, as well as to optimize therapeutical strategies for patients with PCa.

Autophagy is an intricate process that transport intracellular components to the lysosomal compartment for their degradation and subsequent recycling [11]. The critical role of autophagy in cell metabolism, homeostasis, and stress responses has been confirmed in many studies [12]. Functionally, autophagy exerts a dual role in cellular fate: on the one hand, it promotes cell survival and adaptation under stressful microenvironment such as oxidative stress and hypoxia; on the other hand, it can induce type II programmed cell death, commonly referred to as autophagic cell death [13, 14]. Existing evidence further demonstrates extensive crosstalk between autophagic and apoptotic pathways, and disruption of the homeostatic balance between the two pathways is closely implicated in the regulation of malignant cell death and survival [13]. Therefore, in-depth exploration of the regulatory mechanisms governing apoptosis and autophagy in PCa cells is critical for elucidating the molecular basis of PCa malignant progression and identifying novel therapeutic targets.

Long non-coding RNAs (lncRNAs), defined as RNA transcripts exceeding 200 nucleotides in length that lack protein-coding capacity, have emerged as key epigenetic and post-transcriptional regulators implicated in various biological processes, exerting either tumor-suppressive or oncogenic effects in a context-dependent manner [15, 16]. The molecular mechanisms underlying lncRNA-mediated carcinogenesis are highly complex, among which competing endogenous RNA (ceRNA) regulatory network is extensively investigated [17]. Specifically, lncRNAs function as ceRNAs to sequester specific miRNAs, thereby abrogating the miRNA-mediated silencing of downstream target messenger RNAs (mRNAs) and modulating the expression of oncogenes or tumor suppressor genes [18]. MiRNAs are characterized by distinct tissue specificity, plasma stability, and rapid release rate [19]. Mounting evidence has identified a panel of PCa-specific lncRNAs with critical regulatory roles in disease progression. For example, lncRNA LNC565686 represses PCa cell apoptosis while accelerating cell proliferation by preventing the degradation of SND1 [20]. LncRNA MALAT1 promotes PCa metastasis and neuroendocrine differentiation by interacting with miR-216a-5p, thereby upregulating ZFP91 while promoting the degradation of FOXA1 [21]. ELFN1-AS1 is a well-documented oncogenic lncRNA implicated in the malignant development of various types of cancer [22–24]. In colon cancer, silencing ELFN1-AS1 attenuates tumorigenic potential by increasing AURKB expression through competitive binding with miR-4270 [22]. Huang et al. reported that ELFN1-AS1 contributes to cell growth, migration, and invasion in gastric cancer by targeting miR-211-3p and subsequently upregulating TRIM29 [25]. Moreover, ELFN1-AS1 has been shown to display an elevated expression in non-small cell lung cancer tissues, and its high expression is linked to unfavorable outcome and lymph node metastasis status in patients [24]. Nevertheless, the biological role and the mechanism of ELFN1-AS1 in PCa remain largely unexplored.

Preliminary bioinformatic analysis showed significantly upregulated expression of ELFN1-AS1 in prostate adenocarcinoma (PRAD) tissues compared with adjacent normal prostate tissues. On this basis, the study aimed to verify the expression of ELFN1-AS1 in PCa cells and investigate its functional role in regulating malignant cell phenotypes. Our findings identified miR-28-5p as a direct downstream target miRNA for ELFN1-AS1 and further confirmed that basal cell adhesion molecule (BCAM) is a specific downstream target gene of miR-28-5p. Collectively, this study highlights a novel dysregulated lncRNA with potential utility as a diagnostic biomarker for PCa and provides an innovative molecular regulatory axis that may inform the development of targeted therapeutic strategies for PCa.

Materials and methods

Cell culture

Human PCa cell lines (22RV1, PC-3, and DU145) and the normal human prostate stromal cell line (WPMY-1) were purchased from ATCC (Manassas, VA, USA). WPMY-1 cells were cultured in DMEM (iCell Biotechnology, Shanghai, China) with 10% fetal bovine serum (FBS; Procell, Yipu Biotechnology Co. Ltd. Wuhan, China) and 1% penicillin/streptomycin solution (Procell). 22RV1, PC-3 and DU145 cells were maintained in RPMI-1640 (iCell Biotechnology) added with 10% FBS, 1% penicillin/streptomycin, and 2 mM L-Glutamine (TargetMol, Boston, USA). All cell lines were cultured in a humidified incubator at 37℃ with 5% carbon dioxide.

Cell transfection

Short hairpin RNAs targeting ELFN1-AS1 (sh-ELFN1-AS1#1/2), overexpression vectors (pcDNA3.1) carrying full sequence of ELFN1-AS1 or BCAM, miR-28-5p mimics for targeted miRNA overexpression, and corresponding negative controls (sh-NC, empty pcDNA3.1 vector, and NC mimics) were provided by Gene Pharma (Shanghai, China). Cell transfection was performed utilizing Lipofectamine 3000 reagent (Solarbio Life Sciences, Beijing, China) following the product manuals, with the reagent diluted in culture medium prior to transfection. The final working concentrations were set as follows: 40 nM for shRNA plasmids, 10 nM for pcDNA3.1 overexpression vectors, and 50 nM for miRNA mimics. Transfection efficiency was verified 48 h post-transfection via quantitative polymerase chain reaction (qPCR) analysis.

Subcellular fractionation assay

Cytoplasmic and nuclear RNA was isolated from 22RV1 and PC-3 cells using the PARIS™ kit (Yubo Biological, Shanghai, China). Briefly, collected cells were plated in Petri dishes and washed twice with phosphate buffered saline (PBS). Cells were then resuspended in ice-cold cell fractionation buffer and incubated on ice for 5–10 min. Subsequent centrifugation was performed at 500 × g for 3 min at 4℃ to separate nuclear and cytoplasmic fractions. Expression levels of ELFN1-AS1, GAPDH, and U6 in nuclear and cytoplasmic extracts were detected by RT-qPCR. U6 and GAPDH served as nuclear and cytoplasmatic controls, respectively.

Flow cytometry analysis

Apoptosis of transfected PCa cells was assessed using Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme Biotech, Nanjing, China). Cells were seeded into the 6-well plates (2 × 105 cells/well), cultured overnight and washed twice with PBS. After washing, cells were resuspended in 100 µl of 1 × binding buffer, followed by double staining with 10 µL Annexin V-FITC and 10 µL PI in the dark for 10 min each. Based on Annexin V/PI staining patterns, cells were classified into four distinct populations: viable, necrotic, early apoptotic, and late apoptotic cells. The percentages of early and late apoptotic cells were quantified using FACS Calibur flow cytometer (BD Biosciences, Frankin Lakes, USA).

Cell counting kit-8 (CCK-8) assay

Alterations in cell viability were measured using CCK-8 assays. Transfected 22RV1 and PC-3 cells were seeded to the 96-well plates (3000 cells/well) in 100 µL complete medium and incubated at 37℃. At predetermined time points (0, 24, 48, and 72 h post seeding), 10 µL CCK-8 reagent (iCell Biotechnology) was added to each well and incubated for an additional 2 h at 37℃. The optical density at 450 nm was measured using a microplate reader (Bio-Tek Instruments, USA). To ensure reproducibility and reliability of the results, all experiments were performed in triplicate.

Luciferase reporter assay

Wild type (WT) constructs of ELFN1-AS1 and BCAM, designated ELFN1-AS1-Wt and BCAM-WT, were established by subcloning the full-length target sequences into the pmirGLO luciferase reporter vector (Promega, WI, USA). Corresponding mutated constructs (ELFN1-AS1-Mut and BCAM-MUT) were then built via site-directed mutagenesis of the predicted microRNA binding sites, followed by subcloning of the mutated sequences into the same pmirGLO vector. 22RV1 and PC-3 cells were seeded into 24-well plates (5 × 104 cells/well) and incubated for 12 h to allow cell adherence. For co-transfection, BCAM-WT or BCAM-MUT reporter plasmids were transfected with miR-28-5p mimics, NC mimics, or miR-28-5p mimics + ELFN1-AS1 into the cultured cells using Lipofectamine 3000. Separately, ELFN1-AS1-WT/MUT reporters were co-transfected with miR-28-5p mimics or NC mimics into 22RV1 and PC-3 cells using the same transfection reagent. After 48 h of incubation, firefly and Renilla luciferase signals were examined by a Luciferase Reporter Assay System (Promega). Renilla luciferase signals were used for normalization.

Transwell assay

The invasive capacity of PCa cells was evaluated using Transwell chambers (24-well, 8-µm; Corning Costar, Corning, NY, USA). Prior to cell seeding, the upper chamber was coated with Matrigel (BD Biosciences) for 30 min at 37℃. After the gel had fully solidified, 1 × 105 cells resuspended in 200 µL FBS-free medium were seeded into the upper chamber. While culture medium (600 µl) supplemented with 10% FBS was placed in the lower chamber. Following 24 h incubation at 37℃, non-invaded cells remaining on the upper surface were wiped away using a cotton swab, while the cells invaded through membrane pores to the lower chamber were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 15 min. The number of invaded cells was counted under an Olympus fluorescence microscope (Tokyo, Japan) in five representative microscopic fields. All invasion assays were performed in triplicate and repeated three independent times to ensure reproducibility.

RNA immunoprecipitation (RIP) assay

RIP assays were performed using the EZ-Magna RIP RNA-binding Protein Immunoprecipitation Kit (BersinBio, Guangzhou, China). Briefly, 2 × 107 PCa cells were collected and lysed in RIP buffer added with RNase and protease inhibitors. The cell lysates were then incubated with magnetic beads conjugated with anti-Ago2 antibody (ab156870, Abcam, UK) overnight at 4℃ with gentle rotation. Magnetic beads conjugated with non-specific anti-IgG antibody (ab172730, Abcam, UK) were processed in parallel as a negative control. After immunoprecipitation, the resulting protein-RNA complexes were treated with proteinase K treatment to remove proteins. Following RNA extraction using TRIzol reagent (mlBio, Shanghai, China), relative expression levels of ELFN1-AS1, miR-28-5p, and BCAM were quantified by RT-qPCR.

Western blot analysis

Total protein was extracted from cultured PCa cells using a total protein extraction kit (BestBio, Shanghai, China). A BCA kit (Vazyme) was adopted to assess protein concentration. The protein samples (30–50 µg per lane) were separated by SDS-PAGE using precast gels (Yeasen, Shanghai, China) and then electrotransferred onto PVDF membranes (Amersham, USA). The membranes were blocked with 5% non-fat milk at room temperature for 1 h, then incubated overnight with primary antibodies at 4℃ with slow shaking. The membranes were washed by PBST thrice (10 min each time). Primary antibodies are shown as follows: LC3A/B (ab62721, 1:1,000, Abcam, UK), Cleaved-caspase-3 (ab2302, 1:1,000, Abcam), total caspase-3 (ab32351, 1:1,000, Abcam), Cleaved-PARP (#5625, 1:1,000, Cell Signaling Technology), total RARP (#9542, 1:1,000, Cell Signaling Technology), BCAM (ab134110, 1:1,000, Abcam), and GAPDH (ab9485, 1:1,000, Abcam). After primary antibody incubation, membranes were washed three times with PBST buffer, then incubated with goat anti-rabbit IgG secondary antibody (ab6721, 1:2,000, Abcam) at 37℃ for 1 h. Protein bands were visualized using an enhanced chemiluminescence detection system, and images were captured using a luminescent image analyzer (GE Healthcare, USA). Band intensity was quantified using ImageJ software, with GAPDH as the internal reference for normalization.

Monodansylcadaverine (MDC) staining method

MDC is a fluorescent marker that accumulates in acidic autophagic vacuoles, enabling direct visualization of autophagic activity in cells [26]. Intracellular autophagy was detected using a commercial cell autophagy staining detection kit (MDC method, Solarbio Life Sciences, Beijing, China). 22RV1 and PC-3 cells were seeded into 12-well plates and cultured overnight to reach appropriate confluence. After transfection, cells were washed twice with 1× washing buffer, and cell concentration was adjusted to 1 × 106 cells/mL. Next, cell suspension was incubated with 10 µL MDC staining solution in darkness at 37℃ for 30 min. After washing, images of autophagic particles within the cells were captured under a fluorescence microscope (Olympus, Japan).

RT-qPCR analysis

Extraction of total RNAs was achieved utilizing TRIzol reagent (mlBio) following the product manuals. NanoDrop Spectrophotometer was used to determine the concentration of extracted RNA. RNA (1 µg) was reverse transcribed into cDNA utilizing the iScript Reverse Transcription Supermix for RT-qPCR (BioRad, Hercules, USA). PCR was performed using SYBR Premix Ex Taq (Takara, Japan) on a LightCycler 480 Real-Time PCR system (Roche, Shanghai, China). Amplification was performed under the following thermal cycling conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s. The endogenous reference for lncRNA and mRNAs were GAPDH, while that for miRNAs was U6. Relative RNA expression was determined via the 2−ΔΔCT method. The sequences of primers are presented in Table 1.

Table 1.

Sequences of primers used for reverse transcription-quantitative PCR

Gene Sequence
ELFN1-AS1 Forward: 5’-AAAGTCAAGCCAGAGGGAG-3’
Reverse: 5’-TGGAGATTGTGTCTGGAGG-3’
miR-28-5p Forward: 5’-GCGCATTGCACTTGTCTCG-3’
Reverse: 5’-AGTGCAGGGTCCGAGGTATT-3’
BCAM Forward: 5’-CTCAACGTGTTTGCAAAGC-3’
Reverse: 5’-TCCTCCTTTATTACCCAGGG-3’
GAPDH Forward: 5’-TCAAGGCTGAGAACGGGAAG-3’
Reverse: 5’-TGGACTCCACGACGTACTCA-3’
U6 Forward: 5’-CTCGCTTCGGCAGCACATATACT-3’
Reverse: 5’-CGCTTCACGAATTTGCGTGT-3’
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Statistical analysis

All experimental data were analyzed using SPSS 9.0 software (IBM, Armonk, NY, USA). Each experiment was performed independently in triplicate. Quantitative data from three repetitions experiments are shown as mean ± standard deviation (SD). Comparisons between two independent groups were performed using Student’s t-test, while differences among multiple groups were analyzed by one-way or two-way analysis of variance followed by Dunnett’s post-hoc test. A p value < 0.05 was considered statistically significant for all analyses.

Results

ELFN1-AS1 expression is significantly increased in PCa cells

Analysis of the UALCAN database revealed significant upregulated expression of ELFN1-AS1 in prostate adenocarcinoma (PRAD) tissues relative to normal samples (Fig. 1A, p = 1.14E-21). Consistently, RT-qPCR confirmed that ELFN1-AS1 expression was higher in PCa cell lines, particularly in 22RV1 and PC-3 cells, than in the human prostate stromal cell line WPMY-1 (Fig. 1B, p < 0.001). The subcellular distribution of lncRNAs is critical for understanding their functional mechanisms [27, 28]. Following nuclear and cytoplasmic RNA separation, ELFN1-AS1 transcripts were found to be primarily localized in the cytoplasm of 22RV1 and PC-3 cells (82 ± 7% and 86 ± 8%) (Fig. 1C). Collectively, these data indicate that ELFN1-AS1 is highly expressed in PCa cells, and its cytoplasmic enrichment suggests potential regulatory roles at the post-transcriptional level.

Fig. 1.

Fig. 1

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ELFN1-AS1 is highly expressed in prostate cancer (PCa) cells. A ELFN1-AS1 expression levels in prostate adenocarcinoma (PRAD) and normal samples were analyzed using the UALCAN database. B RT-qPCR analysis was performed to examine ELFN1-AS1 expression in PCa cell lines and human prostate stromal cell line WPMY-1. C Subcellular distribution of ELFN1-AS1 in 22RV1 and PC-3 cells was determined by subcellular fractionation assays. Data are shown as mean ± SD. ***p < 0.001 versus WPMY-1 group

Knockdown of ELFN1-AS1 inhibits PCa cell viability and invasion while inducing cell apoptosis and autophagy

To explore the effect of ELFN1-AS1 on malignant phenotypes of PCa cells, loss-of-function experiments were conducted using 22RV1 and PC-3 cells transfected with sh-ELFN1-AS1#1/2. As shown by Fig. 2A, ELFN1-AS1 expression was effectively reduced in the sh-ELFN1-AS1#1/2 groups relative to the sh-NC group. CCK-8 assays demonstrated that ELFN1-AS1 deficiency markedly inhibited PCa cell viability relative to the sh-NC group (Fig. 2B, **p < 0.01, ***p < 0.001). In addition, silencing of ELFN1-AS1 impaired the invasive ability of PCa cells, as evidenced by fewer invaded cells in the sh-ELFN1-AS1 groups (22RV1: 84 ± 8 and 71 ± 7; PC-3: 66 ± 6 and 73 ± 7) compared with the sh-NC group (22RV1: 265 ± 22; PC3: 276 ± 23) (Fig. 2C-D). Moreover, ELFN1-AS1 depletion increased cell apoptotic rate in sh-ELFN1-AS1 groups (22RV1: 16.28 ± 1.33 and 15.58 ± 1.28; PC-3: 15.8 ± 1.25 and 15.89 ± 1.26) relative to the sh-NC group (22RV1: 5.66 ± 0.48; PC3: 3.95 ± 0.32) (Fig. 2E-F). Protein expression of autophagic marker (LC3-II/I) and apoptotic markers (cleaved-caspase-3 and cleaved-PARP) was significantly elevated in PCa cells following ELFN1-AS1 silencing (Fig. 2G). MDC staining further revealed enhanced autophagosome formation upon ELFN1-AS1 knockdown, as indicated by increased MDC fluorescence intensity (Fig. 2H). Taken together, silencing ELFN1-AS1 suppresses PCa cell viability and invasion while promoting apoptosis and autophagy.

Fig. 2.

Fig. 2

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Effects of ELFN1-AS1 depletion on PCa cell invasion, apoptosis, and autophagy. A Knockdown efficiency of sh-ELFN1-AS1 was examined by RT-qPCR. Effects of ELFN1-AS1 knockdown on PCa cell viability B, invasion C-D, and apoptosis E-F were measured by CCK-8 assays B, Transwell assays C-D, and flow cytometry analysis E-F. G Protein levels of apoptosis- and autophagy-related markers in PCa cells were detected by western blot analysis. H Cell autophagy was examined using MDC staining method. Data are presented as mean ± SD. **p < 0.01, ***p < 0.001 versus sh-NC group

ELFN1-AS1 binds to miR-28-5p in PCa cells

To investigate the mechanism by which ELFN1-AS1 functions in PCa cells, the bioinformatic tool LncBase v3 was utilized to predict potential miRNAs that might bind to ELFN1-AS1. The top five candidate miRNAs were selected for further analysis. As shown in Fig. 3A, ELFN1-AS1 depletion led to the upregulation of miR-28-5p expression (4.61- and 4.33-fold) without significantly affecting the expression of the other four miRNAs in both 22RV1 and PC-3 cells. Subsequently, miR-28-5p expression was enhanced via transfection of miR-28-5p mimics into 22RV1 and PC-3 cells (Fig. 3B). The putative binding site between ELFN1-AS1 and miR-28-5p, along with the mutated ELFN1-AS1 sequence, is illustrated in Fig. 3C. Importantly, luciferase activity of the ELFN1-AS1-Wt reporter was significantly reduced in the miR-28-5p mimics group relative to the NC mimics group (Fig. 3D, ***p < 0.001). RT-qPCR analysis further showed that miR-28-5p expression was downregulated in PCa cells compared with WPMY-1 cells (Fig. 3E, p < 0.001). Collectively, ELFN1-AS1 binds to miR-28-5p and inversely regulates its expression in PCa cells.

Fig. 3.

Fig. 3

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ELFN1-AS1 interacts with miR-28-5p in 22RV1 and PC-3 cells. A Expression levels of candidate miRNAs in PCa cells transfected with sh-ELFN1-AS1#1 were measured by RT-qPCR. ***p < 0.001 versus sh-NC group. B Overexpression efficiency of miR-28-5p mimics was confirmed by RT-qPCR. ***p < 0.001 versus NC mimics group. C Putative binding sequences between ELFN1-AS1 and miR-28-5p were predicted from LncBase v3 (https://diana.e-ce.uth.gr/lncbasev3). D Luciferase reporter assays were conducted to measure the interaction between ELFN1-AS1 and miR-28-5p. ***p < 0.001 versus NC mimics group. E RT-qPCR analysis was conducted to assess miR-28-5p expression in PCa cell lines and WPMY-1 cells. Data are presented as mean ± SD. ***p < 0.001 versus WPMY-1 group

ELFN1-AS1 elevates BCAM expression via interaction with miR-28-5p

To further explore the ceRNA network mediated by ELFN1-AS1, the miRDB database was employed to predict target genes of miR-28-5p, and those with a binding score > 90 were selected. Figure 4A showed that miR-28-5p upregulation significantly reduced BCAM expression without altering the expression of other candidate genes in 22RV1 cells. Consistently, BCAM expression was also reduced by miR-28-5p mimics in another PCa cell line (PC-3) (0.42 ± 0.04 vs. 1 ± 0.1) (Fig. 4B). BCAM protein levels were notably reduced in both 22RV1 and PC-3 cells overexpressing miR-28-5p (Fig. 4C). The binding site between miR-28-5p and BCAM was obtained from the TargetScan database, and the complementary sequence of BCAM is presented in Fig. 4D. RT-qPCR analysis demonstrated that ELFN1-AS1 expression was dramatically increased following transfection with pcDNA3.1/ELFN1-AS1 (Fig. 4E, p < 0.001). Upon miR-28-5p overexpression, the luciferase activity of BCAM-WT was significantly reduced, and this effect was partially reversed by ELFN1-AS1 upregulation (Fig. 4F). However, neither miR-28-5p mimics nor pcDNA3.1/ELFN1-AS1 significantly affected the luciferase activity of BCAM-MUT (Fig. 4F). RIP assays showed high enrichment of ELFN1-AS1, miR-28-5p, and BCAM in anti-Ago2 group compared with the anti-IgG group (Fig. 4G-H, p < 0.001). Furthermore, silencing ELFN1-AS1 evidently reduced BCAM expression levels (Fig. 4I-J). BCAM expression was significantly elevated in PCa cells relative to WPMY-1 cells (Fig. 4K). UALCAN database also shows upregulated BCAM expression in PRAD tissues compared with normal samples (Fig. 4L, p < 1E-12). In summary, BCAM is upregulated in PCa cells and serves as a target of miR-28-5p. ELFN1-AS1 increases BCAM expression by interacting with miR-28-5p.

Fig. 4.

Fig. 4

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ELFN1-AS1 interacts with miR-28-5p to regulate BCAM expression. A RT-qPCR analysis was performed to measure mRNA levels of candidate targets (predicted by miRDB with a binding score > 90) in 22RV1 cells transfected with NC mimics or miR-28-5p mimics. B-C RT-qPCR and western blot analyses were performed to measure BCAM expression in PC-3 cells transfected with NC mimics or miR-28-5p mimics. ***p < 0.001 versus NC mimics group. D The predicted binding site (253–259) of BCAM 3’UTR with miR-28-5p from TargetScan is shown. E Overexpression efficiency of ELFN1-AS1 vectors was evaluated by RT-qPCR analysis. ***p < 0.001 versus vector group. F Luciferase reporter assay was performed to assess the interaction among ELFN1-AS1, miR-28-5p, and BCAM. ***p < 0.001 versus NC mimics group. ###p < 0.001 versus miR-28-5p mimics. G-H The interaction among ELFN1-AS1, miR-28-5p, and BCAM was examined by RIP assay. ***p < 0.001 versus anti-IgG group. I-J The effect of ELFN1-AS1 depletion on BCAM expression were assessed by RT-qPCR and western blotting. K BCAM expression in PCa cell lines and WPMY-1 cells was detected by RT-qPCR and western blotting. L BCAM expression in PRAD and normal tissue was analyzed using the UALCAN database. Data are expressed as mean ± SD

BCAM upregulation reverses the inhibitory effects of ELFN1-AS1 silencing on PCa cell malignancy

To further validate that ELFN1-AS1 modulates PCa cell malignancy by upregulating BCAM expression, rescue experiments were conducted. BCAM mRNA and protein levels were first elevated in PCa cells through transfection of pcDNA3.1-BCAM vectors (Fig. 5A, p < 0.001). ELFN1-AS1 deficiency suppressed PCa cell viability and invasion, and these inhibitory effects were counteracted by BCAM overexpression (Fig. 5B and C). Moreover, compared with the sh-ELFN1-AS1#1 group, the sh-ELFN1-AS1#1 + BCAM group exhibited decreased apoptotic rates and reduced autophagosome numbers (Fig. 5D and E). These results indicate that BCAM upregulation reversed the ELFN1-AS1 depletion-induced activation of apoptosis and autophagy in PCa cells. The above findings demonstrate that ELFN1-AS1 promotes malignant phenotypes in PCa cells by upregulating BCAM.

Fig. 5.

Fig. 5

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BCAM

Discussion

Although the morbidity and mortality associated with PCa have progressively declined, PCa remains one of the most prevalent urological malignancies affecting men globally [3, 5]. Recently, accumulating evidence has emphasized the critical role of ceRNA networks in the initiation and progression of various cancers [29, 30]. Therefore, there is an urgent need to identify novel biomarkers based on the ceRNA network to improve prognosis and therapeutic strategies for PCa. In this study, we explored the function of ELFN1-AS1 in PCa cells and found that ELFN1-AS1 acts as a competing endogenous RNA (ceRNA) to modulate cell apoptosis and autophagy through the miR-28-5p/BCAM axis (Fig. 6). The oncogenic role of ELFN1-AS1 is consistent with findings in other malignancies, including esophageal cancer [31], colorectal cancer [32], and gastric cancer [33].

Fig. 6.

Fig. 6

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ELFN1-AS1 inhibits PCa cell apoptosis and autophagy by positively regulating BCAM via interaction with miR-28-5p

Dysregulated apoptosis represents a hallmark of cancer, and the induction of programmed cell death plays a central role in anti-tumor therapy [34]. Autophagy exhibits a dual role in cancer biology: under certain conditions, it acts as a pro-survival mechanism protecting cancer cells from apoptosis, whereas in other contexts, sustained autophagy can promote cell death, often in coordination with apoptotic pathways [14, 35]. As previously reported, transmission electron microscopy (TEM) analysis has revealed that autophagy can be induced through increased LC3-I to LC3-II conversion, enhanced autophagosome formation, and accumulation of acidic vesicular organelles (AO puncta) [36]. In the present study, we examined the effects of ELFN1-AS1 knockdown on PCa cell functions. Through a series of functional assays, we demonstrated that silencing ELFN1-AS1 significantly suppressed cell viability and invasion while promoting apoptosis and autophagy. These findings support the oncogenic role of ELFN1-AS1 in PCa.

The ceRNA function of ELFN1-AS1 has previously been documented in colon cancer, where its downregulation promotes apoptosis and inhibits proliferation and migration via the miR-4270/AURKB axis [22]. In our study, ELFN1-AS1 silencing led to a marked upregulation of miR-28-5p in PCa cells. Earlier studies have shown that overexpression of miR-28-5p suppresses metastasis and proliferation in colon cancer by modulating the CAMTA2/Wnt/β-catenin signaling pathway [37]. Additionally, miR-28-5p expression is downregulated in cholangiocarcinoma tissues, and its restoration inhibits tumor growth and metastasis by directly targeting CD44 [38]. Our results confirmed a significant decrease in miR-28-5p expression in PCa cell lines. Importantly, we validated the direct interaction between ELFN1-AS1 and miR-28-5p in both 22RV1 and PC-3 cell lines.

BCAM, also known as Lutheran, is a member of the immunoglobulin superfamily and functions as a receptor for laminin α5 [39]. Several studies have reported elevated BCAM expression in hepatocellular carcinoma [40], pancreatic cancer [41] and ovarian cancer [42], whereas others have demonstrated its downregulation in malignant thyroid cancer [43] and colon cancer [44]. Recent evidence indicates that high BCAM expression is associated with poor survival outcomes in patients with gastric cancer, and BCAM inhibition has been shown to impair the invasive and migratory capacities of gastric cancer cells in vitro [45]. In contrast, ectopic expression of BCAM suppresses the migration and invasion of hepatoma cells [46]. In the present study, BCAM was found to be upregulated in PCa cells and was confirmed as a direct downstream target of miR-28-5p. Notably, BCAM expression levels were positively correlated with ELFN1-AS1 levels but negatively correlated with miR-28-5p expression. More importantly, BCAM overexpression reversed the effects of ELFN1-AS1 silencing on cell invasion, apoptosis, and autophagy, suggesting that ELFN1-AS1 promotes PCa progression by modulating BCAM expression.

However, certain limitations of the current study should be acknowledged. The oncogenic role of ELFN1-AS1 requires further validation through in vivo experiments. Moreover, given the complexity of molecular regulatory networks, additional indirect mechanisms by which ELFN1-AS1 may regulate BCAM remain to be elucidated.

In conclusion, silencing the lncRNA ELFN1-AS1 inhibits cell viability and invasion while promoting apoptosis and autophagy through modulation of the miR-28-5p/BCAM axis. Our findings provide novel insights into the dysregulation of gene expression in PCa and suggest that the ELFN1-AS1/miR-28-5p/BCAM signaling axis may serve as a promising therapeutic target for PCa treatment.

Supplementary Information

Supplementary Material 1 (348KB, pptx)

Acknowledgements

None.

Authors' contributions

Shuai Luo conceived and designed the experiments. Shuai Luo, Dingwen Gui, Yankuang Guo, Zuwei Xu, Zheng Fang, Geng Huang and Wenbing Wu analyzed the data. Shuai Luo, Geng Huang and Wenbing Wu drafted the manuscript. All authors agreed to be accountable for all aspects of the work. All authors have read and approved the final manuscript.

Funding

None.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Geng Huang, Email: huanggsir@163.com.

Wenbing Wu, Email: hsszxyy459812113@163.com.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (348KB, pptx)

Data Availability Statement

No datasets were generated or analysed during the current study.

Articles from World Journal of Surgical Oncology are provided here courtesy of BMC

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