Tumor suppressor effects of miR-143 and miR-199 on myelogenous leukemia cells through targeting RNA-binding protein Musashi2

Tumor suppressor effects of miR-143 and miR-199 on myelogenous leukemia cells through targeting RNA-binding protein Musashi2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Tumor suppressor effects of miR-143 and miR-199 on myelogenous leukemia cells through targeting RNA-binding protein Musashi2 Liana Lachinani, Rana Iranpour, Mahboobeh Forouzanfar, Mohammad Hossein Nasr-Esfahani, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7471306/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version Abstract The RNA-binding protein Musashi2 plays a critical role in normal and malignant hematopoiesis. Aberrant expression of Musashi2 is linked to the inappropriate expression of several miRNAs. In this study, we observed downregulation of miR-143 and miR-199 in CML and AML cell lines. Bioinformatic analysis revealed potential interactions between these miRNAs and Musashi2 mRNA, which were also confirmed by our experimental data. In K562 cells, which showed the strongest negative correlation between Musashi2 and the candidate miRNAs, overexpression of miRNAs led to reduced cell proliferation and invasiveness. Overexpression of miR-143 and miR-199 decreased the mRNA and protein levels of Musashi2 in K562 cells, with miR-143 showing a more significant reduction in cell proliferation and invasion compared to miR-199a. Our results indicate that the downregulation of Musashi2 following the overexpression of these tumor suppressor miRNAs is responsible for diminishing the cancerous properties of malignant myeloid cells. According to our findings, these miRNAs could be new potential molecular targets for the treatment of myelogenous leukemias. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Computational biology and bioinformatics Biological sciences/Molecular biology Health sciences/Oncology miRNA MSI2 myeloid leukemia RNA-binding protein tumor suppressor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Leukemia cells exhibit a dysregulated developmental program due to various genetic and epigenetic alterations, which are considered major steps in the progression of leukemogenesis [ 1 , 2 ]. Additionally, post-transcriptional modifications play a crucial role in gene regulation. For instance, mRNA transcripts undergo several processes regulated by non-coding RNAs (ncRNAs) or RNA-binding proteins (RBPs), with dysregulated expression patterns observed in numerous cancer types [ 3 – 5 ]. MSI2, a member of the Musashi RNA-binding protein family, plays a key role in normal hematopoiesis, and its abnormal expression is associated with various hematopoietic malignancies. Unlike MSI1, its primarily discovered homolog expressed in multiple tissues, MSI2 is predominantly expressed in hematopoietic stem cells (HSCs). Elevated expression of MSI2 leads to an increase in the number of progenitor and stem cells, rapid progression of leukemia, and a worse clinical prognosis in leukemic patients [ 6 , 7 ]. According to the Cancer Genome Atlas (TCGA, 2012), genomic and RNA sequencing data of primary tumors showed that MSI genes were not significantly mutated in most cancers but were overexpressed in several tumor types [ 8 ]. Elevated expression of MSI2 proteins has been found in chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL). Conversely, its depletion leads to decreased cellular proliferation, increased apoptosis, and differentiation of human myeloid leukemia cell lines [ 6 , 7 , 9 ]. Numerous studies have reported that dysregulated expression of MSI2 could occur due to dysregulated expression of MSI2-targeting miRNAs [ 10 – 12 ]. MicroRNAs (miRNAs) are small endogenous non-coding RNAs that play critical roles in various biological processes. They regulate their target genes by influencing mRNA stabilization, splicing, or post-transcriptional repression [ 13 , 14 ]. Abnormal expression profiles of miRNAs have been observed in numerous malignancies, including leukemia. Due to their stability in biological fluids, cancer-related miRNAs have emerged as prognostic biomarkers for determining cancer subtypes, stages, and treatment responses [ 15 – 17 ]. In malignancies, miRNAs can impact tumor growth, invasion, apoptosis, and angiogenesis, acting as either tumor suppressor miRNAs or oncomiRs. Stimulation of tumor suppressor miRNAs or inhibition of oncomiRs can result in complete regression of the cancer [ 18 – 20 ]. The first case of microRNA dysregulation was reported in chronic lymphocytic leukemia (CLL), where down-regulation of miR-15 and miR-16 caused by a frequent deletion of the 13q14 region leads to increased expression of their targets including the BCL2 antiapoptotic protein [ 21 , 22 ]. Several critical miRNAs and their target genes involved in hematopoiesis and hematological conditions have been identified. Multiple high-throughput studies have reported that the expression of miR-143 and miR-199a, recognized as tumor suppressor miRNAs, is decreased in leukemia [ 23 , 24 ]. Consequently, these miRNAs have been proposed as potential prognostic biomarkers and/or therapeutic targets in human leukemia [ 25 – 27 ]. Given the negative correlation between the expression patterns of these miRNAs and MSI2, we aimed to investigate whether MSI2 could be a direct target for miR-143 and miR-199a in leukemia. To validate our bioinformatic predictions, we experimentally examined the targeting of MSI2 by miR-143 and miR-199a and assessed the impact of these miRNAs on the proliferation and invasion of K562 cells, a CML cell line. Materials and methods Bioinformatics studies Potential miRNA binding sites in the 3'UTR region of MSI2 were predicted using several bioinformatics prediction websites including TargetScan ( http://www.targetscan.org/ ) [ 28 ], miRDB ( http://www.mirdb.org/ ) [ 29 ], miRWalk ( http://mirwalk.umm.uni-heidelberg.de/ ) [ 30 ], PicTar2 ( https://pictar.mdc-berlin.de/ ) [ 31 ], miRMap ( http://cegg.unige.ch/mirmap ) [ 32 ], miRNAMap 2.0 ( http://mirnamap.mbc.nctu.edu.tw/ ) [ 33 ], miRTarBase 6.0 ( https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/index.php ) [ 34 ], RNAhybrid ( http://bibiserv.techfak.uni-bielefeld.de/rnahybrid ) [ 35 ], and RNA22 ( https://cm.jefferson.edu/rna22/ ) [ 36 ]. The selection of candidate miRNAs was based on factors such as minimum free energy, conservation status and the number of binding sites. MiRNAs that met all criteria and received high scores from multiple websites were selected for further analysis. Moreover, the expression profiles of the candidate miRNAs in normal and malignant hematopoietic cells were assessed using GEO array data, from GSE51908 and GSE28825. The final candidate miRNAs with decreased expression levels in malignant samples were prioritized for further investigation. DNA Constructions Human miR-143 and miR-199a precursors (MIMAT0000435 and MIMAT0000232) were PCR amplified using specific primers (Table 1 A) and pfu enzyme (Thermo Scientific, USA). Each miRNA precursor was subcloned into the Xba I and Sal I sites of the pBud-EGFP vector, a modified version of pBudCE4.1 (Invitrogen) that overexpresses EGFP. The miRNA precursors were placed under the CMV promoter, and the new vectors were named pBud-miR-143 and pBud-miR-199a. Additionally, the pBud-Scr vector containing the universal scramble precursor was utilized as the negative control vector [ 37 ]. Another set of vectors was created for the dual-luciferase assay by subcloning the wild type or mutated 3'UTR of MSI2 into the psiCHECK2 vector (Promega, USA). The 3'UTR region of MSI2 (NM_138962.3), which is 5.1 kb in length, and the mutated fragments lacking the seed-complementary regions were amplified using SOEing PCR. Multiple PCR primer sets were designed using Oligo7 (Table 1 B). The obtained wild type and mutated 3'UTR regions were then subcloned downstream of the Renilla luciferase at the Xho I and Not I sites of psiCHECK2. The accuracy of all constructions was confirmed through restriction enzyme digestion and sequencing analysis. Table 1 The list of primers used in this study A] Primers used for amplification of miRNA precursors Primer name Primer sequence Product length miR-199a precursor F: 5'-GTCGACAGTGGTGGTTTCCTTGGCTGCTCAG-3' 309 bp R: 5'-GTCTAGATCGAATCTTCTATGCGAGGCTCTGC-3' miR-143 precursor F: 5'-GTCGACAGACAGGAAACACAGTTGTGAGG-3' 321 bp R: 5'-GTCTAGAATGGAGTCTGGAAACACTCTGTCC-3' B] Primers used for amplification of normal and mutant 3'UTR regions of MSI2 Primer name Primer sequence Product length MSI2U1 F: 5'-GCTCGAGGCAGGTGCTTTCGTTGCCATCTCACTCTGA GAGCATACCTGGATGTCC-3' 1427 bp R: 5'-CAGGCTTATCCACTCCTGGCCCATG-3' MSI2U2 F: 5'-CTATTGTCCAATCATTTCAGCACC-3' 1457 bp R: 5'-AGACCTTGGGTTAGTTGAAACCAC-3' MSI2U3 F: 5'-TGTAAGCGTGCTGTCCTGGTACTAG-3' 1215 bp R: 5'-GCAAGATGTGTGTAACATTCACTGG-3' MSI2U4 F: 5'-CTCTATCCCAGTCGCCCATTAGCTTG-3' 1615 bp R: 5'-GCGGCCGCAACACATTGATTTCTGGACCACAGTAG-3' mutU1 F: 5'-GCAGGTGCTTTCGTTGCACTCTGAGAGCATACCTGGA TGTC-3' 192 bp R: 5'-GTTGTCTTATATTACAGCAGATTCGTTAGTCAACCCT CGATCCGAAGTG-3' mutF2 F: 5'-TTCGGATCGAGGGTTGACTAACGAATCTGCTGTAAT ATAAGACAACAGC-3' 1254 bp mutR4 R: 5'-TGGGATGGTACATTATGTACAGAATAAGAGAACCTT CGAGGGAGGCTC-3' 1156 bp C] Primers used for RT-qPCR Primer name Accession number Primer sequence GAPDH NM_001357943.2 F: 5'-CCACTCCTCCACCTTTGACG-3' R: 5'-CCACCACCCTGTTGCTGTAG-3' MSI2 NM_138962.4 F: 5'-AACAGGCACAGAGGGTTTG-3' R: 5'-ATGACTTCTTTCGGCTGAG-3' Cell lines and culture media In this study, HEK293T cells, two CML cell lines (K562 and KCL-22), and two AML cell lines (KG-1α and HL-60) obtained from the Pasteur Institute in Iran were used. HEK293T cells were cultured in DMEM high glucose (Gibco, USA), while the CML and AML cell lines were cultured in RPMI1640 (Gibco), all supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.1 mM non-essential amino acids (all from Gibco), and 2 mM L-Glutamine (Invitrogen, USA). All cell lines were maintained in a humidified incubator at 37°C with 5% CO 2 . Establishment of K562 cell lines stably expressing precursor miRNAs K562 cells were electroporated with miRNA or precursor-expressing vectors in four groups: pBud-miR-143 group, pBud-miR-199a group, a co-transfected group with both pBud-miR vectors (miR143/miR-199 group), and a pBud-Scr group as the control. For each electroporation reaction, 7 × 10 6 cells and 20 µg of each plasmid were transferred to electroporation cuvettes (0.2 cm) and electroporated at 155 V, 1000 µF using the Gene Pulser Xcell™ Electroporation System (Bio-Rad, USA). The cells were immediately transferred to a prewarmed culture medium and incubated for 48 h. Subsequently, the electroporated cells were treated with zeocin (Invivogen, USA) at a final concentration of 200 µg/ml to establish stable clones. Dual-luciferase reporter assay For luciferase reporter assays, HEK293T cells were transiently co-transfected with luciferase reporter vectors containing the wild-type or mutant 3'UTR region of MSI2 and corresponding precursor or scramble expressing vectors. The HEK293T cells were plated in a 24-well plate, 24 h before transfection. Transfection reactions were performed using Lipofectamine LTX™ Reagent (Invitrogen) according to the manufacturer’s protocol. The efficiency of transfection was assessed by the percentage of EGFP-positive cells 24 h after transfection. Firefly and Renilla luciferase activities were measured 48 h post-transfection using the Dual-Luciferase Reporter Assay Kit (Promega) following the manufacturer’s instructions. Finally, the relative luciferase activity was calculated by normalizing the Renilla luciferase activity to the firefly luciferase activity. Cell proliferation assay To assess the effect of miRNA overexpression on K562 cell proliferation, an MTS assay was conducted on clones that had been stably transfected with miRNA precursors. Each clone was seeded at a density of 10 5 cells/ml in triplicate wells of a 96-well plate. After 24 h, 20 µl of MTS solution was added to each well, and the plates were incubated for an additional 3 hours. Finally, the absorbance was measured at 490 nm and 630 nm using a Stat Fax 2100 Microplate Reader. Transwell invasion assay After miRNA overexpression in K562 cells, cell invasiveness was assessed using the transwell invasion assay. Transwell inserts with an 8.0 µm pore size (Corning, USA) were pre-coated with 0.3 mg/ml Matrigel (Sigma, Germany) diluted in DMEM and incubated at 37°C for 24 h before cell culture. K562 stable clones at a density of 3 × 10 5 cells/ml were seeded in the serum-free medium in the upper chamber, while the lower chambers were filled with serum-containing medium. The cells were then incubated in a humidified cell culture incubator at 37°C with 5% CO 2 for 48 h. Subsequently, the cells on the lower surface were fixed with 4% paraformaldehyde and stained with a 1% crystal violet solution. Cell counting was conducted in 10 random fields of captured images using a light microscope. RNA isolation and expression quantitation Total RNA was extracted from normal peripheral blood cells, K562, KCL-22, KG-1α, and HL-60 cell lines using TRIzol reagent (Invitrogen) following the manufacturer's protocol. 500 ng of total RNA was treated with DNase I (Thermo Scientific) to eliminate potential genomic DNA contamination, and cDNA synthesis was carried out using a cDNA synthesis kit (Takara, Japan) with random hexamer primers. RT-qPCR was conducted with 50 ng of each cDNA in a Step One Plus Real-Time PCR thermal cycler (Applied Biosystems, USA) under pre-optimized conditions using SYBR premix ExTaqII (Takara) and specific primers listed in Table 1 C. The specific primers for the MSI2 gene were designed using Beacon designer (Version 7.2, USA). Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) was utilized as the reference gene for normalizing the expression of target genes. For miRNA cDNA synthesis, a universal cDNA synthesis kit (Exiqon, Germany) was employed. RNA U6 (Exiqon) served as an internal control for quantifying and normalizing miRNA expression levels. All reactions were performed in triplicate to ensure result reproducibility. The relative quantification of gene or miRNA expression was determined using the threshold cycle difference methods (2 −ΔCt and 2 −ΔΔCt methods). Western blot analysis To evaluate MSI2 protein expression in stably transfected K562 clones, protein extraction was carried out using Trizol reagent (Invitrogen). Approximately 30 µg of total protein lysates were separated by 10–15% SDS-PAGE, transferred onto PVDF membranes, and then cut to a specific size based on the expected band size. The membranes were then probed with primary antibodies against MSI2 (1:4000, Abcam, Ab76148) and GAPDH (1:5000, SANTA CRUZ, sc-2357). Subsequently, the membranes were incubated with HRP-conjugated IgG secondary antibodies specific to MSI2 and GAPDH antibodies: mouse anti-rabbit IgG (1:7500, SANTA CRUZ, sc-2357) and goat anti-mouse IgG (1:5000, Dako, P0447). The protein bands of interest were visualized using the Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, USA), and the protein expression levels were quantified using ImageJ software (version 1.42q). Statistical analysis Data values were presented as the mean ± standard error of the mean (SEM) of replicates. Comparisons between groups were conducted using analysis of variance (ANOVA) and t-tests. Statistical significance was defined as p values < 0.05, 0.001, or 0.0001. Results Prediction of candidate miRNAs Several MSI2-targeting miRNAs were predicted using miRNA-target prediction tools. Among the high-scored miRNAs predicted by most software were miR-143-3p and miR-199a-3p (Fig. 1 A). Hsa-miR143-3p was found to bind at three positions in the 3'UTR of MSI2, with two binding sites overlapping, while hsa-miR199a-3p binds to only one site (Fig. 1 B, C). Additionally, the expression levels of these candidate miRNAs were analyzed in two high-throughput studies, GSE51908 and GSE28825. The analysis of normal and malignant hematopoietic cells in these studies revealed that the expression of both miRNAs was decreased in leukemia cell lines. These findings enhanced the accuracy of the miRNA selection process. Interaction of miR-143-3p and miR-199a-3p with MSI2 mRNA To validate the direct interaction of miR-143-3p and miR-199a-3p with the MSI2 transcript, HEK293T cells were co-transfected with precursors of both miRNAs and luciferase reporter vectors containing the wild-type or mutant 3'UTR region of MSI2. In silico analysis indicated three binding sites for miR-143-3p and one binding site for miR-199a-3p in the 3'UTR region of MSI2 (Fig. 1 B, C). Mutated forms of the 3'UTR region were generated by eliminating the complementary sequences of the seed regions using SOEing PCR for each miRNA. As depicted in Fig. 2 , overexpression of miR-143 and miR-199a led to a notable decrease in relative luciferase activity in the wild-type groups compared to the mutant groups. Surprisingly, the expression of scramble sequences also resulted in reduced luciferase activity. This decrease could attributed to the endogenous expression and functional activity of both miRNAs, as reported in other studies. These results confirmed the direct targeting of the MSI2 transcript by both candidate miRNAs as well [ 38 , 39 ]. Correlation of MSI2 with miR-143-3p and miR-199a-3p in leukemia cell lines The endogenous expression level of MSI2 was measured by RT-qPCR in four leukemia cell lines: KCL-22, K562, KG-1α, and HL-60. Among these cell lines, KG-1α and K562 exhibited the highest expression level of MSI2 mRNA (Fig. 3 A). Conversely, RT-qPCR analysis confirmed the reduced expression levels of miR-143-3p and miR-199a-3p in all leukemia cells compared to normal blood cells. However, the lowest expression levels for both miRNAs were observed in K562 cells (Fig. 3 B). Therefore, we chose K562 cells for further studies. Overexpression effects of miR-143-3p and miR-199a-3p on MSI2 in K562 cells To investigate the impact of miR-143-3p and miR-199a-3p on MSI2 expression, we established K562 clones that stably overexpressed the corresponding miRNAs. We selected the K562 cell line for its high MSI2 expression and low levels of both miRNAs compared to other cell lines (Fig. 3 B). Electroporation of K562 cells with the constructed vectors and subsequent zeocin treatment led to the generation of four stable clones: K562-Scr, K562-miR143, K562-miR-199, and K562-miR143/miR-199. The overexpression of miRNAs was confirmed through RT-qPCR analysis (Fig. 4 ). The results indicated a decrease in both MSI2 mRNA and protein levels in each clone due to the overexpression of the candidate miRNAs (Fig. 5 ). Notably, the reduction in MSI2 protein was most significant in the K562-miR143/miR-199 clone, highlighting the synergistic effect of the two miRNAs in reducing MSI2 protein levels. Overexpression effect of miR-143-3p and miR-199a-3p on cell proliferation and invasion The MTS assay was used to evaluate the proliferation of K562 clones stably expressing miR-143-3p and miR-199a-3p. The results indicated that miR-143-3p overexpression reduced cell proliferation, while miR-199a-3p did not significantly affect cell proliferation (Fig. 6 ). Furthermore, the transwell invasion assay demonstarted that K562 clones with stable overexpression of miR-143-3p and miR-199a-3p exhibited lower invasivness compared to intact K562 cells (Fig. 7 ). In clones co-transfected with both miR-143 and miR-199a, the inhibition of cell invasion was less pronounced than when each miRNA was transfected individually. Interestingly, despite miR-199a-3p not affecting cell proliferation, it had the most significant inhibitory effect on cell invasion. Discussion One of the characteristic features of various malignancies, including leukemogenesis, is the up and down-regulation of oncogenes and tumor suppressor genes [ 40 ]. MSI2 is a well-known RNA-binding protein that plays critical roles in normal and malignant hematopoiesis. In fractionated blood cell populations, hematopoietic stem cells (HSCs) exhibit higher levels of MSI2 compared to progenitor cells, differentiated myeloid cells, and lymphocytes [ 7 ]. In leukemogenesis, the expression of MSI2 is increased, and its knockdown leads to a reduction in proliferation and an increase in apoptosis [ 1 , 7 , 41 ]. In several disorders and malignancies, increased expression of the MSI2 protein is associated with aberrant expression of certain miRNAs [ 10 , 11 , 42 ]. Therefore, we aimed to investigate whether a similar association exists between MSI2 and miRNAs in myeloid leukemia and, if so, which miRNAs could directly interact with MSI2 mRNA. According to in silico studies, we identified several miRNAs that potentially interact with MSI2 mRNA. A negative correlation between some miRNAs and MSI2 was observed by analyzing the expression patterns of predicted miRNAs in various malignant hematopoietic cells [ 26 , 27 ]. Among these, two well-known tumor suppressor miRNAs, miR-143-3p and miR-199a-3p, were identified. The tumor suppressor role of miR-199a-3p has been reported in hepatocellular carcinoma, NSCLC (lung cancer), ovarian cancer and glioma, where it decreases proliferation, migration, and invasion of cancer cells by targeting downstream genes of PAK4/Raf/MEK/ERK and PI3K-mTOR pathways [ 43 – 46 ]. Similarly, the tumor suppressor function of miR-143-3p has been validated in multiple cancer types including bladder cancer, esophageal squamous cell carcinoma, renal cell carcinoma and papillary thyroid carcinoma [ 47 – 50 ]. Both candidate miRNAs are also known as leukemia-associated miRNAs [ 51 , 52 ]. In this study, we confirmed that miR-143-3p and miR-199a-3p directly interact with the 3'UTR region of MSI2 mRNA using a dual luciferase assay. Moreover, overexpression of both miRNAs resulted in decreased expression levels of MSI2 in K562 cells at both the mRNA and protein levels. Previous studies have shown that MSI2 knockdown in K562 cells leads to inhibition of cell growth and cell cycle arrest by targeting p21, cyclin D1, as well as the ERK/MAPK and p38/MAPK pathways [ 53 ]. Additionally, miR-143 has been reported to reduce cell proliferation through various pathways, including PI3K/AKT and MAPK, in leukemia, bladder, and prostate cancers [ 54 , 55 ]. Interestingly, the MAPK pathway is reciprocally regulated by MSI2 and miR-143. In bladder cancer, both MSI2 and miR-143 target the oncogene KRAS , with MSI2 promoting cell proliferation by enhancing KRAS translation, while miR-143 downregulates both genes [ 56 ]. Consistent with these findings, our study demonstrated that overexpression of miR-143 resulted in MSI2 depletion, leading to reduced cell proliferation and viability in K562 cells. Surprisingly, overexpression of miR-199a did not impact the cell proliferation rate despite MSI2 depletion. Previous research on bladder cancer cells has shown that miR-199a-3p promotes cell proliferation and functions as an oncomiR by inhibiting the expression of ZHX1 [ 57 ]. Therefore, we hypothesize that the overexpression of miR-199a-3p in K562 cells may compensate for the reduction in MSI2-induced cell proliferation through a similar mechanism. After reducing MSI2 levels by overexpressing miR-143/199a in K562 cells, a significant decrease in cell invasiveness was observed. Surprisingly, co-expression of miR-143/199a did not reduce cell invasion as effectively as the overexpression of a single miRNA. The anti-invasive role of miR-143 and miR-199a has been previously reported in various cancers such as hepatocellular carcinoma, breast and ovarian cancers [ 58 – 60 ]. In K562 cells, a myelogenous leukemia cell line, both miRNAs target MSI2 as a key gene in hematopoiesis. This data suggests a synergistic inhibitory effect of miR-143/199a specifically on MSI2 expression and K562 cell invasion, but not on cell proliferation. Overall, both miR-143-3p and miR-199a-3p could be considered tumor suppressor miRNAs in myelogenous leukemia. Further investigation into the pathways and target genes affected by the inactivation of MSI2 by the both miRNAs could lead to their potential use as effective molecular targets in leukemia treatment. In summary, our findings suggest that miR-143 and miR-199 could be considered as potential biomarkers or therapeutic targets for treating leukemogenesis. This is supported by the inverse relationship observed between the expression of these miRNAs and MSI2. Upon reducing MSI2 levels, the effects of these two miRNAs on cell proliferation and invasion differed in K562 cells. Notably, miR-143 effectively reduced both cell proliferation and invasion, whereas miR-199a only decreased cell invasion. The present study has some limitations, with the most significant one being the identification and evaluation of direct target genes of MSI2 within key signaling pathways following the overexpression of miR-143 and miR-199 in K562 cells. The downstream pathways contributing to the observed reduction in invasion and proliferation also remain unclear. Additionally, to enhance the understanding of the anti-cancer effects of these miRNAs in leukemia, similar investigations should be carried out using AML cell lines, and clinical samples from patients with myelogenous leukemia. These limitations indicate the need for further studies to validate these findings in more relevant AML models and to elucidate the signaling pathways involved in miRNA/MSI2 interactions. Declarations Ethics approval and consent to participate This study was approved by the Ethical Committee of Royan Institute (Approval ID: IR.ACECR.ROYAN.REC.1396.103. All methods were performed in accordance with the relevant guidelines and regulations. Competing interests The authors declare no competing interests. Funding This work was supported by a grant from Royan Institute (95000295). Author Contribution Liana Lachinani: Conceptualization, Methodology, Investigation, Writing–review, Editing and Supervision. Rana Iranpour: Investigation, Visualization and Writing–original draft. Mahboobeh Forouzanfar: Conceptualization and Methodology. Mohammad Hossein Nasr-Esfahani: Funding acquisition and Supervision. Kianoush Dormiani: Methodology, Writing–review, Editing, Funding acquisition and Supervision. Acknowledgement We acknowledge the assistance of Royan Institute for Biotechnology for its services and facilities. Data Availability All data generated or analyzed during this study are included in this published article. The datasets analyzed during the current study are available in the Gene Expression Omnibus (GEO) repository (GSE51908 and GSE28825). References Park, S-M. et al. Musashi2 sustains the mixed-lineage leukemia–driven stem cell regulatory program. J. Clin. Invest. 125 (3), 1286–1298 (2015). Thol, F. et al. Prognostic significance of expression levels of stem cell regulators MSI2 and NUMB in acute myeloid leukemia. Ann. Hematol. 92 , 315–323 (2013). Wurth, L. Versatility of RNA-binding proteins in cancer. Int. J. Genomics . 2012 (1), 178525 (2012). Ciafrè, S. A. & Galardi, S. microRNAs and RNA-binding proteins: a complex network of interactions and reciprocal regulations in cancer. RNA Biol. 10 (6), 934–942 (2013). Schuschel, K. et al. 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10:11:08","extension":"png","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":217813,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/c68d3a343587a850c942ae4e.png"},{"id":96710457,"identity":"52d77285-241e-4147-9391-531bb6a278cf","added_by":"auto","created_at":"2025-11-25 10:10:41","extension":"png","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48308,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/5dab86bba54a637a696f2224.png"},{"id":96709679,"identity":"1283070a-6888-468d-a782-62c0b3bc8977","added_by":"auto","created_at":"2025-11-25 10:09:30","extension":"png","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":198566,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/3c5b020382fc61414a5354cf.png"},{"id":96709444,"identity":"347b7516-851b-4880-814e-8704bf69d517","added_by":"auto","created_at":"2025-11-25 10:09:01","extension":"xml","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125577,"visible":true,"origin":"","legend":"","description":"","filename":"8d39d809a3d34e36b53f5c909393e30e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/0048623b0b2540f51b545149.xml"},{"id":96659045,"identity":"d3f74850-8d8a-44bc-b2f2-14dcb7e73986","added_by":"auto","created_at":"2025-11-24 17:48:27","extension":"html","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139551,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/2881b95870b507068778b8fd.html"},{"id":96659003,"identity":"10c6699a-875f-4fc4-977d-842a9f556aa8","added_by":"auto","created_at":"2025-11-24 17:48:26","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":157572,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted miRNA-binding sites in the 3'UTR region of MSI2 mRNA. (A) Bioinformatic tools were used to predict miRNA/MSI2 interactions. (B) The interaction of miR-143-3p at three positions in MSI2-3'UTR. miR-143-3p binds to three distinct sites: 18-24 bp, 173-179 bp, and 180-187 bp, with the last two sites overlapping. (C) Interaction of miR-199a-3p at position 4649-4655 bp of MSI2-3'UTR. The bold nucleotides that complement seed regions were deleted to create mutated 3'UTR fragments.\u003c/p\u003e","description":"","filename":"Figure1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/754be12e8da9f8acc3696ccc.jpeg"},{"id":96659004,"identity":"2b508eee-a9d6-44b2-ac00-1ea06d2e69fb","added_by":"auto","created_at":"2025-11-24 17:48:26","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91571,"visible":true,"origin":"","legend":"\u003cp\u003emiR-143-3p and miR-199a-3p directly interact with the 3'UTR of MSI2. Luciferase activity decreased in HEK293T cells co-transfected with miRNA precursors and the normal 3'UTR compared to the mutant 3'UTR. Error bars represent the mean ± SEM for three independent repeats (****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). Scr: Scramble.\u003c/p\u003e","description":"","filename":"Figure2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/d5d3e0126aaf5ca979edc5c9.jpeg"},{"id":96710176,"identity":"71d99a93-35e2-4664-9932-bcb83dcb041e","added_by":"auto","created_at":"2025-11-25 10:10:16","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":170878,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of MSI2 and MSI2-targeting miRNAs in different leukemia cell lines. (A) The relative expression levels of MSI2 were compared in two AML cell lines (HL-60 and KG-1α) and two CML cell lines (K562 and KCL22) with normal blood cells. (B) The expression levels of miR-143-3p and miR-199a-3p in leukemia cell lines were compared to normal blood cells. Data were analyzed using the 2\u003csup\u003e-∆∆Ct\u003c/sup\u003e method and are shown as log10 of means ± SEM (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/7671989ccc5754f015db7b3e.jpeg"},{"id":96709814,"identity":"75bcb2cc-0949-4241-b6ed-a43c6b8eccc2","added_by":"auto","created_at":"2025-11-25 10:09:42","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":97791,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of miR-143-3p and miR-199a-3p in K562 stable clones. K562 cells were electroporated with vectors containing miR-143 and miR-199a precursors. The expression levels of miR-143-3p and miR-199a-3p in three stable lines were compared with the scramble group. The data were analyzed using the 2\u003csup\u003e-∆∆Ct\u003c/sup\u003e method, and error bars represent means ± SEM (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/d47872ab351aff63ce2874ce.jpeg"},{"id":96659008,"identity":"7ad71c17-7def-4150-a8f3-7be836be83d4","added_by":"auto","created_at":"2025-11-24 17:48:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":126619,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression levels of MSI2 after overexpression of miR-143-3p or miR-199a-3p in K562 cells. (A) The mRNA levels of MSI2 were reduced in K562 cells stably transfected with miRNA precursors (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). (B) A representative western blot image displays the MSI2 protein expression levels in stably transfected K562 cells. This figure is a composite image created by cropping and combining two other images and then resized for consistency. The data were normalized to GAPDH as an internal control and quantified using ImageJ software (version 1.42q) (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05). Scr: Scramble.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/12087db3b65dcc9b8226d9e0.jpg"},{"id":96710015,"identity":"9825fc92-86fa-4a6b-894c-76756fb2164c","added_by":"auto","created_at":"2025-11-25 10:09:53","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59640,"visible":true,"origin":"","legend":"\u003cp\u003eThe cell proliferation of K562 clones after downregulation of MSI2. Proliferation was decreased in K562 cells transfected with miR-143-3p or miR-143/199a precursors. However, overexpression of miR-199a-3p did not significantly impact on K562 cell proliferation (***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). Scr: Scramble.\u003c/p\u003e","description":"","filename":"Figure6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/9bae834cce8234790620f9a3.jpeg"},{"id":96659015,"identity":"a9128d85-5993-4d65-b135-f4264ff993cd","added_by":"auto","created_at":"2025-11-24 17:48:26","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":533964,"visible":true,"origin":"","legend":"\u003cp\u003eThe transwell invasion assay was performed on K562 clones following the downregulation of MSI2 by overexpressing miR-143-3p or miR-199a-3p, as well as a combination of both miRNAs. The downregulation of MSI2 via miR-143-3p or miR-199a-3p overexpression resulted in a decrease in the invasion of K562 cells. (A) K562 cells transfected with scramble or miRNA precursors were allowed to migrate through the Matrigel membrane, and the migrated cells were stained with crystal violet. (B) The total number of migrated cells through the Matrigel membrane was quantified for each Transwell insert (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). Scr: Scramble.\u003c/p\u003e","description":"","filename":"Figure7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/a4895ac710f53578bbabd503.jpeg"},{"id":96913672,"identity":"1be8b038-0bfe-4303-9cdd-e7a688341f48","added_by":"auto","created_at":"2025-11-27 14:03:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2313650,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/13cdcbb8-e2a7-4e86-bda4-09e8f994ddac.pdf"},{"id":96710103,"identity":"57f01ad8-f0cd-4ac9-838d-ef07b7c2264d","added_by":"auto","created_at":"2025-11-25 10:10:04","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":131779,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471306/v1/d3a638faaa320f770a27ec72.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tumor suppressor effects of miR-143 and miR-199 on myelogenous leukemia cells through targeting RNA-binding protein Musashi2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLeukemia cells exhibit a dysregulated developmental program due to various genetic and epigenetic alterations, which are considered major steps in the progression of leukemogenesis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Additionally, post-transcriptional modifications play a crucial role in gene regulation. For instance, mRNA transcripts undergo several processes regulated by non-coding RNAs (ncRNAs) or RNA-binding proteins (RBPs), with dysregulated expression patterns observed in numerous cancer types [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. MSI2, a member of the Musashi RNA-binding protein family, plays a key role in normal hematopoiesis, and its abnormal expression is associated with various hematopoietic malignancies. Unlike MSI1, its primarily discovered homolog expressed in multiple tissues, MSI2 is predominantly expressed in hematopoietic stem cells (HSCs). Elevated expression of MSI2 leads to an increase in the number of progenitor and stem cells, rapid progression of leukemia, and a worse clinical prognosis in leukemic patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. According to the Cancer Genome Atlas (TCGA, 2012), genomic and RNA sequencing data of primary tumors showed that \u003cem\u003eMSI\u003c/em\u003e genes were not significantly mutated in most cancers but were overexpressed in several tumor types [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Elevated expression of MSI2 proteins has been found in chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL). Conversely, its depletion leads to decreased cellular proliferation, increased apoptosis, and differentiation of human myeloid leukemia cell lines [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Numerous studies have reported that dysregulated expression of MSI2 could occur due to dysregulated expression of MSI2-targeting miRNAs [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMicroRNAs (miRNAs) are small endogenous non-coding RNAs that play critical roles in various biological processes. They regulate their target genes by influencing mRNA stabilization, splicing, or post-transcriptional repression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Abnormal expression profiles of miRNAs have been observed in numerous malignancies, including leukemia. Due to their stability in biological fluids, cancer-related miRNAs have emerged as prognostic biomarkers for determining cancer subtypes, stages, and treatment responses [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In malignancies, miRNAs can impact tumor growth, invasion, apoptosis, and angiogenesis, acting as either tumor suppressor miRNAs or oncomiRs. Stimulation of tumor suppressor miRNAs or inhibition of oncomiRs can result in complete regression of the cancer [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The first case of microRNA dysregulation was reported in chronic lymphocytic leukemia (CLL), where down-regulation of miR-15 and miR-16 caused by a frequent deletion of the 13q14 region leads to increased expression of their targets including the BCL2 antiapoptotic protein [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Several critical miRNAs and their target genes involved in hematopoiesis and hematological conditions have been identified. Multiple high-throughput studies have reported that the expression of miR-143 and miR-199a, recognized as tumor suppressor miRNAs, is decreased in leukemia [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consequently, these miRNAs have been proposed as potential prognostic biomarkers and/or therapeutic targets in human leukemia [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven the negative correlation between the expression patterns of these miRNAs and MSI2, we aimed to investigate whether MSI2 could be a direct target for miR-143 and miR-199a in leukemia. To validate our bioinformatic predictions, we experimentally examined the targeting of MSI2 by miR-143 and miR-199a and assessed the impact of these miRNAs on the proliferation and invasion of K562 cells, a CML cell line.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBioinformatics studies\u003c/h2\u003e\u003cp\u003ePotential miRNA binding sites in the 3'UTR region of MSI2 were predicted using several bioinformatics prediction websites including TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.targetscan.org/\u003c/span\u003e\u003cspan address=\"http://www.targetscan.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], miRDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.mirdb.org/\u003c/span\u003e\u003cspan address=\"http://www.mirdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], miRWalk (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirwalk.umm.uni-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://mirwalk.umm.uni-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], PicTar2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pictar.mdc-berlin.de/\u003c/span\u003e\u003cspan address=\"https://pictar.mdc-berlin.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], miRMap (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cegg.unige.ch/mirmap\u003c/span\u003e\u003cspan address=\"http://cegg.unige.ch/mirmap\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], miRNAMap 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mirnamap.mbc.nctu.edu.tw/\u003c/span\u003e\u003cspan address=\"http://mirnamap.mbc.nctu.edu.tw/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], miRTarBase 6.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/index.php\u003c/span\u003e\u003cspan address=\"https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], RNAhybrid (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bibiserv.techfak.uni-bielefeld.de/rnahybrid\u003c/span\u003e\u003cspan address=\"http://bibiserv.techfak.uni-bielefeld.de/rnahybrid\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and RNA22 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cm.jefferson.edu/rna22/\u003c/span\u003e\u003cspan address=\"https://cm.jefferson.edu/rna22/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The selection of candidate miRNAs was based on factors such as minimum free energy, conservation status and the number of binding sites. MiRNAs that met all criteria and received high scores from multiple websites were selected for further analysis. Moreover, the expression profiles of the candidate miRNAs in normal and malignant hematopoietic cells were assessed using GEO array data, from GSE51908 and GSE28825. The final candidate miRNAs with decreased expression levels in malignant samples were prioritized for further investigation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDNA Constructions\u003c/h3\u003e\n\u003cp\u003eHuman miR-143 and miR-199a precursors (MIMAT0000435 and MIMAT0000232) were PCR amplified using specific primers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and \u003cem\u003epfu\u003c/em\u003e enzyme (Thermo Scientific, USA). Each miRNA precursor was subcloned into the \u003cem\u003eXba\u003c/em\u003eI and \u003cem\u003eSal\u003c/em\u003eI sites of the pBud-EGFP vector, a modified version of pBudCE4.1 (Invitrogen) that overexpresses EGFP. The miRNA precursors were placed under the CMV promoter, and the new vectors were named pBud-miR-143 and pBud-miR-199a. Additionally, the pBud-Scr vector containing the universal scramble precursor was utilized as the negative control vector [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Another set of vectors was created for the dual-luciferase assay by subcloning the wild type or mutated 3'UTR of MSI2 into the psiCHECK2 vector (Promega, USA). The 3'UTR region of MSI2 (NM_138962.3), which is 5.1 kb in length, and the mutated fragments lacking the seed-complementary regions were amplified using SOEing PCR. Multiple PCR primer sets were designed using Oligo7 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The obtained wild type and mutated 3'UTR regions were then subcloned downstream of the Renilla luciferase at the \u003cem\u003eXho\u003c/em\u003eI and \u003cem\u003eNot\u003c/em\u003eI sites of psiCHECK2. The accuracy of all constructions was confirmed through restriction enzyme digestion and sequencing analysis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe list of primers used in this study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003eA] Primers used for amplification of miRNA precursors\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003ePrimer sequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eProduct length\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003emiR-199a precursor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eF: 5'-GTCGACAGTGGTGGTTTCCTTGGCTGCTCAG-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e309 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eR: 5'-GTCTAGATCGAATCTTCTATGCGAGGCTCTGC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003emiR-143 precursor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eF: 5'-GTCGACAGACAGGAAACACAGTTGTGAGG-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e321 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eR: 5'-GTCTAGAATGGAGTCTGGAAACACTCTGTCC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eB] Primers used for amplification of normal and mutant 3'UTR regions of MSI2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePrimer name\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e\u003cb\u003ePrimer sequence\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eProduct length\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eMSI2U1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eF: 5'-GCTCGAGGCAGGTGCTTTCGTTGCCATCTCACTCTGA\u003c/p\u003e\u003cp\u003eGAGCATACCTGGATGTCC-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1427 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eR: 5'-CAGGCTTATCCACTCCTGGCCCATG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eMSI2U2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eF: 5'-CTATTGTCCAATCATTTCAGCACC-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1457 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eR: 5'-AGACCTTGGGTTAGTTGAAACCAC-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eMSI2U3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eF: 5'-TGTAAGCGTGCTGTCCTGGTACTAG-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1215 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eR: 5'-GCAAGATGTGTGTAACATTCACTGG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eMSI2U4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eF: 5'-CTCTATCCCAGTCGCCCATTAGCTTG-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1615 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eR: 5'-GCGGCCGCAACACATTGATTTCTGGACCACAGTAG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003emutU1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eF: 5'-GCAGGTGCTTTCGTTGCACTCTGAGAGCATACCTGGA\u003c/p\u003e\u003cp\u003eTGTC-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e192 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eR: 5'-GTTGTCTTATATTACAGCAGATTCGTTAGTCAACCCT\u003c/p\u003e\u003cp\u003eCGATCCGAAGTG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003emutF2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eF: 5'-TTCGGATCGAGGGTTGACTAACGAATCTGCTGTAAT\u003c/p\u003e\u003cp\u003eATAAGACAACAGC-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1254 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003emutR4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eR: 5'-TGGGATGGTACATTATGTACAGAATAAGAGAACCTT\u003c/p\u003e\u003cp\u003eCGAGGGAGGCTC-3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1156 bp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC] Primers used for RT-qPCR\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePrimer name\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eAccession number\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cb\u003ePrimer sequence\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eGAPDH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNM_001357943.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eF: 5'-CCACTCCTCCACCTTTGACG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eR: 5'-CCACCACCCTGTTGCTGTAG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u003cp\u003eMSI2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNM_138962.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eF: 5'-AACAGGCACAGAGGGTTTG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eR: 5'-ATGACTTCTTTCGGCTGAG-3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eCell lines and culture media\u003c/h3\u003e\n\u003cp\u003eIn this study, HEK293T cells, two CML cell lines (K562 and KCL-22), and two AML cell lines (KG-1α and HL-60) obtained from the Pasteur Institute in Iran were used. HEK293T cells were cultured in DMEM high glucose (Gibco, USA), while the CML and AML cell lines were cultured in RPMI1640 (Gibco), all supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 0.1 mM non-essential amino acids (all from Gibco), and 2 mM L-Glutamine (Invitrogen, USA). All cell lines were maintained in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eEstablishment of K562 cell lines stably expressing precursor miRNAs\u003c/h3\u003e\n\u003cp\u003eK562 cells were electroporated with miRNA or precursor-expressing vectors in four groups: pBud-miR-143 group, pBud-miR-199a group, a co-transfected group with both pBud-miR vectors (miR143/miR-199 group), and a pBud-Scr group as the control. For each electroporation reaction, 7 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells and 20 \u0026micro;g of each plasmid were transferred to electroporation cuvettes (0.2 cm) and electroporated at 155 V, 1000 \u0026micro;F using the Gene Pulser Xcell\u0026trade; Electroporation System (Bio-Rad, USA). The cells were immediately transferred to a prewarmed culture medium and incubated for 48 h. Subsequently, the electroporated cells were treated with zeocin (Invivogen, USA) at a final concentration of 200 \u0026micro;g/ml to establish stable clones.\u003c/p\u003e\n\u003ch3\u003eDual-luciferase reporter assay\u003c/h3\u003e\n\u003cp\u003eFor luciferase reporter assays, HEK293T cells were transiently co-transfected with luciferase reporter vectors containing the wild-type or mutant 3'UTR region of MSI2 and corresponding precursor or scramble expressing vectors. The HEK293T cells were plated in a 24-well plate, 24 h before transfection. Transfection reactions were performed using Lipofectamine LTX\u0026trade; Reagent (Invitrogen) according to the manufacturer\u0026rsquo;s protocol. The efficiency of transfection was assessed by the percentage of EGFP-positive cells 24 h after transfection. Firefly and Renilla luciferase activities were measured 48 h post-transfection using the Dual-Luciferase Reporter Assay Kit (Promega) following the manufacturer\u0026rsquo;s instructions. Finally, the relative luciferase activity was calculated by normalizing the Renilla luciferase activity to the firefly luciferase activity.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell proliferation assay\u003c/h2\u003e\u003cp\u003eTo assess the effect of miRNA overexpression on K562 cell proliferation, an MTS assay was conducted on clones that had been stably transfected with miRNA precursors. Each clone was seeded at a density of 10\u003csup\u003e5\u003c/sup\u003e cells/ml in triplicate wells of a 96-well plate. After 24 h, 20 \u0026micro;l of MTS solution was added to each well, and the plates were incubated for an additional 3 hours. Finally, the absorbance was measured at 490 nm and 630 nm using a Stat Fax 2100 Microplate Reader.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTranswell invasion assay\u003c/h3\u003e\n\u003cp\u003eAfter miRNA overexpression in K562 cells, cell invasiveness was assessed using the transwell invasion assay. Transwell inserts with an 8.0 \u0026micro;m pore size (Corning, USA) were pre-coated with 0.3 mg/ml Matrigel (Sigma, Germany) diluted in DMEM and incubated at 37\u0026deg;C for 24 h before cell culture. K562 stable clones at a density of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/ml were seeded in the serum-free medium in the upper chamber, while the lower chambers were filled with serum-containing medium. The cells were then incubated in a humidified cell culture incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 48 h. Subsequently, the cells on the lower surface were fixed with 4% paraformaldehyde and stained with a 1% crystal violet solution. Cell counting was conducted in 10 random fields of captured images using a light microscope.\u003c/p\u003e\n\u003ch3\u003eRNA isolation and expression quantitation\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from normal peripheral blood cells, K562, KCL-22, KG-1α, and HL-60 cell lines using TRIzol reagent (Invitrogen) following the manufacturer's protocol. 500 ng of total RNA was treated with DNase I (Thermo Scientific) to eliminate potential genomic DNA contamination, and cDNA synthesis was carried out using a cDNA synthesis kit (Takara, Japan) with random hexamer primers. RT-qPCR was conducted with 50 ng of each cDNA in a Step One Plus Real-Time PCR thermal cycler (Applied Biosystems, USA) under pre-optimized conditions using SYBR premix ExTaqII (Takara) and specific primers listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC. The specific primers for the \u003cem\u003eMSI2\u003c/em\u003e gene were designed using Beacon designer (Version 7.2, USA). \u003cem\u003eGlyceraldehyde 3-phosphate dehydrogenase\u003c/em\u003e (\u003cem\u003eGAPDH\u003c/em\u003e) was utilized as the reference gene for normalizing the expression of target genes. For miRNA cDNA synthesis, a universal cDNA synthesis kit (Exiqon, Germany) was employed. RNA U6 (Exiqon) served as an internal control for quantifying and normalizing miRNA expression levels. All reactions were performed in triplicate to ensure result reproducibility. The relative quantification of gene or miRNA expression was determined using the threshold cycle difference methods (2\u003csup\u003e\u0026minus;ΔCt\u003c/sup\u003e and 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e methods).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\u003cp\u003eTo evaluate MSI2 protein expression in stably transfected K562 clones, protein extraction was carried out using Trizol reagent (Invitrogen). Approximately 30 \u0026micro;g of total protein lysates were separated by 10\u0026ndash;15% SDS-PAGE, transferred onto PVDF membranes, and then cut to a specific size based on the expected band size. The membranes were then probed with primary antibodies against MSI2 (1:4000, Abcam, Ab76148) and GAPDH (1:5000, SANTA CRUZ, sc-2357). Subsequently, the membranes were incubated with HRP-conjugated IgG secondary antibodies specific to MSI2 and GAPDH antibodies: mouse anti-rabbit IgG (1:7500, SANTA CRUZ, sc-2357) and goat anti-mouse IgG (1:5000, Dako, P0447). The protein bands of interest were visualized using the Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, USA), and the protein expression levels were quantified using ImageJ software (version 1.42q).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData values were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) of replicates. Comparisons between groups were conducted using analysis of variance (ANOVA) and t-tests. Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 0.001, or 0.0001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003ePrediction of candidate miRNAs\u003c/h2\u003e\u003cp\u003eSeveral MSI2-targeting miRNAs were predicted using miRNA-target prediction tools. Among the high-scored miRNAs predicted by most software were miR-143-3p and miR-199a-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Hsa-miR143-3p was found to bind at three positions in the 3'UTR of MSI2, with two binding sites overlapping, while hsa-miR199a-3p binds to only one site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Additionally, the expression levels of these candidate miRNAs were analyzed in two high-throughput studies, GSE51908 and GSE28825. The analysis of normal and malignant hematopoietic cells in these studies revealed that the expression of both miRNAs was decreased in leukemia cell lines. These findings enhanced the accuracy of the miRNA selection process.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eInteraction of miR-143-3p and miR-199a-3p with MSI2 mRNA\u003c/h2\u003e\u003cp\u003eTo validate the direct interaction of miR-143-3p and miR-199a-3p with the MSI2 transcript, HEK293T cells were co-transfected with precursors of both miRNAs and luciferase reporter vectors containing the wild-type or mutant 3'UTR region of MSI2. \u003cem\u003eIn silico\u003c/em\u003e analysis indicated three binding sites for miR-143-3p and one binding site for miR-199a-3p in the 3'UTR region of MSI2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Mutated forms of the 3'UTR region were generated by eliminating the complementary sequences of the seed regions using SOEing PCR for each miRNA. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, overexpression of miR-143 and miR-199a led to a notable decrease in relative luciferase activity in the wild-type groups compared to the mutant groups. Surprisingly, the expression of scramble sequences also resulted in reduced luciferase activity. This decrease could attributed to the endogenous expression and functional activity of both miRNAs, as reported in other studies. These results confirmed the direct targeting of the MSI2 transcript by both candidate miRNAs as well [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCorrelation of MSI2 with miR-143-3p and miR-199a-3p in leukemia cell lines\u003c/h2\u003e\u003cp\u003eThe endogenous expression level of MSI2 was measured by RT-qPCR in four leukemia cell lines: KCL-22, K562, KG-1α, and HL-60. Among these cell lines, KG-1α and K562 exhibited the highest expression level of MSI2 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Conversely, RT-qPCR analysis confirmed the reduced expression levels of miR-143-3p and miR-199a-3p in all leukemia cells compared to normal blood cells. However, the lowest expression levels for both miRNAs were observed in K562 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Therefore, we chose K562 cells for further studies.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eOverexpression effects of miR-143-3p and miR-199a-3p on MSI2 in K562 cells\u003c/h2\u003e\u003cp\u003eTo investigate the impact of miR-143-3p and miR-199a-3p on MSI2 expression, we established K562 clones that stably overexpressed the corresponding miRNAs. We selected the K562 cell line for its high MSI2 expression and low levels of both miRNAs compared to other cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Electroporation of K562 cells with the constructed vectors and subsequent zeocin treatment led to the generation of four stable clones: K562-Scr, K562-miR143, K562-miR-199, and K562-miR143/miR-199. The overexpression of miRNAs was confirmed through RT-qPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The results indicated a decrease in both MSI2 mRNA and protein levels in each clone due to the overexpression of the candidate miRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Notably, the reduction in MSI2 protein was most significant in the K562-miR143/miR-199 clone, highlighting the synergistic effect of the two miRNAs in reducing MSI2 protein levels.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eOverexpression effect of miR-143-3p and miR-199a-3p on cell proliferation and invasion\u003c/h2\u003e\u003cp\u003eThe MTS assay was used to evaluate the proliferation of K562 clones stably expressing miR-143-3p and miR-199a-3p. The results indicated that miR-143-3p overexpression reduced cell proliferation, while miR-199a-3p did not significantly affect cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Furthermore, the transwell invasion assay demonstarted that K562 clones with stable overexpression of miR-143-3p and miR-199a-3p exhibited lower invasivness compared to intact K562 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In clones co-transfected with both miR-143 and miR-199a, the inhibition of cell invasion was less pronounced than when each miRNA was transfected individually. Interestingly, despite miR-199a-3p not affecting cell proliferation, it had the most significant inhibitory effect on cell invasion.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOne of the characteristic features of various malignancies, including leukemogenesis, is the up and down-regulation of oncogenes and tumor suppressor genes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. MSI2 is a well-known RNA-binding protein that plays critical roles in normal and malignant hematopoiesis. In fractionated blood cell populations, hematopoietic stem cells (HSCs) exhibit higher levels of MSI2 compared to progenitor cells, differentiated myeloid cells, and lymphocytes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In leukemogenesis, the expression of MSI2 is increased, and its knockdown leads to a reduction in proliferation and an increase in apoptosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In several disorders and malignancies, increased expression of the MSI2 protein is associated with aberrant expression of certain miRNAs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Therefore, we aimed to investigate whether a similar association exists between MSI2 and miRNAs in myeloid leukemia and, if so, which miRNAs could directly interact with MSI2 mRNA. According to in silico studies, we identified several miRNAs that potentially interact with MSI2 mRNA. A negative correlation between some miRNAs and MSI2 was observed by analyzing the expression patterns of predicted miRNAs in various malignant hematopoietic cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Among these, two well-known tumor suppressor miRNAs, miR-143-3p and miR-199a-3p, were identified. The tumor suppressor role of miR-199a-3p has been reported in hepatocellular carcinoma, NSCLC (lung cancer), ovarian cancer and glioma, where it decreases proliferation, migration, and invasion of cancer cells by targeting downstream genes of PAK4/Raf/MEK/ERK and PI3K-mTOR pathways [\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Similarly, the tumor suppressor function of miR-143-3p has been validated in multiple cancer types including bladder cancer, esophageal squamous cell carcinoma, renal cell carcinoma and papillary thyroid carcinoma [\u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Both candidate miRNAs are also known as leukemia-associated miRNAs [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we confirmed that miR-143-3p and miR-199a-3p directly interact with the 3'UTR region of MSI2 mRNA using a dual luciferase assay. Moreover, overexpression of both miRNAs resulted in decreased expression levels of MSI2 in K562 cells at both the mRNA and protein levels. Previous studies have shown that MSI2 knockdown in K562 cells leads to inhibition of cell growth and cell cycle arrest by targeting p21, cyclin D1, as well as the ERK/MAPK and p38/MAPK pathways [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Additionally, miR-143 has been reported to reduce cell proliferation through various pathways, including PI3K/AKT and MAPK, in leukemia, bladder, and prostate cancers [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Interestingly, the MAPK pathway is reciprocally regulated by MSI2 and miR-143. In bladder cancer, both MSI2 and miR-143 target the oncogene \u003cem\u003eKRAS\u003c/em\u003e, with MSI2 promoting cell proliferation by enhancing KRAS translation, while miR-143 downregulates both genes [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Consistent with these findings, our study demonstrated that overexpression of miR-143 resulted in MSI2 depletion, leading to reduced cell proliferation and viability in K562 cells. Surprisingly, overexpression of miR-199a did not impact the cell proliferation rate despite MSI2 depletion. Previous research on bladder cancer cells has shown that miR-199a-3p promotes cell proliferation and functions as an oncomiR by inhibiting the expression of ZHX1 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Therefore, we hypothesize that the overexpression of miR-199a-3p in K562 cells may compensate for the reduction in MSI2-induced cell proliferation through a similar mechanism.\u003c/p\u003e\u003cp\u003eAfter reducing MSI2 levels by overexpressing miR-143/199a in K562 cells, a significant decrease in cell invasiveness was observed. Surprisingly, co-expression of miR-143/199a did not reduce cell invasion as effectively as the overexpression of a single miRNA. The anti-invasive role of miR-143 and miR-199a has been previously reported in various cancers such as hepatocellular carcinoma, breast and ovarian cancers [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In K562 cells, a myelogenous leukemia cell line, both miRNAs target \u003cem\u003eMSI2\u003c/em\u003e as a key gene in hematopoiesis. This data suggests a synergistic inhibitory effect of miR-143/199a specifically on MSI2 expression and K562 cell invasion, but not on cell proliferation. Overall, both miR-143-3p and miR-199a-3p could be considered tumor suppressor miRNAs in myelogenous leukemia. Further investigation into the pathways and target genes affected by the inactivation of MSI2 by the both miRNAs could lead to their potential use as effective molecular targets in leukemia treatment.\u003c/p\u003e\u003cp\u003eIn summary, our findings suggest that miR-143 and miR-199 could be considered as potential biomarkers or therapeutic targets for treating leukemogenesis. This is supported by the inverse relationship observed between the expression of these miRNAs and MSI2. Upon reducing MSI2 levels, the effects of these two miRNAs on cell proliferation and invasion differed in K562 cells. Notably, miR-143 effectively reduced both cell proliferation and invasion, whereas miR-199a only decreased cell invasion. The present study has some limitations, with the most significant one being the identification and evaluation of direct target genes of MSI2 within key signaling pathways following the overexpression of miR-143 and miR-199 in K562 cells. The downstream pathways contributing to the observed reduction in invasion and proliferation also remain unclear. Additionally, to enhance the understanding of the anti-cancer effects of these miRNAs in leukemia, similar investigations should be carried out using AML cell lines, and clinical samples from patients with myelogenous leukemia. These limitations indicate the need for further studies to validate these findings in more relevant AML models and to elucidate the signaling pathways involved in miRNA/MSI2 interactions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eThis study was approved by the Ethical Committee of Royan Institute (Approval ID: IR.ACECR.ROYAN.REC.1396.103. All methods were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by a grant from Royan Institute (95000295).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLiana Lachinani: Conceptualization, Methodology, Investigation, Writing\u0026ndash;review, Editing and Supervision. Rana Iranpour: Investigation, Visualization and Writing\u0026ndash;original draft. Mahboobeh Forouzanfar: Conceptualization and Methodology. Mohammad Hossein Nasr-Esfahani: Funding acquisition and Supervision. Kianoush Dormiani: Methodology, Writing\u0026ndash;review, Editing, Funding acquisition and Supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the assistance of Royan Institute for Biotechnology for its services and facilities.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article. The datasets analyzed during the current study are available in the Gene Expression Omnibus (GEO) repository (GSE51908 and GSE28825).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePark, S-M. et al. Musashi2 sustains the mixed-lineage leukemia\u0026ndash;driven stem cell regulatory program. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cb\u003e125\u003c/b\u003e (3), 1286\u0026ndash;1298 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThol, F. et al. 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Oncol.\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e (5), 2037\u0026ndash;2045 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"miRNA, MSI2, myeloid leukemia, RNA-binding protein, tumor suppressor","lastPublishedDoi":"10.21203/rs.3.rs-7471306/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7471306/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe RNA-binding protein Musashi2 plays a critical role in normal and malignant hematopoiesis. Aberrant expression of Musashi2 is linked to the inappropriate expression of several miRNAs. In this study, we observed downregulation of miR-143 and miR-199 in CML and AML cell lines. Bioinformatic analysis revealed potential interactions between these miRNAs and Musashi2 mRNA, which were also confirmed by our experimental data. In K562 cells, which showed the strongest negative correlation between Musashi2 and the candidate miRNAs, overexpression of miRNAs led to reduced cell proliferation and invasiveness. Overexpression of miR-143 and miR-199 decreased the mRNA and protein levels of Musashi2 in K562 cells, with miR-143 showing a more significant reduction in cell proliferation and invasion compared to miR-199a. Our results indicate that the downregulation of Musashi2 following the overexpression of these tumor suppressor miRNAs is responsible for diminishing the cancerous properties of malignant myeloid cells. According to our findings, these miRNAs could be new potential molecular targets for the treatment of myelogenous leukemias.\u003c/p\u003e","manuscriptTitle":"Tumor suppressor effects of miR-143 and miR-199 on myelogenous leukemia cells through targeting RNA-binding protein Musashi2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 17:48:22","doi":"10.21203/rs.3.rs-7471306/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-05T07:18:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-02T06:00:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43137331931255811328627098012009354326","date":"2026-01-01T00:36:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51864169077176114229900826177424169718","date":"2025-12-31T18:34:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-08T15:08:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172142452296992385171777830019908215111","date":"2025-12-06T10:17:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"207505769726305368680449370124435037182","date":"2025-12-02T14:41:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-25T10:32:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"295596765369679561424786329343837114541","date":"2025-11-20T03:49:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"184393069390676266220083798731180268270","date":"2025-11-13T15:50:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-13T10:16:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-04T14:16:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-05T11:23:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-30T07:02:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-30T06:59:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"31af0fa7-2a1f-4988-8202-da95ff6fad59","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58453080,"name":"Biological sciences/Cancer"},{"id":58453081,"name":"Biological sciences/Cell biology"},{"id":58453082,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":58453083,"name":"Biological sciences/Molecular biology"},{"id":58453084,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-11T05:08:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-24 17:48:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7471306","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7471306","identity":"rs-7471306","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}