Spin Column-based Method Research Articles

MP04-03 EXOSOMAL LNCRNA BCYRN1 CAN BE A TARGET FOR DIAGNOSIS AND THERAPY IN RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023MP04-03 EXOSOMAL LNCRNA BCYRN1 CAN BE A TARGET FOR DIAGNOSIS AND THERAPY IN RENAL CELL CARCINOMA Hirofumi Yoshino, Shuichi Tatarano, and Hideki Enokida Hirofumi YoshinoHirofumi Yoshino More articles by this author , Shuichi TataranoShuichi Tatarano More articles by this author , and Hideki EnokidaHideki Enokida More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Exosomes are 40-100 nm nano-sized extracellular vesicles, released from many cell types into the extracellular space and transfer their component to recipient cells. We recently reported the method to collect exosomes from cell lysates and human serum by a spin column-based method in in a relatively short time, and the clinical significance that MYO15A was significantly upregulated in exosomes from renal cell carcinoma (RCC) patient’s sera compared with normal controls. In this study, we focused on one of long non-coding RNAs, BCYRN1, in RCCs. METHODS: Bioinformatic analysis was performed about clinical significance of BCYRN1 by using the database of The Cancer Genome Atlas (TCGA). Then, we performed RT-qPCR analysis of exosomes from clinical patients’ sera. Loss of function assays were performed in RCC cells (786-O, A498) on cell proliferation, cell migration and cell invasion in vitro and in vivo. RESULTS: The analyses with TCGA database indicated that high expression of BCYRN1 was significantly associated with a poorer outcome in RCC. RT-qPCR analysis of exosomes from clinical patients’ sera showed that BCYRN1 was significantly upregulated (p=0.0134) in RCC patients (n=35) compared to that in healthy controls (n=19). Moreover, BCYRN1 expression in patients with ≥T2a (n=9) was significantly higher (p=0.0404) than that in ≤T1b (n=25). Loss-of-function study by using si-RNAs of BCYRN1 suppressed proliferative, migratory, and invasive activities in RCC cells. In addition, BCYRN1 knockdown inhibited tumor growth in xenograft assay. CONCLUSIONS: It was suggested that exosomal BCYRN1 can be a novel diagnosis marker, and that BCYRN1 can be a new therapeutic target in RCCs. The identification of novel target for diagnosis and therapy may lead to a better understanding of RCC and the development of new therapeutic strategies to treat this disease. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e34 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hirofumi Yoshino More articles by this author Shuichi Tatarano More articles by this author Hideki Enokida More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract 13114: Micro RNA206 Secreted From Growing Skeletal Muscle Promotes Angiogenic Response in Endothelial Cells

Introduction: In addition to aerobic exercise, it has been recognized that resistance exercise has beneficial effects for the patients with peripheral arterial disease. Recently, muscle-derived exosomes are demonstrated to convey many types of signaling molecules including microRNAs (miRNAs). Hypothesis: We assessed the hypothesis that exosomal miRNA secreted by growing muscle promotes angiogenic response in endothelial cells. Methods: We utilized skeletal muscle-specific inducible Akt1 transgenic (Akt1-TG) mice that can induce growth of skeletal muscles without exercise training. Exosomes was purified combined usage of ultracentrifugation, and miRNAs were extracted from purified exosomes by spin column-based method. Real-time PCR array and droplet digital PCR was performed to evaluate exosomal miRNA expression. Results: Akt1-TG mice showed remarkable skeletal muscle growth 2 weeks after gene activation (weight of gastrocnemius muscle: 0.21±0.03 vs 0.16±0.02, g, p<0.01). The amount of exosomal proteins did not different between Akt1-TG mice and WT mice (977.7±11.3 vs 826.0±45.2, μg/mL serum, p=0.07). KEGG pathway frequency analysis for 4665 target genes identified by real-time PCR array of miRNAs revealed a significant increase in Akt and its downstream signaling pathways. Among upregulated miRNAs, droplet digital PCR identified that miR-1, -133 and -206 expression was significantly upregulated in Akt1 TG mice compared with WT mice in serum. miR206 was increased in IGF-1-stimulated myotubes. PKH26-labeled exosomes were taken up by human umbilical vein endothelial cells. Exogenous supplementation of exosomal miRNA206 promoted angiogenesis as revealed by spheroid assay, and increased expression of HIF-1 and VEGF in endometrial cells. Conclusions: Exosomal miR206 was upregulated in blood stream in Akt1-TG mice and IGF-stimulated cultured myotubes. Exogenous supplementation of miR206 promoted angiogenic response in endothelial cells. Our data suggest that miR206 secreted from growing muscle act on endothelial cells and promote angiogenesis.

Exosomal microRNA-1 and MYO15A as a target for therapy and diagnosis in renal cell carcinoma

Exosomes are 40–100 nm nano-sized extracellular vesicles and are receiving increasing attention as novel structures that participate in intracellular communication. We previously found that miRNA-1 (miR-1) functions as a tumor suppressor in renal cell carcinoma (RCC). In this study, we investigated the function of exosomal miR-1 and the possibility that the exosome constitutes a tumor maker in RCC. First, we established the method to collect exosomes from cell lysates and human serum by a spin column-based method. Next, we assessed exosomes using Nanosight nanoparticle tracking analysis and Western blot analysis with exosome marker CD63. We confirmed that exosomes labeled with PKH26 fused with recipient cells. Moreover, miR-1 expression was elevated in RCC cells treated with exosomes derived from miR-1-transfected cells. Functional analyses showed that exosomal miR-1 significantly inhibited cell proliferation, migration and invasion compared to control treatment. Our analyses with TCGA database of RCCs showed that miR-1 expression was significantly downregulated in clinical RCC samples compared to that in normal kidney samples, and patients with low miR-1 expression had poorer overall survival in comparison to patients with high expression. Furthermore, RNA sequence analyses showed that expression levels of several genes were altered by exposure to exosomal miR-1. The analyses with TCGA database indicated that high expression of MYO15A was associated with a poorer outcome in RCC. In addition, RT-qPCR analysis of exosomes from clinical patients’ sera showed that MYO15A was significantly upregulated in RCC patients compared to that in healthy controls. This study showed that treatment with exosomal miR-1 might be an effective approach to treating RCCs. In addition, exosomal MYO15A could be a diagnostic tumor marker in RCCs.

MP39-13 FUNCTION OF EXOSOMAL ANTITUMOR MICRORNA-1 IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (MP39)1 Sep 2021MP39-13 FUNCTION OF EXOSOMAL ANTITUMOR MICRORNA-1 IN RENAL CELL CARCINOMA Issei Kawakami, Hirofumi Yosino, Hideki Enokida, and Masayuki Nakagawa Issei KawakamiIssei Kawakami More articles by this author , Hirofumi YosinoHirofumi Yosino More articles by this author , Hideki EnokidaHideki Enokida More articles by this author , and Masayuki NakagawaMasayuki Nakagawa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002054.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: MicroRNAs, a class of small non-coding RNAs about 22 nucleotides in length, are involved in many cellular processes and aberrantly expressed in cancer tissues. For example, miR-1 functions as a tumor suppressor in renal cell carcinoma (RCC) as demonstrated in our previous study. Exosomes are 40–100 nm nano-sized extracellular vesicles, and are receiving increasing attention as novel structures participating in intracellular communication. We hypothesized that miR-1 functions as a tumor suppressor through exosomes in RCC, and investigated that in this study. METHODS: First, we performed statistical analyses of miR-1 based on the TCGA database of clear cell RCC (ccRCC) patients. We also checked miR-1 expression in serum exosomes between healthy controls and RCCs. Exosomes in cell lysate and human serum were isolated by a spin column-based method, and determined using Nanosight nanoparticle tracking analysis and western blot analysis with exosome marker CD63. Exosomes labeled with PKH26 were used to indicate that exosomes were fused into recipient cells, and miR-1 expressions were investigated by RT-PCR in recipient RCC cells with the addition of miR-1 transfected cell derived exosomes. By using the recipient RCC cells, we performed functional analyses such as proliferation, migration and invasion. We also performed functional analyses by using serum exosomes derived from RCC patients with IVC invasion or metastasis. In addition, we performed RNA sequence analyses to identify the mechanism in RCC cell with the addition of exosomal miR-1. RESULTS: MiR-1 expression was significantly downregulated in clinical ccRCC samples compared to that in normal kidney samples (p<0.001), and patients with low miR-1 expression (n=455) had poor overall survival (p=0.0447) in comparison to patients with high expression (n=50). Exosomes in cell lysate and human serum were detected by Nanosight and western blot analysis with CD63. Exosomes labeled with PKH26 were detected in the recipient cells, and miR-1 expressions were elevated nearly 40 and 10 times in exosomal miR-1 fused 786o and A498 cells respectively compared to in exosomal miR-control fused cells. Exosomal miR-1 also significantly inhibited cell proliferation, migration and invasion compared to exosomal miR-control. Furthermore, RNA sequence analyses showed that several genes and pathways might be regulated by exosomal miR-1. CONCLUSIONS: This study showed that exosomal miR-1 might exogenously function as a therapeutic target in the treatment of RCCs. In addition, exosomal miR-1 and/or other miRNAs could be a diagnostic tumor marker of RCCs. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e709-e709 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Issei Kawakami More articles by this author Hirofumi Yosino More articles by this author Hideki Enokida More articles by this author Masayuki Nakagawa More articles by this author Expand All Advertisement Loading ...

Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater

The primary concentration and molecular process are critical to implement wastewater-based epidemiology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the previously developed methods were optimized for nonenveloped viruses. Few studies evaluated if the methods are applicable to the efficient recovery of enveloped viruses from various types of raw sewage. This study aims (1) to compare the whole process recovery of Pseudomonas phage φ6, a surrogate for enveloped viruses, among combinations of primary concentration [ultrafiltration (UF), electronegative membrane vortex (EMV), and polyethylene glycol precipitation (PEG)] and RNA extraction methods (spin column-based method using QIAamp Viral RNA Mini Kit and acid guanidinium thiocyanate–phenol–chloroform extraction using TRIzol reagent) for three types of raw sewage and (2) to test the applicability of the method providing the highest φ6 recovery to the detection of SARS-CoV-2 RNA. Among the tested combinations, PEG+TRIzol provided the highest φ6 recovery ratio of 29.8% to 49.8% (geometric mean). UF + QIAamp Viral RNA Mini Kit provided the second highest φ6 recovery of 6.4% to 35.8%. The comparable φ6 recovery was observed for UF + TRIzol (13.8–30.0%). PEG + QIAamp Viral RNA Mini Kit provided only 1.4% to 3.0% of φ6 recovery, while coliphage MS2, a surrogate for nonenveloped viruses, was recovered comparably with PEG + TRIzol. This indicated that the nonenveloped surrogate (MS2) did not necessarily validate the efficient recovery for enveloped viruses. EMV + QIAamp Viral RNA Mini Kit provided significantly different φ6 recovery (1.6–21%) among the types of raw sewage. Then, the applicability of modified PEG + TRIzol was examined for the raw sewage collected in Tokyo, Japan. Of the 12 grab samples, 4 were positive for SARS-CoV-2 CDC N1 and N3 assay. Consequently, PEG + TRIzol provided the highest φ6 recovery and allowed for the detection of SARS-CoV-2 RNA from raw sewage.

Rapid and economical protocols for genomic and metagenomic DNA extraction from oak (Quercus brantii Lindl.)

Two new efficient, fast and low-cost metagenomic DNA extraction methods were developed for different Persian oak tissues (leaf, stem, root, and rhizospheric) and soil samples. The new “omics” studies on the genus Quercus are of importance to help finding efficient strategies for overcoming environmental challenges, and to do this, presence of efficient DNA extraction protocols for different Quercus species are very critical. The objective of the present study was to develop new efficient methods for extraction of metagenomic DNA (mDNA) from of Persian oak (Quercus brantii Lindl.) tissues. The efficiency of two newly developed mDNA extraction methods, including indirect SDS-based (ISB or concentrate method) and one spin column-based method (SCB) were compared to that of two classical direct methods, including CTAB-based and SDS-based methods, and two commercial mDNA extraction kits. The maximum average yield of mDNA for all samples (leaf, stem, root, bulk, and rhizospheric soils) was obtained by SCB (258 ng/μl) and ISB (189 ng/μl) methods, respectively. Successful PCR amplification for 16SrRNA and ITS sequence was consistently observed for ISB, SCB, and kit-extracted mDNAs, which confirmed the high purity of mDNA extracted by these methods. The new methods showed more than 96% quantitative PCR efficiency, and partial restriction digestion and metagenomic library construction confirmed the high efficiency of the newly developed methods. It could be concluded that two new protocols enhanced efficiency (yield, purity, and cost) of mDNA extraction from different tissues of Persian oak.

A rapid silica spin column-based method of RNA extraction from fruit trees for RT-PCR detection of viruses

Efficient recovery of high quality RNA is very important for successful RT-PCR detection of plant RNA viruses. High levels of polyphenols and polysaccharides in plant tissues can irreversibly bind to and/or co-precipitate with RNA, which influences RNA isolation. In this study, a silica spin column-based RNA isolation method was developed by using commercially available silica columns combined with the application of a tissue lysis solution, and binding and washing buffers with high concentration guanidinium thiocyanate (GuSCN, 50% w/v), which helps remove plant proteins, polysaccharides and polyphenolic compounds. The method was successfully used to extract high quality RNA from citrus (Citrus aurantifolia), grapevine (Vitis vinifera), peach (Prunus persica), pear (Pyrus spp.), taro (Colocosia esculenta) and tobacco (Nicotiana benthamiana) samples. The method was comparable to conventional CTAB method in RNA isolation efficiency, but it was more sample-adaptable and cost-effective than commercial kits. High quality RNA isolated using silica spin column-based method was successfully used for the RT-PCR and/or multiplex RT-PCR amplification of woody fruit tree viruses and a viroid. The study provided a useful tool for the detection and characterization of plant viruses.

A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing.

When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich for pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000–3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs.

PWE-108 Assessment of the Mucosal Microbiota in Inflammatory Bowel Disease

IntroductionGreat progress has been made in understanding the pathophysiology of Crohn’s disease (CD) and ulcerative colitis (UC), and it is now widely believed that dysregulation of the relationship between the...

Evaluation of Cell-free DNA in Urine as a Marker for Bladder Cancer Diagnosis

The diagnosis and follow-up of bladder cancer are mainly based on cystoscopy, an invasive method which could be negative in case of flat malignancies such as carcinoma in situ. Other noninvasive diagnostic methods have not yet given satisfactory results. There is a need for a reliable yet noninvasive method for the detection of bladder cancer. Our aim was to investigate whether cell-free DNA quantified in urine (ucf-DNA) could be a useful marker for the diagnosis of bladder cancer. A standard urine test was performed in 150 naturally voided morning urine samples that were processed to obtain a quantitative evaluation of ucf-DNA. Leukocyturia and/or bacteriuria were found in 18 subjects, who were excluded from the study. Statistical analysis was performed on 45 bladder cancer patients and 87 healthy subjects. Ucf-DNA was extracted from urine samples by a spin column-based method and quantified using four different methods: GeneQuant Pro (Amersham Biosciences, Pittsburg, PA, USA), Quant-iT DNA high-sensitivity assay kit (Invitrogen, Carlsbad, CA, USA), Real-Time PCR (Applied Biosystems, Foster City, CA, USA), and NanoDrop 1000 (NanoDrop Technologies, Houston, TX, USA). Median free DNA quantification did not differ statistically between bladder cancer patients and healthy subjects. A receiver-operating characteristic (ROC) curve was developed to evaluate the diagnostic performance of ucf-DNA quantification for each method. The area under the ROC curve was 0.578 for GeneQuant Pro, 0.573 for the Quant-iT DNA high-sensitivity assay kit, 0.507 for Real-Time PCR, and 0.551 for NanoDrop 1000, which indicated that ucf-DNA quantification by these methods is not able to discriminate between the presence and absence of bladder cancer. No association was found between ucf-DNA quantification and tumor size or tumor focality. In conclusion, ucf-DNA isolated by a spin column-based method and quantified by GeneQuant Pro, Quant-iT DNA high-sensitivity assay kit, Real-Time PCR or NanoDrop 1000 does not seem to be a reliable marker for the diagnosis of bladder cancer.

Evaluation of cell-free DNA in urine as a marker for bladder cancer diagnosis

The diagnosis and follow-up of bladder cancer are mainly based on cystoscopy, an invasive method which could be negative in case of flat malignancies such as carcinoma in situ. Other noninvasive diagnostic methods have not yet given satisfactory results. There is a need for a reliable yet noninvasive method for the detection of bladder cancer. Our aim was to investigate whether cell-free DNA quantified in urine (ucf-DNA) could be a useful marker for the diagnosis of bladder cancer. A standard urine test was performed in 150 naturally voided morning urine samples that were processed to obtain a quantitative evaluation of ucf-DNA. Leukocyturia and/or bacteriuria were found in 18 subjects, who were excluded from the study. Statistical analysis was performed on 45 bladder cancer patients and 87 healthy subjects. Ucf-DNA was extracted from urine samples by a spin column-based method and quantified using four different methods: GeneQuant Pro (Amersham Biosciences, Pittsburg, PA, USA), Quant-iT DNA high-sensitivity assay kit (Invitrogen, Carlsbad, CA, USA), Real-Time PCR (Applied Biosystems, Foster City, CA, USA), and NanoDrop 1000 (NanoDrop Technologies, Houston, TX, USA). Median free DNA quantification did not differ statistically between bladder cancer patients and healthy subjects. A receiver-operating characteristic (ROC) curve was developed to evaluate the diagnostic performance of ucf-DNA quantification for each method. The area under the ROC curve was 0.578 for GeneQuant Pro, 0.573 for the Quant-iT DNA high-sensitivity assay kit, 0.507 for Real-Time PCR, and 0.551 for NanoDrop 1000, which indicated that ucf-DNA quantification by these methods is not able to discriminate between the presence and absence of bladder cancer. No association was found between ucf-DNA quantification and tumor size or tumor focality. In conclusion, ucf-DNA isolated by a spin column-based method and quantified by GeneQuant Pro, Quant-iT DNA high-sensitivity assay kit, Real-Time PCR or NanoDrop 1000 does not seem to be a reliable marker for the diagnosis of bladder cancer.

Detection of cows' milk in goats' cheeses inferred from mitochondrial DNA polymorphism.

A new polymerase chain reaction (PCR)-based method was developed to detect cows milk in goat cheese. This method is based on mitochondrial DNA (mtDNA) control region sequence variations. DNA extractions from 150 mg of cheese were carried out using a spin column-based method. Subsequent PCR amplifications of DNA were performed with cow specific primers, demonstrating the ability to detect cows' milk in a variety of cheeses. This simple approach provides high quality DNA, and is shown to be very sensitive, with a detection limit of less than 0.1% of cows' milk. Analysis of an agarose gel digital image allows a rough estimation of the percentage of cows' milk used in adulteration.