miR-142-3p suppresses uveal melanoma by targeting CDC25C, TGFβR1, GNAQ, WASL, and RAC1

Dewei Peng,1,2 Jing Dong,1,2 Yunping Zhao,1,2 Xiaomei Peng,1,2 Jingjing Tang,1,2 Xiaoyan Chen,1,2 Lihua Wang,1,2 Dan-Ning Hu,2,3 Peter S Reinach,1,2 Jia Qu,1,2 Dongsheng Yan1,21School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, People’s Republic of China; 2State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, Zhejiang, People’s Republic of China; 3Tissue Culture Center, New York Eye and Ear Infirmary, New York Medical College, New York, NY, USAPurpose: Uveal melanoma (UM) is the most frequent metastatic ocular tumor in adults. Therapeutic intervention remains ineffective since none of the novel procedures used to treat this disease increased survival rates. To deal with this limitation, additional studies are required to clarify its pathogenesis. The current study focused on describing how epigenetic modulation by miR-142-3p affects changes in some cellular functions underlying UM pathogenesis.Methods and results: Microarray analysis identified 374 miRNAs which were differentially expressed between UM cells and uveal melanocytes. miR-142-3p was one of the 10 most downregulated miRNAs. Quantitative RT-PCR analysis confirmed that miR-142-3p expression levels were significantly decreased in both UM cell lines and clinical specimens. The results of the MTS, clone formation, scratch wound, transwell assays, and in vivo biofluorescence imaging showed that miR-142-3p overexpression significantly inhibited cell proliferation, migration, and invasiveness. Nevertheless, miR-142-3p did not affect cell apoptotic activity or sensitivity to doxorubicin. Cell cycle and EdU analysis showed that miR-142-3p overexpression induced G1/G2 cell cycle arrest and reduced DNA synthesis in UM cells. Microarray analysis showed that miR-142-3p mainly regulates the TGFβ signaling pathway, and those in which MAPK and PI3K-Akt are constituents. Functional interactions between miR-142-3p and CDC25C, TGFβR1, GNAQ, WASL, and RAC1 target genes were confirmed based on the results of the luciferase reporter assay and Western blot analysis. CDC25C or RAC1 downregulation is in agreement with cell cycle arrest and DNA synthesis disorder induction, while downregulation of TGFβR1, GNAQ, WASL, or RAC1 accounts for declines in cell migration.Conclusion: miR-143-3p is a potential therapeutic target to treat UM since overriding its declines in expression that occur in this disease reversed the pathogenesis of this disease. Such insight reveals novel biomarker for decreasing UM vitality and for improved tracking of tumor progression.Keywords: miR-142-3p, uveal melanoma, tumor suppressor, molecular mechanism

Comparison of Diagnostic Tests in Distinct Well-Defined Conditions Related to Dry Eye Disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Purpose: This study compares signs, symptoms and predictive tools used to diagnose dry eye disease (DED) and ocular surface disorders in six systemic well-defined and non-overlapping diseases. It is well known that these tests are problematic because of a lack of agreement between them in identifying these conditions. Accordingly, we provide here a comparative clinical profile analysis of these different diseases. Methods: A spontaneous and continuous sample of patients with Sjogren's syndrome (SS) (n = 27), graft-versus-host-disease (GVHD) (n = 28), Graves orbitopathy (n = 28), facial palsy (n = 8), diabetes mellitus without proliferative retinopathy (n = 14) and glaucoma who chronically received topical drugs preserved with benzalkonium chloride (n = 20) were enrolled. Evaluation consisted of a comprehensive protocol encompassing: (1) structured questionnaire - Ocular Surface Disease Index (OSDI); (2) tear osmolarity (TearLab Osmolarity System - Ocusense); (3) tear film break-up time (TBUT); (4) fluorescein and lissamine green staining; (5) Schirmer test and (6) severity grading. Results: One hundred and twenty five patients (aged 48.8 years-old +/- 14.1, male:female ratio = 0.4) were enrolled in the study, along with 24 age and gender matched controls. Higher scores on DED tests were obtained in Sjogren Syndrome (P<0.05), except for tear film osmolarity that was higher in diabetics (P<0.001) and fluorescein staining, that was higher in facial palsy (P<0.001). TFBUT and OSDI correlated better with other tests. The best combination of diagnostic tests for DED was OSDI, TBUT and Schirmer test (sensitivity 100%, specificity 95% and accuracy 99.3%). Conclusions: DED diagnostic test results present a broad range of variability among different conditions. Vital stainings and TBUT correlated best with one another whereas the best test combination to detect DED was: OSDI/TBUT/Schirmer.95Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Apoio ao Ensino, Pesquisa e AssistAancia do Hospital das Clinicas da Faculdade de Medicina de Ribeirao Preto da Universidade de Sao Paulo (FAEPA)NAP-FTO USPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP