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Targeting Mir-9 in Gastric Cancer Cells Using Locked Nucleic Acid Oligonucleotides

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Targeting Mir-9 in Gastric Cancer Cells Using Locked Nucleic Acid Oligonucleotides Lima et al. BMC Molecular Biol (2018) 19:6 https://doi.org/10.1186/s12867-018-0107-6 BMC Molecular Biology RESEARCH ARTICLE Open Access Targeting miR‑9 in gastric cancer cells using locked nucleic acid oligonucleotides Joana Filipa Lima1,2,3,4* , Joana Carvalho3,4, Inês Pinto‑Ribeiro4,5, Carina Almeida6, Jesper Wengel7, Laura Cerqueira1,2, Céu Figueiredo3,4,5, Carla Oliveira3,4,5† and Nuno Filipe Azevedo1† Abstract Background: Gastric cancer is the third leading cause of cancer-related mortality worldwide. Recently, it has been demonstrated that gastric cancer cells display a specifc miRNA expression profle, with increasing evidence of the role of miRNA-9 in this disease. miRNA-9 upregulation has been shown to infuence the expression of E-cadherin- encoding gene, triggering cell motility and invasiveness. Results: In this study, we designed LNA anti-miRNA oligonucleotides with a complementary sequence to miRNA-9 and tested their properties to both detect and silence the target miRNA. We could identify and visualize the in vitro uptake of low-dosing LNA-based anti-miRNA oligonucleotides without any carrier or transfection agent, as early as 2 h after the addition of the oligonucleotide sequence to the culture medium. Furthermore, we were able to assess the silencing potential of miRNA-9, using diferent LNA anti-miRNA oligonucleotide designs, and to observe its subse‑ quent efect on E-cadherin expression. Conclusions: The administration of anti-miRNA sequences even at low-doses, rapidly repressed the target miRNA, and infuenced the expression of E-cadherin by signifcantly increasing its levels. Keywords: MicroRNA, miR-9, E-cadherin, LNA-AMOs, Locked nucleic acid, FISH Background so other mechanisms may occur to explain the overall Gastric cancer (GC) is responsible for nearly 1 million defects on E-cadherin expression, such as miRNA post- deaths per year worldwide [1]. Like other cancers, the transcriptional regulation. development of GC is a multistep process where the MicroRNAs (miRNAs) are small, single-stranded and accumulation of genetic and epigenetic changes occurs non-coding RNAs, with approximately 21 nucleotides [2]. E-cadherin inactivation is the most well-established that regulate gene expression at a posttranscriptional defect in GC [3, 4]. In hereditary difuse-type of GC and level [5–7]. Tey complementarily bind to the 3′UTR of in sporadic GC with a difuse component, the mecha- their target mRNAs causing their degradation, transla- nisms that contribute to E-cadherin impairment are asso- tional repression, and/or deadenylation [8]. Upregulated ciated mostly with intragenic mutations, CDH1 locus miRNAs are often associated with protein downregu- deletions and promoter methylation, representing almost lation being implicated in cancer and often displaying 50% of the cases. However, E-cadherin protein expression oncogenic activity [9–14]. Several studies have demon- occurs in most GCs of both difuse and intestinal type, strated that miR-9 is signifcantly upregulated in breast, liver, and colorectal cancer [15–20]. Our analysis of the *Correspondence: [email protected] miRNA profle of 37 cancer and 4 normal gastric tissue †Carla Oliveira and Nuno Filipe Azevedo are Joint last authors and specimens, has also confrmed that miR-9 was overex- contributed equally to this study. pressed in gastric cancer tissues [4]. MiR-9 was shown to 1 Department of Chemical Engineering, LEPABE – Laboratory for Process Engineering, Environment, Biotechnology and Energy, be responsible for the negative regulation of the CDH1 Faculty of Engineering of the University of Porto, Rua Dr. Roberto Frias, gene, leading to tumor progression and invasiveness [21]. 4200‑465 Porto, Portugal Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lima et al. BMC Molecular Biol (2018) 19:6 Page 2 of 13 Te targeting of aberrant miRNAs with antisense oligo- the American Type Culture Collection (ATCC ®, Rock- nucleotides (AMOs) has been put forward as a promising ville, MD, USA). MKN74 cells were maintained in RPMI approach for post-transcriptional gene control in cancer medium 1640 Glutamax I (Gibco) supplemented with therapy [22]. AMOs can act as competitive inhibitors 10% (v/v) fetal bovine serum (FBS, HyClone, Termo of miRNAs, impairing their ability to interact with and Fisher Scientifc) and 1% (v/v) Penicillin/Streptomycin repress cellular target mRNAs [23–31]. Locked Nucleic (Gibco). HeLa and HEK293 cells were cultured in Dul- Acid (LNA) oligonucleotides are being used as AMOs becco’s Modifed Eagle Medium (DMEM, Gibco, Termo due to their increased thermal stability and improved Fisher Scientifc, Inc, UK) supplemented with 10% (v/v) selective power with respect to their nucleic acid targets fetal bovine serum (FBS, HyClone, Termo Fisher Sci- [23–27] to detect and to silence aberrant miRNAs in sev- entifc, Inc, UK) and 1% (v/v) Penicillin/Streptomycin eral human diseases, including cancer. Furthermore, they (Gibco). All cultured cells were maintained at 37 °C in a show low toxicity efects, being prone to be used in vivo humidifed 5% CO2 incubator and the medium was sub- for in situ localization of miRNAs in cells and tissues [27, stituted every 2–3 days. 28]. In this work, we pursued an approach that enables LNA anti‑miR design miRNA antagonism in GC using nucleic acid mimics Te selection of the miR-9 target sequence was achieved (Fig. 1). Our main goal was to design AMOs capable of by searching on the database TargetScan v7.1 [29–32]. detecting and silencing miR-9, with the aim of reestab- Complementary LNA-based AMOs were designed tak- lishing E-cadherin expression, using the model of GC ing into consideration optimal sequence length, speci- as a proof-of-concept. For that, two LNA-based AMOs fcity to the target, and theoretical melting temperature. with a complementary sequence to miR-9 were designed, Two LNA-based AMOs were synthesized with a phos- their cellular uptake and cytotoxicity assessed, and their phorothioate backbone (PS): an LNA/DNA anti-miR efects on the expression of miR-9 and of E-cadherin oligonucleotide sequence (AM_ LNA_PS) and another determined. sequence harboring a 2′O-Methyl substitution (AM_ LNA_2OMe_PS) (Fig. 1a). Additionally, two oligonu- Methods cleotide sequences were used as a control: a scramble Cell lines and culture conditions LNA sequence similar to the LNA/DNA AMO designed Human gastric cancer cell line MKN74 was purchased with 2 mismatches in the stretch targeting the seed from the Japanese Collection of Research Bioresources region of mR-9 (SC_LNA_PS), and a commercial scram- Cell Bank (JCRB025, JCRB Cell Bank, Tokyo, Japan). ble sequence (SC_LNA_PS*). After the selection of the HeLa (ATCC® CCL-2™, derived from human cervix car- AMOs, a nucleotide alignment with Basic Local Align- cinoma) and human embryonic kidney 293 (HEK293, ment Search Tool (BLAST) was performed to ensure ATCC ® CRL-1573™) cell lines were purchased from that the sequences were not targeting a similar region in a b c Fig. 1 Schematic representation of the strategy applied in this work. a Design of the LNA‑AMOs used in this manuscript. b Uptake and cytotoxic assessment of LNA‑AMOs in cancer cell lines by fuorescence in situ hybridization (FISH) and MTT assay, respectively. c Silencing efect of the developed AMOs to the target miR‑9 and its consequences on the expression of the E‑cadherin by real‑time PCR and Western blot analysis Lima et al. BMC Molecular Biol (2018) 19:6 Page 3 of 13 Cellular uptake another gene. Te theoretical melting temperature (Tm) of the AMO sequences was determined using the avail- Cellular uptake of the FAM_AM_LNA_PS sequence was able online platforms, as an indicator of the hybridiza- monitored by Fluorescence in situ hybridization (FISH) tion temperature (TH). Te Tm predictions for LNA/ as described by Guimarães et al. [34] with modifcations. DNA sequences were evaluated using the Biophysics MKN74 cells were cultured in a 24-well plate until reach- tool of IDT (available at http://bioph ysics .idtdn a.com/). ing 70% of confuence. Ten, the medium was discarded Predictions for the AM_LNA_2OMe_PS sequence were and the cells were fxed using a 4% paraformaldehyde determined with the 2′OMeRNA/calculator (available at solution, followed by a fxation with 50% ethanol. Each http://rnach emlab .ibch.pozna n.pl/calcu lator 1.php. fxation was performed for 10 min at RT. After the fxa- tion, it was added a hybridization solution containing the FAM_AM_LNA_PS at a fnal concentration of 5, 50 and LNA anti‑miR synthesis 100 nM. Plates were then incubated at 37 °C for 2 h. Two Te oligonucleotide sequences used in this study were types of hybridization solutions were tested: one con- synthesized at the University of Southern Denmark as taining 50% (v/v) formamide (Across Organic, NJ, USA), described elsewhere [33]. Briefy, the oligonucleotides 10% (v/v) dextran sulphate (Fisher Scientifc, MA, USA), were synthesized under anhydrous conditions using 0.1% (v/v) Triton-X (Panreac, Barcelona, Spain), 5 mM a standard automated nucleic acid synthesizer (Expe- of EDTA disodium salt 2-hydrate (Panreac), 50 mM dite 8909 instrument, PerSpective Biosystems Inc., MA, Tris–HCl (Fisher Scientifc NJ, USA), 10 mM NaCl (Pan- USA). LNA monomers were commercially available at reac); and another containing urea (VWR BHD Prolabo, Exiqon (Vedbaek, Denmark), and 2′-OMe were avail- Haasrode, Belgium) with diferent concentrations (0.5 able at RiboTask ApS (Odense, Denmark).
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