Tumor Suppressive MicroRNA-193b Promotes Breast Cancer Progression Via Targeting DNAJC13and RAN22A

Breast cancer is still a leading cause of cancer deaths in women.Despite improvements in therapeutic approaches in local control,metastatic relapse is almost always incurable, underlining theimportance to better understand the biological bases that contribute todisease progression.Breast cancer is the second leading cause of cancer deaths inwomen worldwide, with~1.7million (11.9%) new cases diagnosed and~522000deaths in2012. Since2008, breast cancer incidence hasincreased by more than20%and mortality has increased by14%[1].With breast cancer screening and therapeutic ad-vances, approximately80%of patients with a localized disease will experience a prolongedsurvival. However, early detection of metastatic relapse has notdemonstrated evidence of survival benefit, the development ofmetastatic relapse is almost always incurable[2], underlining theimportance to better understand the mechanisms that contribute todisease progression and developing novel therapeutic strategies.MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs,which play an essential role in the negatively regulation of geneexpression through translational repression or degradation of mRNAstargets[3]. miRNAs have been described to regulate a number ofbiological processes, including development, differentiation, apoptosisand cancer[4]. MicroRNAs can function either as oncogenes or tumor suppressors and have unique ability to regulate many protein-codinggenes[5]. A single miRNA can potentially regulate several hundreds orthousands of genes[6]. In recent years, the role of miRNAs in humancancer has been thoroughly investigated, and many human cancers haveaberrant expression of miRNAs. Extensive evidence suggests that somemiRNAs may act as selective tools for high-risk patients or miRNAsthemselves can be considered as therapeutic targets[4]. Dysregulation ofmiRNAs in breast cancer has been well documented. miR-10b is the firstdescripted breast cancer-related miRNAs, which highly expresses inmetastatic breast cancer cells and positively regulates breast cancermigration and invasion[7]. MiR-155and miR-21have been shown toinduce chemoresistance through their regulation of keyresistance-associated proteins[8]. MiR-221/222has been shown to bekey regulators of antiendocrine resistance in vitro through the targetingof cell-cycle inhibitor p27-Kip1and Era[9]. Dysregulation of miR-193bhas been previously reported in lymphoma[10], head and neck squamouscell carcinomas[11], non-small cell lung cancer[12]and prostate cancer[13],however, understanding the interaction between miR-193b and theirtargets leading to the disease remains largely unknown. Herein, wereported down-regulation of miR-193b in primary human breast cancercell lines and identified DNAJC13(HPS40) and RAB22A as novel,direct targets of miR-193b, which in turn, regulated RAS oncogenepathway, highlighting the biological importance of miR-193b in breastcancer progression.Objective:To detect the level of mir-193b expression in human MCF-10A,MCF-7and MBA-MD-231cell lines. To observe the results ofreconstitution of miR-193b expression in cell proliferation, clonogenicity, migration and invasion. By using in silico predictionalgorithms approach for target identification,Using Luciferase reporterassays confirmed the direct interaction of miR-193b.Methods:Quantitative real-time PCR for miRNAs and mRNAs expression:QRT-PCR analyses were performed human MCF-10A, MCF-7andMBA-MD-231cell lines to detect the level of mir-193b expression.Cell transfections：LipofectAMINE2000(Invitrogen) was used totransfect the tumor cells with either negative-scramble (SC) control ormimic pre-miR-193b (Ambion) at a final concentration of40nmol/L byusing the reverse transfection protocol, according to the manufacturer’sinstructions.Cell migration and invasion assays：Cell migration and invasionwere assessed using BD BioCoat Matrigel Invasion Chambers andControl Inserts (BD Biosciences). The cells were transfected with eitherpre-miR-193b or SC (40nM), then seeded on either control inserts(polyethylene terephthalate membrane) or trans-well chambers withMatrigel. Two ml RPMI supplemented with15%FBS was added to thelower chamber, served as the chemo-attractant.1×105transfected cellswere re-suspended in RPMI plus1%FBS, added to the upper chamber(0.5ml). Twenty hours later, migrating or invading cells attached to thelower surface of the membrane insert were fixed and stained, thencounted under a microscope. Relative migration was calculated bycomparison with cells transfected with the negative control. Thepercentage invasion was calculated based on the number of cells whichhave invaded through the Matrigel insert, divided by the number of cellswhich have migrated through the control insert.Cell proliferation and clonogenic assay：The soluble tetrazolium dye (MTT)3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideassay was used to assess cell proliferation and cytotoxicity ofintroducing mimic pre-miR-193b in MDA-MB-231cells. Cells werereverse transfected with SC control, pre-miR-193b or Lipofectamine2000and seeded onto96-well plates (5×103cells per well). Cellviability was measured at24,48and72hours after transfection.A clonogenic assay was used to evaluate the effects ofpre-miR-193b on MDA-MB-231cells. Briefly, transfected cells wereseeded into12-well plates.48hours later, cells were harvested, and500cells per milliliter were reseeded into6-well plates in triplicate. After14days incubation, the plates were fixed and stained, and the number ofcolonies was then counted. The fraction of surviving cells was calculatedby comparing with cells treated with SC.To characterize the mechanism by which miR-193b regulation inbreast cancer, we used6miRNA target-prediction software--miRanda,mi-RDB, miRWalk, PICTAR5, RNA-22and TargetScan to predictpotential miR-193b targets.Luciferase reporter assay：Because of the downstream target geneshave putative miR-193b binding sites in their3’-UTR regions, a regionof DNAJC13(HPS40) and RAB22A genes were amplified by PCR andconstructed in a pMIRREPORT luciferase vector (Ambion) as adownstream of the firefly luciferase gene. A mutant sequence was alsocloned as a validation plasmid. Either pMirluciferase orpMir-luciferase_gene specific vectors were co-transfected withpre-miR-193b or SC in MDA-MB-231cell lines. pRL-SV vector(Promega) containing Renilla luciferase was also transfected with eachcondition as a reference control. At48hours post-transfection, luciferaseactivity of Firefly and Renilla luciferase activities were then measured using the Dual-Luciferase Reporter Assay (Promega).Result：Expression of miR-193b in breast cancer cell lines：The expressionof miR-193b was evaluated in human breast cancer cell lines: MCF-7and MDA-MB-231, compared to that of the normal mammary epithelialcell line MCF-10A. miR-193b was significantly down-regulated in twocell lines70%of reduction in MCF-7cells and82%of reduction inMDA-MB-231cells, consistent with a previously published proteinmicroarray identification of microRNAs study.Overexpression of miR-193b caused significantly reduction of cellviability and decreasing of clonogenicity： In order to assess thebiological significance of miR-196b down-regulation, mimic enablemi-RNA functional analysis was performed. Cancer cells weretransfected with40nmol/L scramble controls (SC) or mimicpre-miR-193b. Sustained up-regulation of miR-193b expression wasobserved at48hours after transfection with either more than30foldsincreasing in MCF-7cells or18folds increasing in MDA-MB-231cells.Transfection with pre-miR-193b into MDA-MB-231cells led tosignificantly decreased cell viability compared to controls at48(65%)and72(50%) hours post-transfection. miR-196b over expression alsoresulted in significant reductions in clonogenicity in MDA-MB-231cells;survival fraction was22compared to45of SC.Mir-193b regulated cell migration and invasion：To determine ifmiR-193b was involved in regulating cell migration or invasion, in vitrotrans-well migration and invasive assays were used. Compared withtheir corresponding negative controls, transfection with pre-miR-193bsignificantly reduced migration of MDA-MB-231cells by72%.Moreover, pre-miR-193b transfection resulted in reduction in invasion of MDA-MB-231cells.Mir-193b potential mRNA targets identification：To characterize themechanism by which miR-193b regulation in breast cancer, we used6miRNA target-prediction software--miRanda, mi-RDB, miRWalk,PICTAR5, RNA-22and TargetScan to predict potential miR-193btargets. Three top mRNAs were selected to further validation. DNAJC13(Hsp40or RME-8) is a DNA Domain-Containing Protein, a criticalregulator of Hsp70; ERBB4(Viral Oncogene Homolog-Like4) relate tocell mitogenesis and differentiation; RAB22A (member RAS oncogenefamily) plays a role in endocytosis and intracellular protein transport.Initially, we assessed the basal levels of DNAJC13, ERBB4andRAB22A expression by using qRT-PCR methods in two breast cancercell lines. DNAJC13and RAB22A were significant up-regulated in bothMDA-MB-231and MCF-7cells, compared with normal control (NC).However, ERBB4was only up-regulated in MDA-MB-231cells but wasnot shown the consistence in MCF-7cells, showing down-regulationcompared to normal. After transfected pre-miR-193b (40nM) to thosebreast cancer cells, DNAJC13and RAB22A expression levels werereduced in both cell lines, comparing to normal. Next, to determinewhether miR-193b regulates DNAJC13and RAB22A by binding to its3’ UTR, we constructed several reporter vectors carrying the predictedbinding site(s) down stream of a firefly luciferase gene in thepMIR-Report vector as shown. For the luciferase assay, MDA-MB-231cells were co-transfected with pre-miR-193b (or a scramble control) andpmiR-DNAJC13-3’ UTR (or a mutated pmiR-DNAJC13-3’ UTR).The luciferase reporter that contained the DNAJC133’ UTR wassignificantly suppressed by pre-miR-193b, whereas the mutated reporterwas not affected. Similar results were obtained when we used pmiR-RAB22A-3’ UTR (or a mutated pmiR-RAB22A) on the cells.Conclusion:our experiments have shown that miR-193b is a novel tumorsuppressor in breast cancer through regulation of at least thetranscription factor DNAJC13and the expression of oncogene RAB22A.The resulting phenotype of miR-193b down-regulation includesincreased cell proliferation, clonogenicity, migration and invasion. Thesefindings suggest other mechanisms such as, miR-193b~DNAJC13ormiR-193b~RAB22A axis, which could account for the aggressivebehaviour of breast cancer.