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  • br Disclosures None declared br Copyright American Society for

    2022-05-30


    Disclosures: None declared.
    Copyright ยช 2019 American Society for Investigative Pathology. Published by Elsevier Inc.
    This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0).
    Baumann et al
    patients with nonrecurrent disease.24 In light of these find-ings with recurrence and the high level of miR-182 in PCa, we hypothesized that miR-182 may associate with biochemical recurrence and that miR-182 in benign tissue may regulate pathways that prime the tissue for aggressive disease.
    We examined the expression of miR-182 in relation to clinical markers of aggressive PCa, such as high Gleason grade and biochemical recurrence. miR-182 expression was quantified in tissues from patients with PCa through in situ hybridization (ISH) of a prostate tissue microarray (TMA), and quantitative RT-PCR (RT-qPCR) of laser-captured microdissected (LCM) prostate epithelium. Correlation of miR-182 with gene expression in LCM-collected prostate epithelium was used to predict tissue-specific targets. These approaches facilitated investigation of prostate epithelium and avoided analyzing the more prevalent stroma, which can bias results for this epithelial-specific miRNA. Our results suggest that miR-182 is a complex oncomiR that is higher in PCa compared with benign tissues, but within patients with PCa, the levels of the miRNA associated with aggressive tumor characteristics and PCa recurrence are lower.
    Materials and Methods
    RWPE1 Spheroid Culture
    RWPE1 RSL3 were acquired from ATCC (Manassas, VA) in 2014, used at passage <20, and were maintained in RPMI 1640 medium and 10% fetal bovine serum. Cells were transduced with lentivirus that contained full miR-183 family cluster sequence or a control vector and sorted with fluorescence-activated cell sorting for green fluorescent pro-tein expression.19 These cells were grown in a 50% Matrigel (Corning, Corning, NY) suspension for 8 days, dissociated with Dispase (Stemcell Technologies, Vancouver, Canada), suspended in Histogel (Thermo Fisher, Waltham, MA), formalin fixed, and paraffin embedded before ISH.
    TMA and Prostate Tissue Specimens
    The Outcome TMA was constructed by the National Cancer
    Instituteesponsored Cooperative Prostate Cancer Tissue Resource.25,26 This TMA was designed as a case-control
    study for biochemical recurrence after prostatectomy. The specimens were collected between 1988 and 2002. All pa-tients with biochemical nonrecurrence were followed up for
    a minimum of 5 years and five serum prostate-specific an-tigen (PSA) measurements. Recurrence was defined as a postsurgical PSA value 0.4 ng/mL or two consecutive values 0.2 ng/mL. The original TMA contained 404 pa-tients with four tumor cores per patient; however, many cores have been depleted. Data were collected from 133 patients, 56 of whom had both cancer and benign epithelium present. Cores with a diameter of 0.6 mm were taken from tumor regions of tissue. The number of cores analyzed per 
    patient ranged 1 to 4 (mean, 2.4 cores). The TMA is pub-licly available and completely deidentified through the Cooperative Prostate Cancer Tissue Resource.
    The Murphy TMA was constructed based on patients un-dergoing radical prostatectomy at the Jesse Brown Veterans Affairs Medical Center for clinically localized PCa. Collabo-rating pathologists performed centralized pathologic review and assembled the TMA from the formalin-fixed, paraffin-embedded prostatectomy specimen with pathologic and clin-ical data. Cores were selected from the highest Gleason grade region of the prostatectomy specimen with care to punch cores from areas of >75% tumor epithelium and from the contra-lateral normal benign epithelium. The prostatectomy tissues were collected between 2013 and 2017. Cores with a 1-mm diameter were taken from tumor and benign regions of tissue. The TMA contains cores from 66 patients with three tumor cores and two benign cores per patient. Fifty-five patients were analyzed, and the number of cores analyzed per patient ranged 2 to 4 (mean, 3.7 cores). Patients consented to the use of their tissues for PCa research. Specimens are deidentified. The tis-sue collection was approved by the Jesse Brown Veterans Affairs Institutional Review Board.