In Vitro Validation of Micro-RNAs (miRNAs) Associated to Treatment Failure in Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: Micro-RNAs (miRNAs), have been shown to be one of the main regulators of gene expression and other biological processes. Recently, attention is focused on their potential role as prognostic predictors in different types of cancer, including diffuse large B cell lymphoma (DLBCL). We previously reported several miRNAs associated to progression/refractoriness in DLBCL (miR-1244, miR-193b-5p and miR-1231). The aim of this study was to validate these resultsin vitro, exploring the molecular pathways involved. Material and methods: The DLBCL cell line, U-2932 (activated B-cell (ABC) subtype) (ACC 633) was obtained from DSMZ (Germany). Transient reverse transfection of DLBCL cells for 48 or 72 h with mirVana miRNA mimics or inhibitors (miR-1244, miR-193b-5p and miR-1231) or with a miRNA negative control (100 nM) was performed using Viromer®GREEN reagent (Lipocalyx). In some experiments, after 24 h of transfection cells were treated with vehicle (1% DMSO) or with increasing concentrations of CHOP (cyclophosphamide, adriamycin, vincristine sulfate, and prednisone) (Sigma-Aldrich) for 48 h. The ratio of the four drugs was 80 mg/ 5.33 mg/ 0.16 mg/ 5.77 mg. After 48 h of transfection or treatment, cell viability was measured using the CellTiter 96®AQueous One Solution and total RNA was isolated using the RNeasy Mini Kit from Qiagen. Gene expression profiling (GEP) was performed using GeneChip human Clarion S of Affymetrix-ThermoFisher. Results: Reverse transfection of U-2932 with the miRNA mimic negative control (100 nM) for 48 h did not alter the cell viability compared to non-transfected (NT) cells, showing that the transfection method did not affect viability in these DLBCL cells. Transfection of mimic miR-1244, miR-193b-5p or miR-1231 (100 nM) did not significantly alter the viability compared to cells transfected with the miRNA mimic negative control. However, inhibition of these endogenous miRNA molecules for 48 h significantly decreased by 24.1 ± 5.7%, 28.9 ± 6.5% and 30.9 ± 3.3% cell viability, respectively, suggesting that these miRNAs could be involved in the survival of these tumor cells. In these experiments, the percentage of cell transfection was 89.6%. The effect of miRNA 1244, 193b-5p or 1231 on the response of DLBCL cells to CHOP treatment was also evaluated. Transfection of U-2932 cells with miRNA mimic negative control did not alter its sensibility to CHOP, which dose-dependently decreased its viability with an IC50 of 1.45 μg/ml in NT or transfected cells. Transfection of U-2932 cells with mimic miR-1244 or miR-193b-5p (100 nM) increased cell viability in the presence of vehicle (1% DMSO) and also reverted the inhibitory effect of CHOP (0.3 μg/ml) after 48 h of treatment. However, transfection of U-2932 cells with mimic miR-1231 (100 nM) did not alter the inhibitory effect of CHOP (0.3, 1 and 3 μg/ml) on cell viability at any of the concentrations studied. GEP studies showed that these miRNAs reduced the expression of genes associated to chemosensitivity (MCTP1) in the case of miR-1244 and miR-193-5p or tumoral suppressor genes (NRN1) in the case of miR-1231. Conclusions: We validatedin vitroour previously reported miRNAs about the role of miR-1244, miR-193b-5p and miR-1231 associated to treatment failure. The inhibition of endogenous miR-1244, miR-193b-5p or miR-1231 molecules significantly decreased cell viability. Transfection of U-2932 cells with mimic miR-1244 or miR-193b-5p (100 nM) increased cell viability of cells treated with CHOP. GEP studies showed that these miRNAs were linked to chemoresistance and inhibition of tumoral suppressor genes. Future works will have to translate these results to clinical practice in DLBCL. Disclosures Salar: Celgene:Speakers Bureau;Janssen:Speakers Bureau;Roche:Speakers Bureau.

Abstract 12: The Etiology Of Inverse Salt Sensitivity Of Blood Pressure: Mirna-485-5p Binds To The Dopamine Type 2 Receptor (d 2 R) Snp Rs6276 And Decreases D 2 R Expression

Inverse salt sensitivity is the paradoxical increase in blood pressure of individuals to a low sodium diet. rs6276, a single nucleotide polymorphism found in the 3’ untranslated region of the D2R gene (DRD2), which is associated with decreased expression and is also associated with the inverse salt sensitive phenotype (ISS). Urine derived renal proximal tubule cells grown from ISS participants with homozygous rs6276 SNPs vs salt resistant (SR) wild type for rs6276 SNPs have decreased expression of D2R as measured by receptor binding studies using BodipyFL-Spiperone (100 nM for 2 hrs, -36.9% ± 2.6% reduction in ISS-10 vs SR-6, n=5, p<0.01). miRNA-485-5p potentially binds to the rs6276 SNP in the 3’ UTR site and therefore could be a member of a newly identified classification of SNPs called miRSNPs. We transfected a miRNA-485-5p miRNA mimic and a miRNA blocker (20 nM for 72 hrs) to determine the effect on D2R expression on both wild type and homozygous variant SNP participant cell lines. Transfection of mir-485-5p only alters the expression of D2R in the ISS cell lines with SNPs (52619 ± 2001 RFU ISS control miRNA vs 69496 ± 2108 RFU ISS-10 miRNA blocker and vs 30434 ± 1824 RFU ISS-10 miRNA mimic, n=9, p<0.01, one-way ANOVA). The miRNA-485-5p blocker was able to return the D2R expression levels back to levels found in SR cells. Further studies are necessary to determine if the miRNA-485-5p mimic enhances and miRNA-485-5p blocker reverses any of the ISS cell phenotypes identified.

Computational and in vitro Investigation of miRNA-Gene Regulations in Retinoblastoma Pathogenesis: miRNA Mimics Strategy

Purpose Retinoblastoma (RB), a primary pediatric intraocular tumor, arises from primitive retinal layers. Several novel molecular strategies are being developed for the clinical management of RB. miRNAs are known to regulate cancer-relevant biological processes. Here, the role of selected miRNAs, namely, miR-532-5p and miR-486-3p, has been analyzed for potential therapeutic targeting in RB. Methods A comprehensive bioinformatic analysis was performed to predict the posttranscriptional regulators (miRNAs) of the select panel of genes [Group 1: oncogenes (HMGA2, MYCN, SYK, FASN); Group 2: cancer stem cell markers (TACSTD, ABCG2, CD133, CD44, CD24) and Group 3: cell cycle regulatory proteins (p53, MDM2)] using Microcosm, DIANALAB, miRBase v 18, and REFSEQ database, and RNA hybrid. The expressions of five miRNAs, namely, miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p, were analyzed by qRT-PCR on primary RB tumor samples (n = 30; including 17 invasive RB tumors and 13 noninvasive RB tumors). Detailed complementary alignment between 5’ seed sequence of differentially expressed miRNAs and the sequence of target genes was determined. Based on minimum energy level and piCTAR scores, the gene targets were selected. Functional roles of these miRNA clusters were studied by using mimics in cultured RB (Y79, Weri Rb-1) cells in vitro. The gene targets (SYK and FASN) of the studied miRNAs were confirmed by qRT-PCR and western blot analysis. Cell proliferation and apoptotic studies were performed. Results Nearly 1948 miRNAs were identified in the in silico analysis, From this list, only 9 upregulated miRNAs (miR-146b-5p, miR-305, miR-663b, miR-299, miR-532-5p, miR-892b, miR-501, miR-142-5p, and miR-513b) and 10 downregulated miRNAs (miR-1254, miR-328, miR-133a, miR-1287, miR-1299, miR-375, miR-486-3p, miR-720, miR-98, and miR-122*) were found to be common with the RB serum miRNA profile. Downregulation of five miRNAs (miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p) was confirmed experimentally. Predicted common oncogene targets (SYK and FASN) of miR-486-3p and miR-532-5p were evaluated for their mRNA and protein expression in these miRNA mimic-treated RB cells. Experimental overexpression of these miRNAs mediated apoptotic cell death without significantly altering the cell cycle in RB cells. Conclusion Key miRNAs in RB pathogenesis were identified by an in silico approach. Downregulation of miR-486-3p and miR-532-5p in primary retinoblastoma tissues implicates their role in tumorigenesis. Prognostic and therapeutic potential of these miRNA was established by the miRNA mimic strategy.