Prognostic value of miR-26b-5p in prostate cancer and its regulatory effect on tumor progression

Abstract

Background

The diagnosis and treatment of prostate cancer (PCa) continue to encounter numerous. The specific regulatory mechanism is not yet clear. Studies have shown that miRNA plays a role in the progression of PCa. This research was the first to systematically explore the regulatory mechanism of the miR-26b-5p/ADAM17 pathway in PCa. It reveals the role of the “miR-26b-5p/ADAM17” signaling axis in inhibiting the malignant phenotype of PCa cells and the tumor inflammatory microenvironment.

Methods

This study recruited serum samples from 138 PCa patients and 138 healthy controls. RT-qPCR was employed to quantify mRNA expression. The interaction was validated using a dual-luciferase assay. Cell proliferation was assessed by the CCK-8 assay, while cell migration and invasion were evaluated via Transwell. Flow cytometry was utilized to measure cell apoptosis, and enzyme-linked immunosorbent assay (ELISA) was performed to determine inflammatory cytokines levels.

Results

miR-26b-5p was significantly downregulated in PCa. Its low expression was strongly correlated with poor prognosis, and it functioned as an independent protective prognostic factor. Functional experiments demonstrated that the overexpression of miR-26b-5p markedly suppressed proliferation, migration, and invasion while promoting apoptosis and inhibiting the secretion of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Mechanistically, miR-26b-5p exerted its anti-tumor effects by directly targeting and suppressing ADAM17 expression. Furthermore, the overexpression of ADAM17 partially reversed the tumor-suppressive and anti-inflammatory effects mediated by miR-26b-5p.

Conclusion

miR-26b-5p functioned in PCa by directly targeting ADAM17.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12957-026-04228-3.

Introduction

Approximately 80% of prostate cancer (PCa) cases are classified as localized prostate cancer at the time of diagnosis [1]. Extensive clinical evidence indicates that patients with localized PCa can achieve a relatively high 5-year survival rate following active treatment [2]. To date, the precise mechanisms underlying PCa pathogenesis remain incompletely understood. Consequently, early diagnosis to detect localized PCa and timely intervention before the onset of distant metastasis are critical steps in halting disease progression and reducing mortality.

microRNAs (miRNAs) play critical roles in apoptosis, and differentiation [3]. Specifically, miR-26b-5p suppresses the proliferation of triple-negative breast cancer cells [4]. In rheumatoid arthritis (RA) synovial tissues, miR-26b-5p is significantly lower and contributes to the modulation of inflammatory responses in RA [5]. Other studies have demonstrated that miR-26b-5p inversely correlates with tumor malignancy [6]. Studies have shown that miR-26b-5p has strong predictive capabilities as a new candidate for predicting PCa metastasis and is a promising minimally invasive detection tool in the clinical management of PCa [7, 8]. Nevertheless, the precise regulatory mechanisms of miR-26b-5p in PCa remain to be elucidated.

A Disintegrin and Metalloproteinase 17 (ADAM17) is a transmembrane protease [9, 10]. Aberrant expression or activation of ADAM17 has been linked to multiple diseases, such as autoimmune disorders, inflammatory conditions, cancers, and cardiovascular diseases [11]. In chronic lung injuries, the excessive activation of the ADAM17/EGFR signaling axis promotes the release of pro-inflammatory mediators and contributes to airway remodeling [12]. ADAM17 activates the EGFR signaling pathway by shedding EGFR ligands, such as TGFα and HB-EGF, thereby driving tumor cell proliferation and epithelial-mesenchymal transition (EMT) [13]. Furthermore, siRNA targeting ADAM17 effectively inhibits the proliferation of PC-3 cells, highlighting its potential value in gene therapy for PCa [14]. By integrating prediction results from TargetScanHuman_8.0 and ENCORI through bioinformatics analysis, we identified ADAM17 as a potential target gene of miR-26b-5p. Furthermore, we found a conserved binding site for miR-26b-5p in the 3’UTR region of ADAM17. Previous studies have reported that ADAM17 has potential application value in the gene therapy of PCa, further supporting its biological relevance [14]. More importantly, in the results section of this study, we directly confirmed through dual-luciferase reporter gene experiments that miR-26b-5p can specifically bind to the 3’UTR of ADAM17 and significantly inhibit its reporter gene activity, thereby providing experimental evidence for this targeting relationship. Based on these findings, we hypothesize that the miR-26b-5p/ADAM17 axis may be implicated in PCa progression.

This study, through cell experiments and clinical samples, has for the first time systematically revealed the role of the “miR-26b-5p/ADAM17” signaling axis in inhibiting the malignant phenotype of PCa cells and the inflammatory tumor microenvironment, thereby providing new ideas for clarifying the molecular mechanism of PCa.

Methods

Study participants

This study received ethical approval from the Ethics Committee of Xuzhou Medical University Affiliated Xuzhou Municipal Hospital. All participants provided written informed consent.

This study enrolled 138 PCa patients from Xuzhou Medical University Affiliated Xuzhou Municipal Hospital and 138 healthy volunteers recruited from the health examination center. The inclusion criteria: pathological confirmation of PCa via prostate biopsy, no prior exposure to chemotherapy, radiotherapy, endocrine therapy, surgical castration, or other related treatments before enrollment, and age of 18 years or older. The exclusion criteria: brain or spinal cord metastases that could interfere with the assessment of study endpoints; abnormal blood pressure or glucose levels; acute prostatitis or uncontrolled systemic infections; history of prostatectomy, radiotherapy, or other radical local therapies (e.g., cryotherapy); psychiatric disorders or refusal to provide informed consent.

The 138 healthy control volunteers recruited for this study came from the physical examination center of our hospital. The inclusion criteria were as follows: their age range was similar to that of the PCa patient group, and through physical examination and medical history inquiry, it was confirmed that they had no history of any malignant tumors (especially urinary system tumors), no acute or chronic prostate diseases, no severe autoimmune diseases, systemic infections or other major systemic diseases that might affect the expression level of serum miRNA. The physical examination indicators (such as blood pressure, blood sugar, liver and kidney function, and blood routine) showed no obvious abnormalities. The prostate-specific antigen (PSA) level was within the normal reference range, and they had no clinical symptoms of prostate cancer or other prostate diseases, in order to ensure the consistency of the baseline of the two groups and enhance the reliability of the research results.

According to miR-26b-5p level, PCa patients were categorized into low-expression group (expression level below the mean) and high-expression group (expression level above the mean).

Immediately after the diagnosis of prostate cancer, 5 mL of fasting venous blood was collected and promptly centrifuged at a standard speed to separate the serum, which was subsequently stored at −80 °C.

The Gleason score was a grading system that correlates with the aggressiveness of prostate tumors [15]. Scores range from 2 to 10 (score ≤ 7, low grade, score > 7 high grade). Patients were followed up with visits scheduled every three months over a total period of five years.

Cell culture and transfection

In the cell experiments of this study, the normal control group used was the commercial immortalized human normal prostatic epithelial cell line RWPE-1 cells (Beina Biotechnology Co., Ltd.). The PCa cell lines PC-3 and DU145 were obtained from Wanwu Biotechnology Co., Ltd. The RWPE-1 cells were cultured in a medium without keratinocyte serum (K-SFM, Gibco Company in the United States), and this medium was supplemented with bovine pituitary extract (BPE) and human recombinant epidermal growth factor (EGF). The PCa cells (PC-3 and DU145) were maintained in F-12 K nutrient medium (produced by Gibco, USA). All media were supplemented with 10% fetal bovine serum (FBS) under a humidified atmosphere of 37 °C and 5% CO₂.

miR-26b-5p mimics, miR-26b-5p inhibitor, and ADAM17 overexpression plasmid (pcDNA3.1-ADAM17) were synthesized by the Shanghai Gemma Company. PC-3 and DU145 cells were seeded into 6-well plates and incubated. Lipofectamine™ 2000 reagent, miR-26b-5p mimics, miR-26b-5p inhibitor, and ADAM17 overexpression plasmid were transfected into cells. To ensure the consistency and reliability of the experimental conditions, the transfection efficiency was systematically verified based on the standard transfection concentration (50 nM) used in the functional experiments.

RT-qPCR

Total RNA was extracted using the TRIzol LS (Thermo Fisher). TRIzol LS can efficiently lyse cells and inhibit RNase activity, thereby enabling the stable extraction of high-quality RNA. It can also simultaneously separate DNA and proteins from the same sample. It is mainly used for liquid samples, such as serum, virus preparations, blood, etc. All the extracted RNA samples were quantified and purity evaluated using the NanoDrop™ micro spectrophotometer. The RNA quality control standards were set as follows: the A260/A280 ratio should be between 1.8 and 2.1, and the A260/A230 ratio should be greater than 2.0, to ensure that the RNA was highly pure and to exclude the interference of proteins, phenols, or salts, etc. The extracted RNA was then reverse-transcribed into cDNA. RT-qPCR amplification was subsequently performed using TaqMan Universal PCR Master Mix II and 7900HT Real-Time PCR System (Applied Biosystems). This process utilized the obtained cDNA as a template. For normalization, GAPDH was selected as the internal reference gene for mRNA, while U6 was chosen as the internal reference gene. The Ct value of the internal reference gene U6 snRNA remained stable and showed little variation across all samples. The amplification efficiency was within the standard range, indicating that the extracted miRNA had good integrity and amplifiability. And the quality of the extracted miRNAs was sufficient to meet the requirements of subsequent quantitative detection and functional analysis.

Cell counting Kit-8 (CCK-8) assay

Logarithmic phase transfected cells were harvested 48 h post-transfection and subsequently counted using a hemocytometer. Cells were then seeded into 96-well plates. Proliferation capacity was assessed using a CCK-8 assay kit.

Migration and invasion

The cell was inoculated into the upper chamber of the Transwell, while 500 µL of medium supplemented with 10% FBS was added to the lower chamber. Following incubation, the Transwell insert was removed, gently rinsed twice, and fixed and stained.

For invasion assay, diluted Matrigel was evenly applied onto the surface of the Transwell membrane in the upper chamber. All subsequent steps were identical to those performed in the migration assay. The statistical analysis of this experiment was based on the average value of cell counts from 5 randomly non-overlapping fields. The entire experiment was repeated five times.

Apoptosis assay

For the flow cytometry-based apoptosis assay, cells were labeled with Annexin V-FITC/PI double staining and incubated. Cellular fluorescence signals were acquired using flow cytometry. The proportions of cells at each stage of apoptosis were subsequently quantified.

Enzyme-linked immunosorbent assay (ELISA)

Samples were added and incubated to form antibody-antigen-detection antibody complexes. After another wash, streptavidin-conjugates were added to specifically bind with the biotin-labeled antibodies. Finally, a substrate solution was added to initiate an enzyme-catalyzed color reaction. The absorbance was measured to determine the concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6).

Dual-luciferase reporter assay

The TargetScanHuman_8.0 and ENCORI databases predicted that ADAM17 was a potential target gene for miR-26b-5p, and it was found that there was a conserved miR-26b-5p binding site in its 3’UTR region. The predicted binding site and its mutant sequence were cloned into the pmirGLO vector to generate ADAM17- wild type (WT) and ADAM17- mutant (MUT) reporter vectors. Thereafter, Lipofectamine™ 2000 was employed to co-transfect these vectors along with miR-26b-5p mimics or mimics-NC into cultured cells. Following a 48-hour incubation period, luciferase activity was assessed to confirm the direct binding interaction.

Statistical analysis

Data statistical analysis and visualization were conducted using SPSS 26.0 and GraphPad Prism 9. For continuous variables, the Mann-Whitney U test was applied; for categorical variables, the chi-square test was utilized. The survival probability was assessed via Kaplan-Meier survival analysis, while the COX regression was employed to identify independent risk factors influencing PCa prognosis. In this study, we analyzed miR-26b-5p expression levels in all 138 PCa patients and found the distribution to be approximately normal with low dispersion. Under these conditions, the mean effectively reflects the central tendency of the data and closely approximates the median (mean: 0.61, median: 0.63). Using the mean as the cutoff value provides a more intuitive interpretation—classifying patients as having “above-average” or “below-average” expression—which enhances clinical interpretability. All cell experiments were independently repeated at least three times (n ≥ 3), and 5 replicate wells were set up for each experiment. A P-value < 0.05 was considered statistically significant.

Results

miR-26b-5p was associated with PCa prognosis

miR-26b-5p was significantly lower in PCa patients (Fig. 1A). miR-26b-5p expression was not significantly associated with patient age or tumor size. Conversely, low miR-26b-5p expression was significantly correlated with lymph node metastasis, Gleason score, bone metastasis, and advanced tumor staging in PCa (Table 1). Notably, miR-26b-5p was significantly lower in PCa patients with poor prognosis compared to those with favorable outcomes (Fig. 1B). Furthermore, miR-26b-5p expression was significantly associated with survival probability. Compared with the group with high expression of miR-26b-5p, the overall survival period of patients in the low-expression group was significantly shortened (HR = 0.080, 95% CI: 0.030–0.215, P < 0.0025) (Fig. 1C). Additionally, lymph node metastasis, Gleason score, bone metastasis, and tumor staging were independent risk factors for poor prognosis, whereas miR-26b-5p served as an independent protective prognostic factor (Table 2).

Fig. 1.

Expression and prognostic significance of miR-26b-5p in PCa. A. Differential expression of miR-26b-5p between PCa patients and healthy controls. B. Correlation between miR-26b-5p expression and clinical prognosis in PCa patients. C. Impact of miR-26b-5p expression on survival probability in PCa patients. *** indicates P < 0.001 compared to the control group

Table 1.

The correlations between miR-26b-5p expression levels and clinicopathological characteristics in prostate cancer

Characteristics	Total (n = 138)	Lower miR-26b-5p (n = 76)	Higher miR-26b-5p (n = 62)	P-value
Age (years)				0.596
≤ 55	70	37	33
> 55	68	39	29
Gleason score				0.043
≤ 7	107	54	53
> 7	31	22	9
Tumor size (cm)				0.154
≤ 3	62	30	32
> 3	76	46	30
Lymph node metastasis				0.024
No	103	51	52
Yes	35	25	10
Osseous metastasis				0.024
No	125	65	60
Yes	13	11	2
Tumor stage				0.034
I–III	94	46	48
Ⅳ	44	30	14

Table 2.

Cox regression of the prognosis of prostate cancer patients

Characteristics	HR	95% CI	P-value
Age	1.231	0.458–3.309	0.680
Gleason score	2.848	1.059–7.656	0.038
Tumor size	2.466	0794-7.659.659	0.119
Lymph node metastasis	3.099	1.130–8.499	0.028
Osseous metastasis	3.611	1.157–11.277	0.027
Tumor stage	2.866	1.062–7.735	0.038
miR-26b-5p	0.084	0.011–0.639	0.017

HR Hazard Ratio, CI Confidence Interval

Regulatory effect of miR-26b-5p on biological functions in PCa

miR-26b-5p expression in PCa cell lines was markedly lower compared to the normal control group (Fig. 2A). miR-26b-5p mimic could significantly upregulated the miR-26b-5p expression (Fig. 2B-C). Overexpression of miR-26b-5p substantially suppressed the proliferation capacity (Fig. 2D-E), attenuated migration (Fig. 2F-G), reduced invasion potential (Figure. 2 H-I), and markedly enhanced the apoptosis rate (Fig. 2J-K).

Fig. 2.

Functional regulation of PCa cells by miR-26b-5p. A Compared with the control group, miR-26b-5p expression was significantly downregulated in PCa cells. B-C Validation of miR-26b-5p overexpression efficiency. D-E Suppressive effects of miR-26b-5p on cell proliferation. F-G Inhibitory effects of miR-26b-5p on cell migration. H-I Inhibitory effects of miR-26b-5p on cell invasion. J-K Promotive effects of miR-26b-5p on cell apoptosis. *** indicates P < 0.001 compared to the control group

Overexpression of miR-26b-5p significantly suppressed TNF-α and IL-6 secretion in PCa cells (Figure. 3 A-B).

Fig. 3.

Regulation of inflammatory cytokine secretion (TNF-α, IL-6) in PCa cells by miR-26b-5p. A-B Significant suppression of TNF-α and IL-6 secretion in PCa cells (PC-3, DU145) by miR-26b-5p. *** indicates P < 0.001 compared to the control group

Targeting regulatory relationship

In the WT-ADAM17 group, miR-mimic significantly suppressed luciferase activity, whereas miR-inhibitor markedly enhanced luciferase activity. In contrast, in the MUT-ADAM17 group, no significant differences in luciferase activity were observed among the groups (Fig. 4A-C). Furthermore, ADAM17 was significantly elevated in PCa patients (Fig. 4D). Mechanistic investigations revealed that miR-26b-5p directly targeted and suppressed ADAM17 expression, and transfection with exogenously overexpressed ADAM17 partially rescued the inhibitory effect of miR-26b-5p on ADAM17 (Fig. 4E-F).

Fig. 4.

Targeted regulatory relationship between miR-26b-5p and ADAM17. A Prediction of direct binding between miR-26b-5p and ADAM17. B-C Dual-luciferase reporter assay confirming the targeted interaction between miR-26b-5p and ADAM17. D Basal expression of ADAM17 in PCa. E-F Regulatory effects of miR-26b-5p on ADAM17 expression. *** indicates P < 0.001 compared to the control group. ns stands for not significant

Overexpression of ADAM17 could reverse the effects mediated by miR-26b-5p

Overexpression of ADAM17 partially reversed the miR-26b-5p-induced inhibition of cell proliferation (Fig. 5A-B), migration, and invasion (Fig. 5C-F), as well as the miR-26b-5p-mediated promotion of cell apoptosis (Fig. 5G-H).

Fig. 5.

miR-26b-5p regulated PCa cell functions through targeting and inhibiting ADAM17. A-B Overexpression of ADAM17 reversed the suppressive effect of miR-26b-5p on cell proliferation. C-D Overexpression of ADAM17 reversed the suppressive effect of miR-26b-5p on cell migration. E-F Overexpression of ADAM17 reversed the suppressive effect of miR-26b-5p on cell invasion. G-H Overexpression of ADAM17 reversed the promotive effect of miR-26b-5p on cell apoptosis. *** indicates P < 0.001 compared to the control group

Overexpression of ADAM17 partially reversed the inhibitory effects of miR-26b-5p on TNF-α and IL-6 (Fig. 6A-B).

Fig. 6.

miR-26b-5p regulated the secretion of inflammatory cytokines (TNF-α, IL-6) in PCa cells via targeting ADAM17. A-B Overexpression of ADAM17 reversed the suppressive effect of miR-26b-5p on TNF-α and IL-6 secretion. *** indicates P < 0.001 compared to the control group

Discussion

The diagnosis and treatment of PCa continue to encounter numerous significant challenges [16]. Existing studies have demonstrated that miR-26b-5p mitigates intestinal mucosal cell apoptosis and alleviates intestinal ischemia-reperfusion injury [17]. In a rat model of chronic heart failure (CHF), overexpression of miR-26b-5p significantly enhances cardiac function and suppresses inflammatory responses [18]. miR-26b-5p exhibits low expression levels in hepatocellular carcinoma (HCC), and its reduced abundance is strongly associated with poorer survival outcomes [19]. This study enrolled 138 PCa patients and 138 healthy controls. Research has found miR-26b-5p levels negatively correlated with tumor malignancy and invasion-metastasis potential. Patients with low expression experienced shorter overall survival, suggesting that miR-26b-5p served as an independent protective prognostic factor for PCa.

In multiple myeloma (MM), miR-26b-5p exerts anti-tumor effects by directly targeting JAG1 [20]. Additionally, miR-26b-5p treatment mitigates the progression of osteoarthritis (OA) by alleviating synovitis and cartilage degeneration [21]. Our research indicated that low miR-26b-5p expression enhanced proliferation, migration, and invasion capabilities, as well as inhibited apoptosis, and was significantly correlated with the activation of the inflammatory microenvironment. Furthermore, studies have demonstrated that ADAM17 knockout markedly enhances the recognition and clearance efficiency of CAR-T cells against solid tumor cells, strengthens anti-tumor activity, and prolongs the survival time of mice [22]. Further investigations revealed that low miR-26b-5p expression resulted in abnormal activation of ADAM17, which in turn promoted the proliferation, migration, and invasion of PCa cells, as well as the secretion of inflammatory factors, thereby driving tumor progression. Moreover, high expression of ADAM17 in PCa patients was directly linked to the diminished tumor-suppressive function of miR-26b-5p.

Detecting miR-26b-5p and ADAM17 expression in clinical settings may serve as auxiliary evidence for diagnosing PCa and evaluating its prognosis. Furthermore, the development of drugs or gene therapy approaches that enhanced miR-26b-5p expression or inhibited ADAM17 activity was anticipated to effectively suppress the proliferation and inflammatory responses of cancer cells, thus offering novel strategies for targeted PCa therapy.

This study possessed certain limitations that warrant acknowledgment. The experiments predominantly rely on cell line models. Second, this study focused exclusively on a single pathway and did not thoroughly investigate its interactions with other signaling networks. Furthermore, the precise downstream molecular mechanisms by which miR-26b-5p regulated cancer cell functions via targeting ADAM17 remain to be elucidated. Future research could expand clinical sample size, integrate animal models with clinical data, and comprehensively validate the diagnostic and therapeutic potential of this pathway. Simultaneously, it was essential to deeply analyze the synergistic or antagonistic relationships between the miR-26b-5p/ADAM17 pathway and other signaling pathways, construct a more comprehensive regulatory network, and thereby facilitate the translation of fundamental research findings into clinical applications.

In conclusion, miR-26b-5p targeted and inhibited ADAM17, thereby suppressing PCa progression. This study may offer novel insights for a deeper understanding of the molecular pathological mechanisms underlying PCa.

Authors’ contributions

CG and XLC conceived and designed the experiments. LL and LM performed the experiments. SM and CL contributed sample collection and statistical analysis. CG and XLC wrote the manuscript. All authors revised it critically for important intellectual content. All authors read and approved the final manuscript.

Funding

No funding was received to assist with the preparation of this work.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study protocol was approved by The Ethics Committee of Xuzhou Medical University Affiliated Xuzhou Municipal Hospital and followed the principles outlined in the Declaration of Helsinki.All participants provided written informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.