Acoustic Enrichment of Heterogeneous Circulating Tumor Cells and Clusters from Metastatic Prostate Cancer Patients.

BACKGROUND: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on the microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. METHODS: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility), resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. RESULTS: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogeneous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding a higher number of CTCs using acoustophoresis. CONCLUSION: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Quantification of microRNA editing using two-tailed RT-qPCR for improved biomarker discovery.

Even though microRNAs have been viewed as promising biomarkers for years, their clinical implementation is still lagging far behind. This is in part due to the lack of RT-qPCR technologies that can differentiate between microRNA isoforms. For example, A-to-I editing of microRNAs through adenosine deaminase acting on RNA (ADAR) enzymes can affect their expression levels and functional roles, but editing isoform-specific assays are not commercially available. Here, we describe RT-qPCR assays that are specific for editing isoforms, using microRNA-379 (miR-379) as a model. The assays are based on two-tailed RT-qPCR, and we show them to be compatible both with SYBR Green and hydrolysis-based chemistries, as well as with both qPCR and digital PCR. The assays could readily detect different miR-379 editing isoforms in various human tissues as well as changes of editing levels in ADAR-overexpressing cell lines. We found that the miR-379 editing frequency was higher in prostate cancer samples compared to benign prostatic hyperplasia samples. Furthermore, decreased expression of unedited miR-379, but not edited miR-379, was associated with treatment resistance, metastasis, and shorter overall survival. Taken together, this study presents the first RT-qPCR assays that were demonstrated to distinguish A-to-I-edited microRNAs, and shows that they can be useful in the identification of biomarkers that previously have been masked by other isoforms.

Two-Step Acoustophoresis Separation of Live Tumor Cells from Whole Blood.

There is an unmet clinical need to extract living circulating tumor cells (CTCs) for functional studies and in vitro expansion to enable drug testing and predict responses to therapy in metastatic cancer. Here, we present a novel two-step acoustophoresis (A2) method for isolation of unfixed, viable cancer cells from red blood cell (RBC) lysed whole blood. The A2 method uses an initial acoustofluidic preseparation step to separate cells based on their acoustic mobility. This acoustofluidic step enriches viable cancer cells in a central outlet, but a significant number of white blood cells (WBCs) remain in the central outlet fraction due to overlapping acoustophysical properties of these viable cells. A subsequent purging step was employed to remove contaminating WBCs through negative selection acoustophoresis with anti-CD45-functionalized negative acoustic contrast particles. We processed 1 mL samples of 1:1 diluted RBC lysed whole blood mixed with 10 000 DU145 cells through the A2 method. Additional experiments were performed using 1000 DU145 cells spiked into 1.5 × 106 WBCs in 1 mL of buffer to further elucidate the dynamic range of the method. Using samples with 10 000 DU145 cells, we obtained 459 ± 188-fold depletion of WBC and 42% recovery of viable cancer cells. Based on spiked samples with 1000 DU145 cells, our cancer cell recovery was 28% with 247 ± 156-fold WBC depletion corresponding to a depletion efficacy of ≥99.5%. The novel A2 method provides extensive elimination of WBCs combined with the gentle recovery of viable cancer cells suitable for downstream functional analyses and in vitro culture.

Regulation of cell-cell adhesion in prostate cancer cells by microRNA-96 through upregulation of E-Cadherin and EpCAM.

Prostate cancer is one of the most common cancers in men, yet the biology behind lethal disease progression and bone metastasis is poorly understood. In this study, we found elevated levels of microRNA-96 (miR-96) in prostate cancer bone metastasis samples. To determine the molecular mechanisms by which miR-96 deregulation contributes to metastatic progression, we performed an Argonaute2-immunoprecipitation assay, in which mRNAs associated with cell-cell interaction were enriched. The expression of two cell adhesion molecules, E-Cadherin and EpCAM, was upregulated by miR-96, and potential targets sites were identified in the coding sequences of their mRNAs. We further showed that miR-96 enhanced cell-cell adhesion between prostate cancer cells as well as their ability to bind to osteoblasts. Our findings suggest that increased levels of miR-96 give prostate cancer cells an advantage at forming metastases in the bone microenvironment due to increased cell-cell interaction. We propose that miR-96 promotes bone metastasis in prostate cancer patients by facilitating the outgrowth of macroscopic tumours in the bone.

Acoustic Enrichment of Extracellular Vesicles from Biological Fluids.

Extracellular vesicles (EVs) have emerged as a rich source of biomarkers providing diagnostic and prognostic information in diseases such as cancer. Large-scale investigations into the contents of EVs in clinical cohorts are warranted, but a major obstacle is the lack of a rapid, reproducible, efficient, and low-cost methodology to enrich EVs. Here, we demonstrate the applicability of an automated acoustic-based technique to enrich EVs, termed acoustic trapping. Using this technology, we have successfully enriched EVs from cell culture conditioned media and urine and blood plasma from healthy volunteers. The acoustically trapped samples contained EVs ranging from exosomes to microvesicles in size and contained detectable levels of intravesicular microRNAs. Importantly, this method showed high reproducibility and yielded sufficient quantities of vesicles for downstream analysis. The enrichment could be obtained from a sample volume of 300 µL or less, an equivalent to 30 min of enrichment time, depending on the sensitivity of downstream analysis. Taken together, acoustic trapping provides a rapid, automated, low-volume compatible, and robust method to enrich EVs from biofluids. Thus, it may serve as a novel tool for EV enrichment from large number of samples in a clinical setting with minimum sample preparation.

Clinical-Scale Cell-Surface-Marker Independent Acoustic Microfluidic Enrichment of Tumor Cells from Blood.

Enumeration of circulating tumor cells (CTCs) predicts overall survival and treatment response in metastatic cancer, but as many commercialized assays isolate CTCs positive for epithelial cell markers alone, CTCs with little or no epithelial cell adhesion molecule (EpCAM) expression stay undetected. Therefore, CTC enrichment and isolation by label-free methods based on biophysical rather than biochemical properties could provide a more representative spectrum of CTCs. Here, we report on a clinical-scale automated acoustic microfluidic platform processing 5 mL of erythrocyte-depleted paraformaldehyde (PFA)-fixed blood (diluted 1:2) at a flow rate of 75 µL/min, recovering 43/50 (86 ± 2.3%) breast cancer cell line cells (MCF7), with 0.11% cancer cell purity and 162-fold enrichment in close to 2 h based on intrinsic biophysical cell properties. Adjustments of the voltage settings aimed at higher cancer cell purity in the central outlet provided 0.72% cancer cell purity and 1445-fold enrichment that resulted in 62 ± 8.7% cancer cell recovery. Similar rates of cancer-cell recovery, cancer-cell purity, and fold-enrichment were seen with both prostate cancer (DU145, PC3) and breast cancer (MCF7) cell line cells. We identified eosinophil granulocytes as the predominant white blood cell (WBC) contaminant (85%) in the enriched cancer-cell fraction. Processing of viable cancer cells in erythrocyte-depleted blood provided slightly reduced results as to fixed cells (77% cancer cells in the enriched cancer cell fraction, with 0.2% WBC contamination). We demonstrate feasibility of enriching either PFA-fixed or viable cancer cells with a clinical-scale acoustic microfluidic platform that can be adjusted to meet requirements for either high cancer-cell recovery or higher purity and can process 5 mL blood samples in close to 2 h.

Non-coding RNAs in Prostate Cancer: From Discovery to Clinical Applications.

Prostate cancer is a heterogeneous disease for which the molecular mechanisms are still not fully elucidated. Prostate cancer research has traditionally focused on genomic and epigenetic alterations affecting the proteome, but over the last decade non-coding RNAs, especially microRNAs, have been recognized to play a key role in prostate cancer progression. A considerable number of individual microRNAs have been found to be deregulated in prostate cancer and their biological significance elucidated in functional studies. This review will delineate the current advances regarding the involvement of microRNAs and their targets in prostate cancer biology as well as their potential usage in the clinical management of the disease. The main focus will be on microRNAs contributing to initiation and progression of prostate cancer, including androgen signalling, cellular plasticity, stem cells biology and metastatic processes. To conclude, implications on potential future microRNA-based therapeutics based on the recent advances regarding the interplay between microRNAs and their targets are discussed.

miR-145 suppress the androgen receptor in prostate cancer cells and correlates to prostate cancer prognosis.

Androgen signalling through the androgen receptor (AR) is essential for prostate cancer initiation, progression and transformation to the lethal castration-resistant state. The aim of this study was to characterize the mechanisms by which miR-145 deregulation contribute to prostate cancer progression. The miR-145 levels, measured by quantitative reverse transcription-polymerase chain reaction, were found to inversely correlate with occurrence of metastases, survival and androgen deprivation therapy response in a well-characterized prostate cancer cohort. Introduction of ectopic miR-145 in prostate cancer cells generated an inhibitory effect on the AR at both transcript and protein levels as well as its activity and downstream targets prostate-specific antigen (PSA), kallikrein-related peptidase 2 and TMPRSS2. The regulation was shown to be mediated by direct binding using Ago2-specific immunoprecipitation, but there was also indication of synergetic AR activation. These findings were verified in clinical prostate specimens by demonstrating inverse correlations between miR-145 and AR expression as well as serum PSA levels. In addition, miR-145 was found to regulate androgen-dependent cell growth in vitro. Our findings put forward novel possibilities of therapeutic intervention, as miR-145 potentially could decrease both the stem cells and the AR expressing bulk of the tumour and hence reduce the transformation to the deadly castration-resistant form of prostate cancer.

Expression of miR-34c induces G2/M cell cycle arrest in breast cancer cells.

BACKGROUND: MicroRNA-34 is a family of three miRNAs that have been reported to function as tumor suppressor miRNAs and show decreased expression in various cancers. Here, we examine functions of miR-34c in basal-like breast cancer cells. METHODS: Data from The Cancer Genome Atlas (TCGA) were used for evaluation of expression in primary breast cancers. Cellular processes affected by miR-34c were investigated by thymidine incorporation, Annexin V-assays and cell cycle analysis using breast cancer cell lines. Effects on potential targets were analyzed with qPCR and Western blot. RESULTS: TCGA data revealed that miR-34c was expressed at lower levels in basal-like breast cancer tumors and low expression was associated with poor prognosis. Ectopic expression of miR-34c in basal-like breast cancer cell lines resulted in suppressed proliferation and increased cell death. Additionally, miR-34c influenced the cell cycle mainly by inducing an arrest in the G2/M phase. We found that expression levels of the known cell cycle-regulating miR-34 targets CCND1, CDK4 and CDK6, were downregulated upon miR-34c expression in breast cancer cell lines. In addition, the levels of CDC23, an important mediator in mitotic progression, were suppressed following miR-34c expression, and siRNAs targeting CDC23 mimicked the effect of miR-34c on G2/M arrest. However, protein levels of PRKCA, a predicted miR-34c target and a known regulator of breast cancer cell proliferation were not influenced by miR-34c. CONCLUSIONS: Together, our results support the role of miR-34c as a tumor suppressor miRNA also in breast cancer.

miQ--a novel microRNA based diagnostic and prognostic tool for prostate cancer.

Today, the majority of prostate tumors are detected at early stages with uncertain prognosis. Therefore, we set out to identify early predictive markers of prostate cancer with aggressive progression characteristics. We measured the expression of microRNAs (miRNA) using qRT-PCR in formalin fixed and paraffin embedded prostatic tissue samples from a Swedish cohort of 49 patients with prostate cancer and 25 without cancer and found seven of 13 preselected miRNAs to discriminate between the two groups. Subsequently, four discriminatory miRNAs were combined to a quota, denoted the miRNA index quote (miQ); ((miR-96-5p × miR-183-5p)/(miR-145-5p × miR221-5p)). The advantage of using a quote is increased discrimination, no need for house-keepings, and most important it may be an advantage considering the heterogeneity of the disease. miQ was found to successfully predict diagnosis (p < 0.0001) with high accuracy (area under the curve, AUC = 0.931) that was verified in an independent Dutch cohort and three external cohorts, and significantly outperforming prostate-specific antigen. Importantly, miQ also has prognostic power to predict aggressiveness of tumors (AUC = 0.895), metastatic statues (AUC = 0.827) and overall survival (p = 0.0013, Wilcoxon test HR = 6.5, median survival 2 vs. 5 years), verified in the Dutch cohort. In this preliminary study, we propose that miQ has potential to be used as a clinical tool for prostate cancer diagnosis and as a prognostic marker of disease progression.

Two-Tailed RT-qPCR for the Quantification of A-to-I-Edited microRNA Isoforms.

MicroRNAs are short non-coding RNAs with important functions in the regulation of gene expression in healthy and diseased tissues. To optimally utilize the biological and clinical information that is contained in microRNA expression levels, tools for their accurate and cost-effective quantification are needed. While the standard method, qPCR, allows for quick and cheap microRNA quantification, specificity is limited due to the short lengths of microRNAs and the high similarity between closely related microRNA family members. A-to-I editing can further diversify the microRNA pool by altering individual nucleotides. There is currently a lack of protocols for the accurate quantification of A-to-I-edited microRNA isoforms using qPCR. Here, we describe a protocol to quantify microRNA editing isoforms using two-tailed RT-qPCR, with either SYBR Green or hydrolysis probes. The user will perform reverse transcription of RNA samples, generate standard curves, and quantify the resulting cDNA in the following qPCR step. We also give guidelines for primer design and for the evaluation of assays using synthetic oligonucleotides. These tools are expected to be transferable to any A-to-I-edited microRNA and its isoforms. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Two-tailed reverse transcription of A-to-I-edited microRNAs Basic Protocol 2: SYBR Green-based qPCR for A-to-I-edited microRNAs Alternate Protocol: Hydrolysis probe-based qPCR for A-to-I-edited microRNAs Support Protocol: Preparation of standard curves using synthetic RNA oligonucleotides.

Functional consequences of A-to-I editing of miR-379 in prostate cancer cells.

Prostate cancer is the predominant cause of cancer in men, but there is still a lack of biomarkers and treatments for metastatic spread. The initial promise of microRNAs to provide avenues to solve these problems has been dampened by the realisation that microRNAs co-exist in multiple functionally distinct isoforms, for example due to A-to-I editing. We recently found that A-to-I-editing of microRNA-379 (miR-379) was associated with prostate cancer, and that only the unedited isoform was negatively correlated with aggressive disease. Here, we set out to decipher the biological effects of unedited and edited miR-379 in prostate cancer cells. After transfection of four different prostate cancer cell lines with isoform-specific miR-379 mimics, we performed assays for cell growth, colony formation, migration, cell-cell adhesion, and analysed epithelial-mesenchymal transition (EMT) and stemness markers. We found that unedited miR-379 affected cell growth, with a promoting function in androgen receptor (AR)-negative cells and an inhibiting effect in AR-positive cells. This is supported by our in silico analysis that found unedited miR-379 targets are predicted to be predominantly involved in cellular proliferation whereas the targets of edited miR-379 are not. We further found that both miR-379 isoforms could promote colony formation, migration, and cell-cell adhesion. Overall, our data suggests that editing of miR-379 attenuates the growth-suppressive function of unedited miR-379 in androgen-sensitive prostate cancer cells, thereby promoting tumor growth.

Expression of microRNA-379 reduces metastatic spread of prostate cancer.

Introduction: Prostate cancer (PCa) is the most common type of cancer in males, and the metastatic form is a leading cause of death worldwide. There are currently no curative treatments for this subset of patients. To decrease the mortality of this disease, greater focus must be placed on developing therapeutics to reduce metastatic spread. We focus on dissemination to the bone since this is both the most common site of metastatic spread and associated with extreme pain and discomfort for patients. Our strategy is to exploit microRNAs (miRNAs) to disrupt the spread of primary PCa to the bone. Methods: PCa cell lines were transduced to overexpress microRNA-379 (miR-379). These transduced PCa cells were assessed using cell growth, migration, colony formation and adhesion assays. We also performed in vivo intracardiac injections to look at metastatic spread in NSG mice. A cytokine array was also performed to identify targets of miR-379 that may drive metastatic spread. Results: PCa cells with increased levels of miR-379 showed a significant decrease in proliferation, migration, colony formation, and adhesion to bone cells in vitro. In vivo miR-379 overexpression in PC3 cells significantly decreased metastatic spread to bone and reduced levels of miR-379 were seen in patients with metastases. We identified GDF-15 to be secreted from osteoblasts when grown in conditioned media from PCa cells with reduced miR-379 levels. Discussion: Taken together, our in vitro and in vivo functional assays support a role for miR-379 as a tumour suppressor. A potential mechanism is unravelled whereby miR-379 deregulation in PCa cells affects the secretion of GDF-15 from osteoblasts which in turn facilitates the metastatic establishment in bone. Our findings support the potential role of miR-379 as a therapeutic solution for prostate cancer.

Functional In Vivo Screening Identifies microRNAs Regulating Metastatic Dissemination of Prostate Cancer Cells to Bone Marrow.

Distant metastasis is the major cause of cancer-related deaths in men with prostate cancer (PCa). An in vivo functional screen was used to identify microRNAs (miRNAs) regulating metastatic dissemination of PCa cells. PC3 cells transduced with pooled miRZiP™ lentivirus library (anti-miRNAs) were injected intraprostatic to 13 NSG mice followed by targeted barcode/anti-miR sequencing. PCa cells in the primary tumours showed a homogenous pattern of anti-miRNAs, but different anti-miRNAs were enriched in liver, lung, and bone marrow, with anti-miR-379 highly enriched in the latter. The bone metastasis-promoting phenotype induced by decreased miR-379 levels was also confirmed in a less metastatic PCa cell line, 22Rv1, where all mice injected intracardially with anti-miR-379-22Rv1 cells developed bone metastases. The levels of miR-379 were found to be lower in bone metastases compared to primary tumours and non-cancerous prostatic tissue in a patient cohort. In vitro functional studies suggested that the mechanism of action was that reduced levels of miR-379 gave an increased colony formation capacity in conditions mimicking the bone microenvironment. In conclusion, our data suggest that specific miRNAs affect the establishment of primary tumours and metastatic dissemination, with a loss of miR-379 promoting metastases in bone.

Acoustic enrichment of heterogenous circulating tumor cells and clusters from patients with metastatic prostate cancer.

Background: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. Methods: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility) resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. Results: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogenous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC-clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding higher number of CTCs using acoustophoresis. Conclusion: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC-clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables sensitive label-free enrichment of cells with epithelial phenotype in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

High-Throughput and Automated Acoustic Trapping of Extracellular Vesicles to Identify microRNAs With Diagnostic Potential for Prostate Cancer.

Molecular profiling of extracellular vesicles (EVs) offers novel opportunities for diagnostic applications, but the current major obstacle for clinical translation is the lack of efficient, robust, and reproducible isolation methods. To bridge that gap, we developed a microfluidic, non-contact, and low-input volume compatible acoustic trapping technology for EV isolation that enabled downstream small RNA sequencing. In the current study, we have further automated the acoustic microfluidics-based EV enrichment technique that enables us to serially process 32 clinical samples per run. We utilized the system to enrich EVs from urine collected as the first morning void from 207 men referred to 10-core prostate biopsy performed the same day. Using automated acoustic trapping, we successfully enriched EVs from 199/207 samples (96%). After RNA extraction, size selection, and library preparation, a total of 173/199 samples (87%) provided sufficient materials for next-generation sequencing that generated an average of 2 × 106 reads per sample mapping to the human reference genome. The predominant RNA species identified were fragments of long RNAs such as protein coding and retained introns, whereas small RNAs such as microRNAs (miRNA) accounted for less than 1% of the reads suggesting that partially degraded long RNAs out-competed miRNAs during sequencing. We found that the expression of six miRNAs was significantly different (Padj < 0.05) in EVs isolated from patients found to have high grade prostate cancer [ISUP 2005 Grade Group (GG) 4 or higher] compared to those with GG3 or lower, including those with no evidence of prostate cancer at biopsy. These included miR-23b-3p, miR-27a-3p, and miR-27b-3p showing higher expression in patients with GG4 or high grade prostate cancer, whereas miR-1-3p, miR-10a-5p, and miR-423-3p had lower expression in the GG4 PCa cases. Cross referencing our differentially expressed miRNAs to two large prostate cancer datasets revealed that the putative tumor suppressors miR-1, miR-23b, and miR-27a are consistently deregulated in prostate cancer. Taken together, this is the first time that our automated microfluidic EV enrichment technique has been found to be capable of enriching EVs on a large scale from 900 µl of urine for small RNA sequencing in a robust and disease discriminatory manner.

miR-34c is downregulated in prostate cancer and exerts tumor suppressive functions.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression. There have been several reports of miRNA deregulation in prostate cancer (PCa) and the biological evidence for an involvement of miRNAs in prostate tumorigenesis is increasing. In this study, we show that miR-34c is downregulated in PCa (p = 0.0005) by performing qRT-PCR on 49 TURPs from PCa patients compared to 25 from patients with benign prostatic hyperplasia. The miR-34c expression was found to inversely correlate to aggressiveness of the tumor, WHO grade, PSA levels and occurrence of metastases. Furthermore, a Kaplan-Meier analysis of patient survival based on miR-34c expression levels divided into low (< 50th percentile) and high (> 50th percentile) expression, significantly divides the patients into high risk and low risk patients (p = 0.0003, log-rank test). The phenotypic effects of miR-34c deregulation were studied in prostate cell lines, where ectopic expression of miR-34c decreased cell growth, due to both a decrease in cellular proliferation rate and an increase in apoptosis. In concordance to this, miR-34c was found to negatively regulate the oncogenes E2F3 and BCL-2, which stimulates proliferation and suppress apoptosis in PCa cells, respectively. Reversely, we could also show that blocking miR-34c in vitro increases cell growth. Further, ectopic expression of miR-34c was found to suppress migration and invasion. Our findings provide new insight into the role of miR-34c in the prostate, exhibiting tumor suppressing effects on proliferation, apoptosis and invasiveness.

Rapidly evolving marmoset MSMB genes are differently expressed in the male genital tract.

BACKGROUND: Beta-microseminoprotein, an abundant component in prostatic fluid, is encoded by the potential tumor suppressor gene MSMB. Some New World monkeys carry several copies of this gene, in contrast to most mammals, including humans, which have one only. Here we have investigated the background for the species difference by analyzing the chromosomal organization and expression of MSMB in the common marmoset (Callithrix jacchus). METHODS: Genes were identified in the Callithrix jacchus genome database using bioinformatics and transcripts were analyzed by RT-PCR and quantified by real time PCR in the presence of SYBR green. RESULTS: The common marmoset has five MSMB: one processed pseudogene and four functional genes. The latter encompass homologous genomic regions of 32-35 kb, containing the genes of 12-14 kb and conserved upstream and downstream regions of 14-19 kb and 3-4 kb. One gene, MSMB1, occupies the same position on the chromosome as the single human gene. On the same chromosome, but several Mb away, is another MSMB locus situated with MSMB2, MSMB3 and MSMB4 arranged in tandem. Measurements of transcripts demonstrated that all functional genes are expressed in the male genital tract, generating very high transcript levels in the prostate. The transcript levels in seminal vesicles and testis are two and four orders of magnitude lower. A single gene, MSMB3, accounts for more than 90% of MSMB transcripts in both the prostate and the seminal vesicles, whereas in the testis around half of the transcripts originate from MSMB2. These genes display rapid evolution with a skewed distribution of mutated nucleotides; in MSMB2 they affect nucleotides encoding the N-terminal Greek key domain, whereas in MSMB3 it is the C-terminal MSMB-unique domain that is affected. CONCLUSION: Callitrichide monkeys have four functional MSMB that are all expressed in the male genital tract, but the product from one gene, MSMB3, will predominate in seminal plasma. This gene and MSMB2, the predominating testicular gene, have accumulated mutations that affect different parts of the translation products, suggesting an ongoing molecular specialization that presumably yields functional differences in accessory sex glands and testis.

Correction: A urinary extracellular vesicle microRNA biomarker discovery pipeline; from automated extracellular vesicle enrichment by acoustic trapping to microRNA sequencing.

[This corrects the article DOI: 10.1371/journal.pone.0217507.].

A urinary extracellular vesicle microRNA biomarker discovery pipeline; from automated extracellular vesicle enrichment by acoustic trapping to microRNA sequencing.

Development of a robust automated platform for enrichment of extracellular vesicles from low sample volume that matches the needs for next-generation sequencing could remove major hurdles for genomic biomarker discovery. Here, we document a protocol for urinary EVs enrichment by utilizing an automated microfluidic system, termed acoustic trap, followed by next-generation sequencing of microRNAs (miRNAs) for biomarker discovery. Specifically, we compared the sequencing output from two small RNA library preparations, NEXTFlex and CATS, using only 130 pg of input total RNA. The samples prepared using NEXTflex was found to contain larger number of unique miRNAs that was the predominant RNA species whereas rRNA was the dominant RNA species in CATS prepared samples. A strong correlation was found between the miRNA expressions of the acoustic trap technical replicate in the NEXTFlex prepared samples, as well as between the acoustic trap and ultracentrifugation enrichment methods. Together, these results demonstrate a robust and automated strategy for biomarker discovery from small volumes of urine.