RESUMO
Convenient, ultrasensitive, and accurate detection of rare variants is essential for early cancer diagnosis and precision medicine, however, despite years of efforts, tools that have all these qualities remain elusive. Here, we developed a one-step CRISPR/Cas12a-based digital diagnostic platform for accurately quantifying mutant alleles, referred to as the CRISPR ASsoaciated Mutation Allele Rapid Test (CASMART). The platform accurately quantifies the variant allele frequency of EGFR L858R within 1 h at 42 °C and can detect mutant targets as low as 0.3 copies/µL (0.498 aM) in mock multiplex cfDNA samples. We further investigated the applicability of CASMART using human genomic samples with confirmed EGFR L858R mutations previously measured variant allele frequency by next-generation sequencing. Comparison across platforms revealed equivalent detection performance (Pearson's correlation coefficient, R2 = 0.9208) and high quantification accuracy for mutation allele frequency (intraclass correlation coefficient = 0.959). Our one-step approach enables easy and accurate variant allele frequency measurement of rare mutant alleles without PCR instrumentation, while the assay time was reduced by approximately half compared to the digital PCR with the shortest turnaround. The CASMART is an alternative to conventional single nucleotide polymorphism detection methods with great potential as a next-generation biosensor for rapidly quantifying the variant allele fraction, especially in resource-limited settings.
Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , Humanos , Alelos , Sistemas CRISPR-Cas/genética , Mutação , Receptores ErbB/genéticaRESUMO
Objective: To investigate the effects of all-trans retinoic acid (ATRA) on prostatic stromal cells during wound repair after prostatectomy in vitro. METHODS: Each of the M1 and M2 types of monocytic macrophage (THP-1) cells were divided into an experimental, a control and a non-activated group, the M1 macrophages of the former two groups activated by PMA and IFNγ, and the M2 macrophages by PMA and IL-4, respectively. The cells in the two experimental groups were treated with all-trans retinoic acid (ATRA) at 100 nmol/L, followed by detection of the expressions of IL-10, IL-12, IL-6, TNF-α and TGF-ß in the supernatant by ELISA. The supernatant was co-cultured with primarily cultured prostatic stromal cells or vascular endothelial cells in different groups. The expressions of RARα, RARß, RARγ, Arg1, Mmp9 and Soat1 in the macrophages were determined by PCR. The influence of the macrophages on the function of the stromal cells was analyzed by gel shrinkage test, scratch test and vascular endothelial cell tubular vascular formation test. The expression levels of Arg1 mRNA were reexamined under the action of RAR receptor subtype inhibitors. RESULTS: Compared with the control, the M2 macrophages treated with ATRA showed dramatically up-regulated expressions of IL-10 (ï¼»213.38 ± 2.02ï¼½ vs ï¼»298.22 ± 1.70ï¼½ pg/ml, P < 0.01) and TGF-ß (ï¼»185.37 ± 1.33ï¼½ vs ï¼»246.00 ± 2.14ï¼½ pg/ml, P < 0.01). The ATRA-treated macrophage supernatant enhanced the contraction and migration of the prostatic stromal cells and tubular formation of the vascular endothelial cells. The mRNA levels of Arg1 and RARß were significantly increased in the experimental group, and RARß was further confirmed to be the key receptor subtype in this process. CONCLUSIONS: ATRA activates prostatic stromal cells and enhances their migration and angiogenesis by acting on macrophages via RARß.
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PURPOSE: To investigate the safety of electrocoagulation and thulium laser (Tm-laser) sealing methods of distal ureter resection during radical nephroureterectomy (NFU) in a porcine model. METHODS: 9 pigs were used in the study: 6 were used to measure the bursting pressure (BP) and 3 were used to measure the highest pressure during NFU. Twelve ureters were to measure BP after being sealed by electrocoagulation or Tm-laser (n = 6, each). Six experimental NFUs were performed in 3 pigs to measure the intraluminal pressure of all procedures. RESULTS: The mean BP in the electrocoagulation group (104.3 ± 25.0 cmH2O) was similar to that of the Tm-laser group (74.8 ± 23.3 cmH2O, P > .05). The peak intraluminal pressure (35.9 ± 7.6 cmH2O) during NFU was significantly lower than the BP (P < .05). CONCLUSION: The effectiveness of the sealing was confirmed using both electrocoagulation and Tm-laser during NFU.
Assuntos
Eletrocoagulação/métodos , Cuidados Intraoperatórios , Fotocoagulação a Laser/métodos , Inoculação de Neoplasia , Nefroureterectomia , Ureter/cirurgia , Animais , Cuidados Intraoperatórios/instrumentação , Cuidados Intraoperatórios/métodos , Modelos Anatômicos , Modelos Animais , Nefroureterectomia/efeitos adversos , Nefroureterectomia/métodos , Suínos , Túlio/uso terapêutico , Neoplasias Urológicas/cirurgiaRESUMO
BACKGROUND: Increased prostatic smooth muscle tone and hyperplastic growth contribute to urethral obstruction and voiding symptoms in benign prostatic hyperplasia (BPH). It has been suggested that different proliferative potential of stromal cells between transition zone (TZ) and adjoining regions of the prostate plays a significant role in the development of BPH. However, the molecular mechanisms of this hyperplastic process remain unclear. We found tumor necrosis factor receptor-associated factor 6 (TRAF6) highly expressed in TZ stromal cells compared to peripheral zone (PZ) stromal cells by gene array analyzes. Therefore, we aim to study the potential mechanisms of stromal TRAF6 in promoting BPH progression. METHODS: Stromal cells obtained from BPH-derived primary cultures. The TRAF6-siRNA vector were constructed and transfected into cultured human BPH primary TZ stromal cells, and TRAF6-overexpressing vector were constructed and transfected into cultured human BPH primary PZ stromal cells. Stromal cells were recombined with BPH-1 cells then subcutaneously inoculated into the kidney capsule of male nude mice. Cell proliferation was evaluated by CCK-8 assay. Multiple proteins in the Akt/mTOR pathway were assessed using western blot. RESULTS: TRAF6 levels were increased in TZ stroma compared with PZ stroma of BPH. The in vitro cell culture and in vivo cell recombination revealed that selective downregulation of TRAF6 in TZ stromal cells led to suppression of the proliferation, while upregulation of TRAF6 in PZ stromal cells enhanced the proliferation. We found that the Phosphorylation and Ubiquitination of Akt as well as the Phosphorylation of mTOR, P70S6K were decreased when TRAF6 was downregulated in primary cultured TZ stromal cells of BPH. CONCLUSIONS: TRAF6 can promote the proliferation of stromal cells of BPH via Akt/mTOR signaling. Our results may make stromal TRAF6 responsible for zonal characteristic of BPH and as a promising therapeutic strategy for BPH treatment.
Assuntos
Proliferação de Células/fisiologia , Próstata/metabolismo , Hiperplasia Prostática/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Estromais/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Nus , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/genéticaRESUMO
BACKGROUND: The wound-healing process is very important for reducing complications after thulium laser resection of the prostate (TmLRP). The retinoic acid (RA) signaling pathway has been well studied in the wound-healing process of the skin and other organs. The goals of this study were to identify the role of RA signaling in the repair of the prostate after TmLRP and to investigate the molecular mechanism of this process. RESULTS: Retinoic acid receptors (RARs) were present in the prostate, and their expression was increased after TmLRP. RARß was expressed in the macrophages and may be related to the role of stromal cells in the wound-healing process. In vitro, RA enhanced the function of anti-inflammatory macrophages and promoted stromal cell activation and angiogenesis. Arg1 was also increased via RARß after treatment with RA. MATERIALS AND METHODS: The expression of RARs was analyzed in vivo by immunohistochemistry (IHC), real time qPCR, and western blot analysis. THP-1 cells were co-treated with or without RA and stimulating factor and then assessed by ELISA and qPCR. The supernatants from these cells were cultured with stromal cells and vascular endothelial cells, and the effects on these cells were analyzed. CONCLUSIONS: We found that RA signaling was involved in the wound-healing process of the prostate after TmLRP. RA treated macrophages activated stromal cells and promoted angiogenesis. RARß was the key isoform in this process.
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BACKGROUND: Complications after a thulium laser resection of the prostate (TmLRP) are related to re-epithelialization of the prostatic urethra. Since prostate growth and development are induced by androgen, the aim of this study was to determine the role and explore the mechanism of androgen in wound healing of the prostatic urethra. METHODS: Beagles that received TmLRPs were randomly distributed into a castration group, a testosterone undecanoate (TU) group, and a control group. The prostate wound was assessed once a week using a cystoscope. Histological analysis was then carried out to study the re-epithelialization of the prostatic urethra in each group. The inflammatory response in the wound tissue and urine was also investigated. RESULTS: The healing of the prostatic urethra after a TmLRP was more rapid in the castration group and slower in the TU group than that in the control group. Castration accelerated re-epithelialization by promoting basal cell proliferation in the wound surface and beneath the wound and by accelerating the differentiation of basal cells into urothelial cells. Castration reduced the duration of the inflammatory phase and induced the conversion of M1 macrophages to M2 macrophages, thus accelerating the maturation of the wound. By contrast, androgen supplementation enhanced the inflammatory response and prolonged the inflammatory phase. Moreover, the anti-inflammatory phase was delayed and weakened. CONCLUSION: Androgen deprivation promotes re-epithelialization of the wound, regulates the inflammatory response, and accelerates wound healing of the prostatic urethra after a TmLRP. Prostate 77:708-717, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Androgênios , Complicações Intraoperatórias , Próstata , Testosterona/análogos & derivados , Ressecção Transuretral da Próstata/efeitos adversos , Uretra , Androgênios/administração & dosagem , Androgênios/efeitos adversos , Androgênios/metabolismo , Animais , Modelos Animais de Doenças , Cães , Complicações Intraoperatórias/metabolismo , Complicações Intraoperatórias/fisiopatologia , Complicações Intraoperatórias/terapia , Macrófagos/patologia , Macrófagos/fisiologia , Masculino , Próstata/patologia , Próstata/cirurgia , Reepitelização/efeitos dos fármacos , Reepitelização/fisiologia , Estatística como Assunto , Testosterona/administração & dosagem , Testosterona/efeitos adversos , Testosterona/metabolismo , Túlio/farmacologia , Ressecção Transuretral da Próstata/métodos , Uretra/lesões , Uretra/patologia , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologiaRESUMO
OBJECTIVE: To study the different proportions of intermediate epithelial cells in human prostate cancer tissue and their clinical significance. METHODS: We performed immunohistochemical staining for Cytokeratin 5 (CK5) and Cytokeratin 8 (CK8) on 60 samples of human prostate cancer, determined the proportions of intermediate epithelial cells in the cancer tissue, and classified the samples into 2 types, one with a majority of intermediate epithelial cells (CaP-INT, n = 32), and the other composed mostly of luminal epithelial cells (CaP-LUM, n = 28). Then we compared the 2 types of prostate cancer in the expression of the androgen receptor (AR), age of the patient, serum t-PSA, prostate volume, Gleason score, clinical stage, androgen resistance, and incidence of distant metastasis. RESULTS: CaP-INT showed a significantly lower expression of AR ([24.42 +/- 11.41] %) and a higher incidence of distant metastasis (n = 14) than CaP-LUM ([77.21 +/- 10.22] % and n = 4) (P < 0.05). In the CaP-INT group, 6 of the 26 endocrinologically treated cases developed into androgen-independent prostate cancer (AIPC), while in the CaP-LUM group, only 1 out of 23 (P < 0.05). The former also showed remarkably higher clinical stages than the latter (P < 0.05), but no significant differences were found in age, serum t-PSA, prostate volume and Gleason score between the two groups (P > 0.05). CONCLUSION: A higher proportion of intermediate epithelial cells may lead to increased invasiveness and metastasis of human prostate cancer.
Assuntos
Células Epiteliais/patologia , Próstata/patologia , Neoplasias da Próstata/patologia , Idoso , Idoso de 80 Anos ou mais , Contagem de Células , Diferenciação Celular , Células Epiteliais/classificação , Humanos , Masculino , Pessoa de Meia-Idade , Receptores Androgênicos/metabolismoRESUMO
OBJECTIVE: To investigate targeted degradation of the androgen receptor (AR) by chimeric molecules (DHT-PROTAC) via the ubiquitin-proteasome pathway in androgen-independent prostate cancer CA-2B cells, and explore the proliferation, secretion and apoptosis of the treated cells. METHODS: C4-2B cells were treated with DHT-PROTAC, and then the expressions of the AR protein and caspase3 in the C4-2B cells were detected by immunohistochemistry and Western blot. The concentration of PSA in the supernatant was examined by ELISA. The cells were counted and their proliferation analyzed by a growth curve. The inhibitory effect on the growth of C4-2B cells was evaluated by MIT assay. RESULTS: Compared with the control group, the DHT-PROTAC-treated group showed an obviously decreased expression of AR proteins with a significant attenuation of the band signals (P < 0.05), a 40% reduction of the AR-positive cells and a 60% decrease of the PSA concentration in the supernatant (P < 0.05). DHT-PROTAC exhibited an inhibitory effect on the C4-2B cells in a time-dependant manner (P < 0.05). CONCLUSION: The chimeric molecule (DHT-PROTAC) can target the degradation of androgen receptors, reduce the secretion of PSA and repress the in vitro growth of C4-2B cells.