Study on the roles of glycolipid metabolism indicators and microRNAs in benign prostatic hyperplasia

Participants and clinical sample processing

The healthy volunteers with normal physical examinations (n=100) and patients with BPH (n=110) came from Zhangjiakou First Hospital. Their basic information was collected in Supplementary Table 1. Their blood samples and basic information were kept intact. Informed consent was obtained from all participants. The Ethics Committee of Zhangjiakou First Hospital approved this study. (Approval Number: 2023040, Date: 2023-06-28).

The blood samples were collected from the anterior elbow veins of the volunteers when they came to our hospital for examination, using EDTA anticoagulant tubes (purple, VACUETTE, Austria), potassium oxalate/sodium fluoride anticoagulant tubes (gray, VACUETTE), and serum tubes (red, VACUETTE). The whole blood samples in the purple tubes were used for HbA1c detection using high performance liquid chromatography in the Mindray glycated hemoglobin analyzer (China), with imprecision (coefficient of variation, CV) of 3.0 %, bias of 3.6 %. The whole blood samples in the grey tubes were centrifuged (1,000 g, 10 min) and the supernatant (plasma) was taken for FBG detection using the hexokinase method in the Beckman Coulter UniCel DxC 800 Synchron fully automatic biochemical analyzer (USA), with CV of 3.0 %, bias of 2.0 %. The whole blood samples in the red tubes were centrifuged (1,000 g, 10 min) and the supernatant (serum) was taken for TC and TG detection using the cholesterol oxidase and glycerol kinase methods in the Beckman Coulter UniCel DxC 800 Synchron fully automatic biochemical analyzer, with CV of 3.0 and 5.0 %, bias of 4.0 and 5.0 %, respectively. For preservation of samples, the separated serum samples were aliquoted into transparent sterile Eppendorf (EP) tubes (Corning, USA) and then stored at −80 °C for sample retention. The blood levels of HbA1c, FBG, TC, and TG were detected when the volunteers were admitted to the hospital. These indicators were collected from clinical laboratory records and defined as GLMI.

Evaluation of diagnostic value for BPH

The predictive value of clinical factors in BPH was evaluated through the ROC curve constructed in SPSS software. The multivariate binary logistic regression analysis was performed to evaluate the risk factors for BPH.

Real-time quantitative PCR (RT-qPCR)

When conducting the research, the serum samples were first placed at −20 °C for 24 h, and then completely thawed at 4 °C for RT-qPCR detection. Trizol reagent (Invitrogen, USA) was used for the RNA extraction of serum samples and cells. Total RNA was reverse transcribed into cDNA using the BeyoRT III cDNA First Strand Synthesis kit (Beyotime, China). RT-qPCR was performed using SYBR^® Green real-time PCR Premix (Toyobo Biologics, Japan) in a transparent 96-well PCR microplate (Corning, USA). U6 Small Nuclear 1 (U6) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal reference genes. The relative expression of genes was measured by the 2^−ΔΔCt. The primer sequences are shown below: miR-96-5p (F/R: 5′-CAG​TCG​TTT​TTA​CAC​GAT​CAC-3′/3′-GGT​CCA​GTT​TTT​TTT​TTT​TTT​TAA​ACC-5′); miR-181a-5p (F/R: 5′-CTC​GCT​TCG​GCA​GCA​CA-3′/5′-AAC​GCT​TCA​CGA​ATT​TGC​GT-3′); FOXP2 (F/R: 5′-TTG​GAT​GAC​CGA​AGC​ACT​G-3′/5′-AGG​TTT​GGG​AGA​TGG​TTT​GG-3′); U6 (F/R: 5′- CTC​GCT​TCG​GCA​GCA​CAT​ATA​CT-3′/5′-ACG​CTT​CAC​GAA​TTT​GCG​TGT​C-3′); GAPDH (F/R: 5′-GTA​ACC​CGT​TGA​ACC​CCA​TT-3′/5′-CCA​TCC​AAT​CGG​TAG​TAG​CG-3′).

Cell culture

Human normal prostatic matrix immortalized cells (WPMY-1) and human prostatic hyperplasia cells (BPH-1) were purchased from SUNNCELL (China) and were cultured using the special medium with catalog numbers SNLM-019 and SNLM-625, respectively, from the same company. The culture environment was 37 °C, containing 5 % CO₂.

Cell transfection

The inhibitors of miR-96/181a-5p were from MedChemExpress (USA). The small interfering RNA for FOXP2 was purchased from RiboBio (China). Opti-MEM (Gibco, USA), RNA/plasmid, and Lipofectamine reagent (Invitrogen) were mixed for 15 min and then added to the cells into 96-well plates. After 8 h, the transfection mixture was removed and a fresh complete medium was added. The subsequent experiments were conducted 24–48 h later.

Evaluation of cell viability

The cell counting kit-8 (CCK-8) kit (Solarbio, China) was used for cell viability assessment. The experiment was carried out in a 96-well plate. After cell treatment, the old medium was replaced with 100 μL fresh medium containing 10 % CCK-8 to incubate for 2 h at 37 °C. Finally, the optical density (OD) at 450 nm was measured with an enzyme-labeled instrument to calculate cell viability according to this formula: Cell viability (%) = [OD (experimental group) - OD (blank)]/[OD (control group) – OD (blank)] × 100.

Evaluation of apoptosis

An Annexin V- fluorescein isothiocyanate (FITC) apoptosis detection kit (Beyotime, China) was used for apoptosis detection. The cell pellet was collected in an eppendorf (EP) tube. Annexin V-FITC binding solution (195 μL), Annexin V-FITC (5 μL), and PI (10 μL) were then added successively. After incubation for 30 min away from light, the apoptosis rate was measured using flow cytometry.

Databases

The targets of miR-96-5p and miR-181a-5p were predicted using the TargetScan8.0 database (https://www.targetscan.org/). The GeneCards database (https://www.genecards.org/) helped identify the genes associated with BPH. The GSE119195 dataset, from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/), provided a gene expression profile by analyzing healthy prostate samples from brain-dead young men and BPH tissue samples from surgery.

Western blotting

The whole cell lysate was obtained by cracking the cells in radio immunoprecipitation assay (RIPA) lysate for 30 min and centrifuging to obtain the protein supernatant. The proteins (50 μg/lane) were isolated using 10 % polyacrylamide gel and then transferred to the polyvinylidene fluoride (PVDF) membrane which was then enclosed with 5 % skim milk for 2 h at room temperature (RT). After incubation with primary antibodies of FOXP2 (1:1000, Abcam, UK) and GAPDH (1:2000, Affinity Biosciences, China) overnight at 4 °C, the PVDF membrane was incubated in the secondary antibody (1:10000, Beyotime, China) at RT for 2 h. After washing with TBST buffer, the protein signal was determined using an enhanced chemiluminescence (ECL) chemiluminescence kit (LABIO, China).

Dual luciferase assay

The luciferase vector of wild-type (wt)/mutant (mut) FOXP2 was co-transfected with the inhibitors of miR-96-5p/miR-181a-5p into cells. After 24 h, the cellular luciferase activity was measured using a Double-Luciferase Reporter Assay Kit (GENE CREATE, China).

Statistical analysis

The one-way ANOVA method was used to analyze more than two groups, while the student’s t-test compared two groups, utilizing GraphPad Prism 5 software. A p value of <0.05 was considered statistically significant.

The diagnostic value of GLMI in BPH

Because of the high correlation between abnormal glucose/lipid metabolism and BPH, we constructed a ROC curve to predict the occurrence and development of BPH based on blood GLMI, which includes FBG, HbA1c, TC, and TG. Comparing blood GLMI levels between healthy volunteers and BPH patients, we found that their combination can predict BPH occurrence, with an AUC of 0.869 (95 % confidence interval (CI): 0.820–0.917) (Figure 1A). Additionally, evaluation of IPSS against blood GLMI in BPH patients shows that GLMI is a good classifier of BPH severity, with an AUC of 0.922 (95 % CI: 0.869–0.976) (Figure 1B). These findings suggest that GLMI has potential for diagnosing BPH.

Figure 1:

The role of GLMI in diagnosing BPH. The ROC curve of GLMI in predicting the occurrence (A) and development (B) of BPH. AUC, area under the curve.

The diagnostic value of miR-96-5p/miR-181a-5p in BPH

MiR-96-5p and miR-181a-5p were found to be overexpressed in the blood of BPH patients (Figure 2A and B), and their blood level showed predictive accuracy for diagnosing BPH, with AUC of 0.845 (95 % CI: 0.791–0.899) and 0.855 (95 % CI: 0.803–0.907), respectively (Figure 2C and D). Combining these two microRNAs resulted in higher diagnostic accuracy with an AUC of 0.901 (95 % CI: 0.857–0.944) (Figure 2E). Elevated miR-96-5p/miR-181a-5p levels were observed in BPH patients with high IPSS scores (Figure 2F and G). The ROC curve analysis of IPSS and the levels of these microRNAs indicates that high miR-96-5p or miR-181a-5p level could predict the malignant progression of BPH (AUC=0.853 [95 % CI: 0.779–0.927]/0.847 [95 % CI: 0.775–0.920]) (Figure 2H and I), with the combined analysis showing even higher diagnostic accuracy, AUC of 0.927 (95 % CI: 0.882–0.972) (Figure 2J). Multivariate logistic regression analysis revealed that these two miRNAs independently raised the risk of both the onset and advancement of BPH (Tables 1 and 2). Additionally, the blood GLMI combined with miR-96-5p and miR-181a-5p provided more accurate predictions for the occurrence and progression of BPH compared to using only a single factor (AUC=0.927 [95 % CI: 0.892–0.962]/0.962 [95 % CI: 0.932–0.991]) (Figure 2K–L). Moreover, the correlation analysis shows that their blood levels were positively correlated with IPSS (Supplementary Figure 1), further demonstrating their clinical value in predicting the occurrence and development of BPH.

Figure 2:

The role of miR-96-5p/miR-181a-5p in diagnosing BPH. (A–B) Levels of miR-96-5p/miR-181a-5p in the blood samples of healthy volunteers and BPH patients. ROC curves for miR-96-5p and miR-181a-5p in predicting BPH occurrence, either (C–D) independently or (E) jointly. (F–G) Levels of miR-96-5p and miR-181a-5p in the blood of BPH patients across different IPSS groups. ROC curves for miR-96-5p and miR-181a-5p predicting BPH development, either (H–I) independently or (J) jointly. ROC curves for miR-96-5p and miR-181a-5p combined with GLMI in predicting BPH incidence (K) and progression (L). ***p<0.001.

Effects of miR-96-5p/miR-181a-5p on WPMY-1 and BPH-1 functions

MiR-96-5p and miR-181a-5p were highly expressed in BPH-1 cells (Figure 3A and B). Inhibition of miR-96-5p or miR-181a-5p significantly reduced their levels (Figure 3C and D). This reduction decreased the viability of WPMY-1 and BPH-1 cells (Figure 3E and F) and promoted cell apoptosis (Figure 3G and H), indicating that miR-96-5p/miR-181a-5p may promote BPH pathogenesis.

Figure 3:

Roles of miR-96-5p and miR-181a-5p in WPMY-1 and BPH-1 cells. (A–B) The expression of miR-96-5p and miR-181a-5p. (C–D) Working efficiency of miR-96-5p and miR-181a-5p inhibitors. Effects of miR-96-5p/miR-181a-5p on the cell viability (E–F) and apoptosis (G–H). NC, negative control. *p<0.05; **p<0.01; ***p<0.001.

Regulation of FOXP2 by miR-96-5p and miR-181a-5p

To further explore the mechanisms by which miR-96-5p and miR-181a-5p affect the functions of WPMY-1 and BPH-1 cells, we used the TargetScan8.0 database to predict their target genes. Through the creation of a Venn diagram that included the targets of miR-96-5p, miR-181a-5p, the genes associated with BPH, and the genes that are differentially expressed in BPH and normal prostate tissues (GSE119195), we identified FOXP2 as a key gene (Figure 4A). The TargetScan8.0 predictions indicated that miR-181a-5p and miR-96-5p both have binding sites in FOXP2 3′UTR (Figure 4B). Transfection with inhibitor of miR-96-5p/miR-181a-5p enhanced the cell luciferase intensity with wild-type (wt) FOXP2 vector transfection, but did not affect the mutant (mut) transfection group (Figures 4C and D). Inhibition of either miR-96-5p or miR-181a-5p significantly increased FOXP2 expression (Figure 4E–H), suggesting that these miRNAs negatively regulate FOXP2 expression by targeting its mRNA.

Figure 4:

Regulation of FOXP2 by miR-96-5p and miR-181a-5p. (A) Venn diagram showing the overlap among targets of miR-96-5p/miR-181a-5p, genes associated with BPH from the GeneCards database, and genes differentially expressed in BPH and normal prostate tissues. (B) The binding site of miR-96-5p or miR-181a-5p with FOXP2 mRNA. Effects of the miR-96-5p or miR-181a-5p inhibitor on cellular luciferase activity transfected with luciferase vector of wt-/mut-FOXP2 (C–D), and on the expression of FOXP2 (E–H). NC, negative control. ***p<0.001; ns, no significant difference.

FOXP2 inhibited the functions of miR-181a-5p and miR-96-5p

FOXP2 was found to be under-expressed in BPH-1 cells and BPH patients (Figure 5A and B). Transfection of small interfering RNA for FOXP2 (si-FOXP2) significantly reduced FOXP2 expression (Figure 5C and D). Moreover, the miR-181a-5p/miR-96-5p inhibitor decreased the viability and induced apoptosis in WPMY-1 and BPH-1 cells. This effect was reversed by co-transfection with si-FOXP2 (Figure 5E–H). These results indicate that miR-181a-5p and miR-96-5p played potential functions in BPH by regulating FOXP2.

Figure 5:

FOXP2 inhibited the functions of miR-96-5p and miR-181a-5p. (A–B) The expression of FOXP2 in WPMY-1/BPH-1 cells and BPH patients. (C–D) The working efficiency of the small interfering RNA of FOXP2. Effects of the co-transfection of miR-96-5p or miR-181a-5p inhibitor with si-FOXP2 on the viability (E–F) and apoptosis (G–H) of WPMY-1 and BPH-1 cells. NC, negative control. *p<0.05; **p<0.01; ***p<0.001.