Danilo Zampini

Circulating microRNAs and kallikreins before and after radical prostatectomy: are they really prostate cancer markers?

The aim of our study was to monitor serum levels of two miRNAs (miR-21 and miR-141) and three KLKs (hK3/PSA, hK11, and hK13) before and 1, 5, and 30 days after radical prostatectomy, in order to characterize their fluctuations after surgery. 38 patients with prostate cancer were included. miR-21 and miR-141 were quantified through real-time PCR, while ELISA assays were used to quantify hK3 (PSA), hK11, and hK13. Both miR-21 and miR-141 showed a significant increase at the 5th postoperative day, after which a gradual return to the preoperative levels was recorded. These findings suggest that miR-21 and miR-141 could be involved in postsurgical inflammatory processes and that radical prostatectomy does not seem to alter their circulating levels. Postoperative serum kallikreins showed a significant decrease, highlighting the potential usefulness of kallikreins apart from PSA as potential prostate cancer markers.

Stability Assessment of Candidate Reference Genes in Urine Sediment of Prostate Cancer Patients for miRNA Applications

We aimed at assessing the stability of candidate reference genes in urine sediments of men subjected to digital rectal examination for suspected prostate cancer (PCa). Two microRNAs (miR-191 and miR-25) and 1 small nucleolar RNA (SNORD48) were assayed in 35 post-DRE urine sediments of men with PCa and in 26 subjects with histologically confirmed benign prostatic hyperplasia (BPH). The stability of candidate reference genes was assessed through BestKeeper algorithm and equivalence test. miR-200b and miR-452 were used to test for the effect of normalization on target genes. Our results proved miR-191 to be the most stable gene, showing the lowest degree of variation and the highest stability value. miR-25 and SNORD48 values fell beyond the cutoff of acceptability. In conclusion, we recommend the use of miR-191 for normalization purposes in post-DRE urine sediments.

Characterization of Kallireins and microRNAs in Urine Sediment for the Discrimination of Prostate Cancer from Benign Prostatic Hyperplasia

Objectives: Prostate Cancer (PCa) and Benign Prostatic Hyperplasia (BPH) are frequently coexisting in elderly men. The measurement of serum PSA together with Digital Rectal Examination (DRE) represents the primary diagnostic tool to suspect PCa, whereas definitive diagnosis is achieved by prostate biopsy. The low specificity of PSA and the modest detection rate of biopsy convict the patient to a quite often unnecessary and uncomfortable clinical itinerary. There is a urgent need for new and more accurate methodologies to diagnosize PCa. In the present study, the expression of 4 mRNAs and 2 miRNAs was evaluated in post DRE urine cell pellets from patients suffering PCa and age-matched subjects affected by BPH with elevated PSA levels. We also evaluated the diagnostic accuracy of markers in predicting PCa. Materials and methods: The expression levels of 4 mRNAs (3 kallikreins - KLK3, KLK11, KLK13 and a prostate cancer antigen - PCA3) and 2 microRNAs (miR-9-3p and miR-19a-3p) were assayed by means of real-time PCR in post DRE urine of 79 men undergoing prostate biopsy for PSA levels > 3 ng/mL. The diagnostic power of tested markers was evaluated through logistic regression analysis. Results: PCA3 was undetectable in 22 out of 38 BPH subjects. KLK3 and KLK11 were significantly upregulated in PCa group (p value < 0.001), while miR-9-3p and miR-19a-3p were up-regulated in BPH group (p value <0.001 and < 0.01, respectively). KLK13 was not differentially expressed between groups. MiR-19a-3p and miR- 9-3p reached the highest specificity (64.29%) and sensitivity (81.08%), respectively. The more accurate bivariate logistic model was obtained combining KLK11 with either miR-9-3p and miR-19a-3p. Conclusions: Our findings demonstrated that selected kallikreins and miRNAs proved to be an accurate diagnostic tool for PCa. Urine cells pellets obtained after DRE represent a reliable biological matrix for minimally invasive gene expression assays.

Next Generation Sequencing of urine exfoliated cells: An approach of prostate cancer microRNAs research

There is emerging evidence that microRNAs (miRNAs) dysregulation is involved in the genesis and the progression of Prostate Cancer (PCa), thus potentially increasing their use in urological clinical practice. This is the first pilot study which utilizes Illumina Deep Sequencing to examine the entire miRNAs spectrum existent in urine exfoliated prostate cells (UEPCs) of PCa patients. A total of 11 male patients with histological diagnosis of PCa were enrolled in the present study. First-catch urine (30 mL) was collected following a prostate massage. Total RNA was extracted from urine and sequenced using an HiSeq2500 System (Illumina). QPCR assay was used to validate the highest NGS results in PCA patients and in age-matched, caucasian men. Remarkably, PCA let-7 family was down-regulated (P < 0.01), compared to the controls. The results of our study support the notion of a relatively high diagnostic value of miRNA family for PCa detection, especially in the let-7 family. The present research confirmed the potential use of miRNAs as non-invasive biomarkers in the diagnosis of PCa, potentially reducing the invasiveness of actual clinical strategy.

Gold nanoparticles approach to detect chondroitin sulphate and hyaluronic acid urothelial coating

This study investigated the location of hyaluronic acid (HA)- and chondroitin sulphate (CS)-coated gold nanoparticles in rabbit bladder and evaluated gene expression of CD44, RHAMM and ICAM-1 receptors involved in HA and CS transport into the cell. Gold nanoparticles were synthesised by reduction of gold salts with HA or CS to form HA-AuNPs and CS-AuNPs. Bladder samples were incubated with CS-AuNPs and HA-AuNPs or without glycosaminoglycans. Transmission electron microscopy, optic microscopy and scanning electron microscopy were used to determine the location of the synthesised AuNPs. Real-time PCR was used to analyse expression of urothelial cell receptors CD44, RHAMM, ICAM-1, after ex vivo administration of CS-AuNPs and HA-AuNPs. We showed that HA-AuNPs and CS-AuNPs were located in the cytoplasm and tight junctions of urothelial umbrella cells; this appearance was absent in untreated bladders. There were no significant differences in gene expression levels for CD44, RHAMM and ICAM-1 receptors in treated versus control bladder tissues. In conclusion, we clearly showed the presence of exogenous GAGs in the bladder surface and the tight junctions between umbrella cells, which is important in the regeneration pathway of the urothelium. The GAGs-AuNPs offer a promising approach to understanding the biophysical properties and imaging of urothelial tissue.