Musashi-1 Research Articles

Expression of Musashi-1 Increases in Bone Healing.

Musashi-1 (MSI1) is an RNA-binding protein that regulates progenitor cells in adult and developing organisms to maintain self-renewal capacities. The role of musashi-1 in the bone healing environment and its relation with other osteogenic factors is unknown. In the current study, we analyze the expression of MSI1 in an experimental model of rat femoral bone fractures. We also analyze the relation between MSI1 expression and the expression of two osteogenic markers: periostin (POSTN) and runt-related transcription factor 2 (RUNX2). We use histological, immunohistochemical, and qPCR techniques to evaluate bone healing and the expression of MSI1, POSTN, and RUNX2 over time (4, 7, and 14 days). We compare our findings with non-fractured controls. We find that in bone calluses, the number of cells expressing MSI1 and RUNX2 increase over time and the intensity of POSTN expression decreases over time. Within bone calluses, we find the presence of MSI1 expression in mesenchymal stromal cells, osteoblasts, and osteocytes but not in hypertrophic chondrocytes. After 14 days, the expression of MSI1, POSTN, and RUNX2 was significantly correlated. Thus, we conclude that musashi-1 potentially serves in the osteogenic differentiation of mesenchymal stromal cells and bone healing. Therefore, further studies are needed to determine the possibility of musashi-1′s role as a clinical biomarker of bone healing and therapeutic agent for bone regeneration.

Deciphering the Role of Intestinal Crypt Cell Populations in Resistance to Chemotherapy.

Intestinal crypts are composed of heterogeneous and highly plastic cell populations. Lgr5high-stem cells (SC) are responsible for homeostatic renewal, but other cells can revert to an SC-like phenotype to maintain epithelial integrity. Despite their distinct roles in orchestrating homeostasis, both populations have been designated as the putative "cell-of-origin" of colorectal cancer. However, their respective involvement in the emergence of drug-resistant cancer SCs (CSC), responsible for tumor relapse and associated with poor outcome of colorectal cancer, remains elusive. In this context, the intestinal SC/progenitor-marker Musashi1 (MSI1) is interesting as it plays important functions in intestinal homeostasis and is frequently overexpressed in human colorectal cancer. Therefore, our aims were: (i) to study the impact of chemotherapy on Lgr5-expressing and MSI1-expressing cell populations, (ii) to explore the effect of increased MSI1 levels in response to treatment, and (iii) to evaluate the relevance in human colorectal cancer. Engineered mouse models treated with the therapeutic agent 5-fluorouracil showed that upon increased MSI1 levels, Lgr5high SCs remain sensitive while Lgr5low progenitors reprogram to a drug-resistant phenotype. This resulted in the expansion of an MSI1-expressing cell subpopulation with improved resistance to DNA damage and increased detoxification, typical properties of dormant-CSCs that can reactivate after chemotherapy. Analysis in patients with colorectal cancer revealed a correlation between MSI1 levels and tumor grading, CSC phenotype, and chemoresistance. Altogether, these results shed new light on the biology and plasticity of normal crypt and cancer cell populations and also open new perspectives to target MSI1 to improve chemotherapy outcome. SIGNIFICANCE: This study unveils paradoxical roles for MSI1, underlining its importance in facilitating intestinal regeneration upon injury but also unraveling its new function in drug-resistant colorectal cancer stem cells.

Musashi 2 (MSI2) expression as an independent prognostic biomarker in non-small cell lung cancer (NSCLC).

BackgroundMusashi-2 (MSI2) is a member of RNA-binding protein family that regulates mRNA translation of numerous intracellular targets and influences maintenance of stem cell identity. This study assessed MSI2 as a potential clinical biomarker in non-small cell lung cancer (NSCLC).MethodsThe current study included 40 patients with NSCLC, of whom one presented with stage 1, 14 presented with stage II, 15 presented with stage III, and 10 patients had stage IV. All patients received standard of care treatments. All patient samples were obtained before treatment started. We used immunohistochemical (IHC) approach to measure MSI2 protein expression in matching specimens of normal lung versus tumor tissues, and primary versus metastatic tumors, followed by correlative analysis in relation to clinical outcomes. In parallel, clinical correlative analysis of MSI2 mRNA expression was performed in silico using publicly available datasets (TCGA/ICGC and KM plots).ResultsMSI2 protein expression in patient samples was significantly elevated in NSCLC primary tumors versus normal lung tissue (P=0.03). MSI2 elevated expression positively correlated with a decreased progression free survival (PFS) (P=0.026) combined for all stages and with overall survival (OS) at stage IV (P=0.013). Elevated MSI2 expression on RNA level was confirmed in primary tumor versus normal tissue samples in TCGA dataset (P<0.0001), and positively correlated with decreased OS (P=0.02). No correlation was observed between MSI2 expression and age, sex, smoking, and treatment type.ConclusionsElevated MSI2 expression in primary NSCLC tumors is associated with poor prognosis and can be used as a novel potential prognostic biomarker in NSCLC patients. Future studies in an extended patient cohort are warranted.

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC)

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.

Potential Role of Musashi-2 RNA-Binding Protein in Cancer EMT.

Local invasion and distant metastasis are the key hallmarks in the aggressive progression of malignant tumors, including the ability of cancer cells to detach from the extracellular matrix overcome apoptosis, and disseminate into distant sites. It is generally believed that this malignant behavior is stimulated by epithelial-mesenchymal transition (EMT). Musashi (MSI) RNA-binding proteins, belonging to the evolutionarily conserved RNA-binding proteins (RBP) family, were originally discovered to regulate asymmetric cell division during embryonic development. Recently, Musashi-2 (MSI2), as a key member of MSI family, has been prevalently reported to be tightly associated with the advanced clinical stage of several cancers. Multiple oncogenic signaling pathways mediated by MSI2 play vital roles in EMT. Here, we systematically reviewed the detailed role and signal networks of MSI2 in regulating cancer development, especially in EMT signal transduction, involving EGF, TGF-β, Notch, and Wnt pathways.

Exosomal circ_IFT80 Enhances Tumorigenesis and Suppresses Radiosensitivity in Colorectal Cancer by Regulating miR-296-5p/MSI1 Axis.

BackgroundExosomal circular RNAs (circRNAs) can act as biomarkers and play crucial roles in colorectal cancer (CRC) and radiosensitivity. The aim of this study was to explore the functions and regulatory mechanism of exosomal circRNA intraflagellar transport 80 (circ_IFT80) in tumorigenesis and radiosensitivity of CRC.MethodsExosomes were detected using transmission electron microscopy (TEM). Protein levels were determined by Western blot assay. The expression of circ_IFT80, microRNA-296-5p (miR-296-5p) and musashi1 (MSI1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell cycle distribution, cell apoptosis, and cell proliferation were detected by flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. Colony formation assay was used to determine the radiosensitivity of cells. The interaction between miR-296-5p and circ_IFT80 or MSI1 was verified by dual-luciferase reporter assay. A xenograft tumor model was established to explore the role of exosomal circ_IFT80 in vivo.ResultsCirc_IFT80 was upregulated in exosomes derived from CRC patient serum and CRC cells. Exosomal circ_IFT80 or circ_IFT80 overexpression facilitated tumorigenesis by increasing cell proliferation and reducing apoptosis, and inhibited radiosensitivity via promoting colony formation and inhibiting apoptosis. Additionally, circ_IFT80 acted as a sponge of miR-296-5p, and miR-296-5p reversed the effects of circ_IFT80 on tumorigenesis and radiosensitivity. Moreover, MSI1 was a direct target of miR-296-5p. Furthermore, miR-296-5p overexpression inhibited tumorigenesis and promoted radiosensitivity by downregulating MSI1. Exosomal circ_IFT80 also accelerated tumor growth in vivo.ConclusionExosomal circ_IFT80 promoted tumorigenesis and reduced radiosensitivity by regulating miR-296-5p/MSI1 axis, which might provide a novel avenue for treatment of CRC.

Musashi 2 influences chronic lymphocytic leukemia cell survival and growth making it a potential therapeutic target

Progression of chronic lymphocytic leukemia (CLL) results from the expansion of a small fraction of proliferating leukemic B cells. When comparing the global gene expression of recently divided CLL cells with that of previously divided cells, we found higher levels of genes involved in regulating gene expression. One of these was the oncogene Musashi 2 (MSI2), an RNA-binding protein that induces or represses translation. While there is an established role for MSI2 in normal and malignant stem cells, much less is known about its expression and role in CLL. Here we report for the first time ex vivo and in vitro experiments that MSI2 protein levels are higher in dividing and recently divided leukemic cells and that downregulating MSI2 expression or blocking its function eliminates primary human and murine CLL and mature myeloid cells. Notably, mature T cells and hematopoietic stem and progenitor cells are not affected. We also confirm that higher MSI2 levels correlate with poor outcome markers, shorter time-to-first-treatment, and overall survival. Thus, our data highlight an important role for MSI2 in CLL-cell survival and proliferation and associate MSI2 with poor prognosis in CLL patients. Collectively, these findings pinpoint MSI2 as a potentially valuable therapeutic target in CLL.

LncRNA TUG1 promotes Ewing's sarcoma cell proliferation, migration, and invasion via the miR-199a-3p-MSI2 signaling pathway.

The aim of this study was to investigate the roles and potential mechanisms of long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) in the proliferation, migration, and invasion of Ewing's sarcoma cells. RT-qPCR was used to detect the expression of TUG1, microRNA-199a-3p (miR-199a-3p), and musashi2 (MSI2) in Ewing's sarcoma tissues and cell lines. Kaplan-Meier overall survival curves showed the survival rates of Ewing's sarcoma patients with high and low expression of TUG1. The association between the expressions of TUG1/MSI2 and miR-199a-3p in Ewing's sarcoma tissues was assessed by Pearson's correlation analysis. Cell proliferation, migration, and invasion were detected by CCK-8 assay and Transwell assay, respectively. The protein level of MSI2 was determined using western blotting. The interaction between TUG1/MSI2 and miR-199a-3p was validated by the dual-luciferase reporter assay. The levels of TUG1 and MSI2 were increased, while the level of miR-199a-3p was decreased in Ewing's sarcoma tissues and cells. High expression of TUG1 or MSI2 indicated a decreased overall survival rate of Ewing's sarcoma patients. TUG1/MSI2 level was negatively correlated with miR-199a-3p level. While TUG1 level was positively correlated with MSI2 level. In Ewing's sarcoma cells, knockdown of TUG1/MSI2 or overexpression of miR-199a-3p inhibited cell proliferation, migration, and invasion, whereas the overexpression of TUG1/MSI2 presented the opposite results. TUG1 functioned as a competing endogenous RNA to regulate MSI2 expression by sponging miR-199a-3p. Finally, miR-199a-3p inhibitor or MSI2 overexpression counteracted the TUG1 knockdown-mediated inhibitory effect on Ewing's sarcoma cell proliferation, migration, and invasion. TUG1 promotes proliferation, migration, and invasion of Ewing's sarcoma cells via sequestering miR-199a-3p to enhance the MSI2 expression, suggesting that TUG1 might be a potential target for treating Ewing's sarcoma.

MBRS-01. DISSECTING REGULATORS OF THE ABERRANT POST-TRANSCRIPTIONAL LANDSCAPE IN MYC-AMPLIFIED GROUP 3 MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common solid malignant pediatric brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that an RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. Unbiased integrative multi-omics analysis of MSI1 function in human G3 MB suggests a paradigm shift beyond traditional gene-based profiling of oncogenes. Here we identify MSI1 as an oncogene in G3 MB driving stem cell self-renewal through stabilization of HIPK1 mRNA, a downstream context-specific therapeutic target for drug discovery.

Musashi-1 promotes cancer stem cell properties of glioblastoma cells via upregulation of YTHDF1

BackgroundGlioblastoma (GBM) is the most lethal brain tumor characterized by high morbidity and limited treatment options. Tumor malignancy is usually associated with the epigenetic marks, which coordinate gene expression to ascertain relevant phenotypes. One of such marks is m6A modification of RNA, whose functional effects are dependent on the YTH family m6A reader proteins.Methods and resultsIn this study, we investigated the expression of fiveYTH family proteins in different GBM microarray datasets from the Oncominedatabase, and identified YTHDF1 as the most highly overexpressed member of thisfamily in GBM. By performing the knockdown of YTHDF1 in a GBM cell line, wefound that it positively regulates proliferation, chemoresistance and cancerstem cell-like properties. Musashi-1 (MSI1) is a postranscriptional geneexpression regulator associated with high oncogenicity in GBM. By knocking downand overexpressing MSI1, we found that it positively regulates YTHDF1expression. The inhibitory effectsimposed on the processes of proliferation and migration by YTHDF1 knockdownwere shown to be partially rescued by concomitant overexpression of MSI1. MSI1and YTHDF1 were shown to be positively correlated in clinical glioma samples,and their concomitant upregulation was associated with decreased survival ofglioma patients. We identified the direct regulation of YTHDF1 by MSI1.ConclusionsGiven the fact that both proteins are masterregulators of gene expression, and both of them are unfavorable factors in GBM,we suggest that in any future studies aimed to uncover the prognostic value andtherapy potential, these two proteins should be considered together.

Down-Regulation of Circ_0032833 Sensitizes Colorectal Cancer to 5-Fluorouracil and Oxaliplatin Partly Depending on the Regulation of miR-125-5p and MSI1.

Background5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX) is an effective chemotherapy for colorectal cancer (CRC) in clinic. It remains unclear regarding the effect of circular RNA (circRNA) circ_0032833 on regulating chemosensitivity in CRC.MethodsDrug resistance analysis was performed by Cell Counting Kit-8 (CCK-8) assay. All RNA and protein levels were, respectively, measured via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Cellular colony capacity, apoptosis and metastasis were evaluated using colony formation assay, Annexin-FITC/PI flow cytometry and transwell migration/invasion assays. The molecular combination was notarized using dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The in vivo experiment was conducted via xenograft tumors in mice.ResultsCirc_0032833 was significantly up-regulated in FOLFOX-resistant CRC and associated with drug resistance. Knockdown of circ_0032833 could sensitize FOLFOX-resistant CRC cells to 5-fluorouracil and oxaliplatin. Circ_0032833 was a miR-125-5p sponge, and miR-125-5p overexpression was responsible for the effect of circ_0032833 knockdown on 5-fluorouracil and oxaliplatin sensitivities. Besides, miR-125-5p targeted Musashi1 (MSI1) to increase the susceptibility of 5-fluorouracil and oxaliplatin in FOLFOX-resistant CRC cells. We found that circ_0032833 generated the regulation on MSI1 by sponging miR-125-5p. Circ_0032833 down-regulation also promoted the 5-fluorouracil and oxaliplatin sensitivities partly through the miR-125-5p/MSI1 axis in vivo.ConclusionThis study illuminated an unambiguous mechanism circ_0032833/miR-125-5p/MSI1 on regulating 5-fluorouracil and oxaliplatin sensitivities in FOLFOX therapy, maybe providing a deep insight of resistance formation and developing a novel strategy to enhance chemosensitivity in CRC.

Interplay among p21Waf1/Cip1, MUSASHI-1 and Kr\xfcppel-like factor 4 in activation of Bmi1-CreER reserve intestinal stem cells after gamma radiation-induced injury

Gamma radiation is a commonly used adjuvant treatment for abdominally localized cancer. Since its therapeutic potential is limited due to gastrointestinal (GI) syndrome, elucidation of the regenerative response following radiation-induced gut injury is needed to develop a preventive treatment. Previously, we showed that Krüppel-like factor 4 (KLF4) activates certain quiescent intestinal stem cells (ISCs) marked by Bmi1-CreER to give rise to regenerating crypts following γ irradiation. In the current study, we showed that γ radiation-induced expression of p21Waf1/Cip1 in Bmi1-CreER cells is likely mitigated by MUSASHI-1 (MSI1) acting as a negative regulator of p21Waf1/Cip1 mRNA translation, which promotes exit of the Bmi1-CreER cells from a quiescent state. Additionally, Bmi1-specific Klf4 deletion resulted in decreased numbers of MSI1+ cells in regenerating crypts compared to those of control mice. We showed that KLF4 binds to the Msi1 promoter and activates its expression in vitro. Since MSI1 has been shown to be crucial for crypt regeneration, this finding elucidates a pro-proliferative role of KLF4 during the postirradiation regenerative response. Taken together, our data suggest that the interplay among p21Waf1/Cip1, MSI1 and KLF4 regulates Bmi1-CreER cell survival, exit from quiescence and regenerative potential upon γ radiation-induced injury.

Musashi1 expression is negatively correlated with numb expression in brain metastases.

The expression of tumor stem cell markers musashi1 (msi1) and numb in brain metastases were detected to explore their roles in the development of brain metastases.A total of 51 cases of brain metastasis, 29 cases of primary tumor and 15 cases of normal brain tissue were selected. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to detect msi1 and numb expression at the protein and mRNA levels. Correlation between msi1 and numb in brain metastases were evaluated.Immunohistochemistry and RT-PCR showed that no significant difference in the expression of msi1 and numb between brain metastases and primary tumors was observed (P > .05); the expression of msi1 and numb in brain metastases was significantly higher than that in normal brain tissues (P < .05); and the expression of msi1 and numb in primary tumors was significantly higher than that in normal brain tissues (P < .05). In general, the expression of msi1 gene was negatively correlated with the expression of numb at mRNA level by Pearson correlation analysis (r = −0.345, P < .05). Additionally, the expression of msi1 and numb in brain metastases was not related to gender, age, and tissue origin (P > .05).Msi1 is highly expressed in brain metastases and primary tumors, while numb is lowly expressed in brain metastases and primary tumors; msi1 and numb are negatively correlated in brain metastases, suggesting that msi1 and numb may have regulatory mechanisms in the development of brain metastases.

Knockdown of MIR4435-2HG Suppresses the Proliferation, Migration and Invasion of Cervical Cancer Cells via Regulating the miR-128-3p/MSI2 Axis in vitro.

PurposeLong non-coding RNAs (lncRNAs) play major roles in the development of several cancers, including cervical cancer (CC). The purpose of the present study is to explore the regulatory mechanism of MIR4435-2HG on CC in vitro.Patients and MethodsFifty-nine pairs of CC tissues and adjacent normal tissues were collected from 59 patients by resection. The expression of lncRNA MIR4435-2HG, microRNA (miR)-128-3p and Musashi 2 (MSI2) in CC tissues and cells was detected by quantitative reverse-transcription PCR (qRT-PCR). The viability of CC cells was detected by 3-(4, 5-Dimethyl-2-Thiazolyl)-2, 5-Diphenyl-2-H-Tetrazolium Bromide (MTT) assay. The ability of migration and invasion in CC cells was measured by wound healing assay and transwell invasion assay, respectively. Starbase software and Targetscan software were utilized to predict the relationship between miR-128 and MIR4435-2HG/MSI2, respectively. The dual-luciferase reporter assay was used to confirm these interactions.ResultsLncRNA MIR4435-2HG expression was significantly up-regulated in CC tissues (P < 0.001) and cells (P < 0.01). Knockdown of MIR4435-2HG inhibited the proliferation, migration and invasion of CC cells (P < 0.01). MiR-128-3p was a target of MIR4435-2HG and was negatively modulated by MIR4435-2HG (P < 0.0001, r = −0.6331). Up-regulation of miR-128-3p suppressed the proliferation, migration and invasion of CC cells (P < 0.01). In addition, MSI2 was the target gene of miR-128-3p and negatively regulated by miR-128-3p (P < 0.0001, r = −0.4775). Both down-regulation of miR-128-3p and up-regulation of MSI2 reversed the inhibitory effects of MIR4435-2HG knockdown on the proliferation, migration and invasion of CC cells (P < 0.05).ConclusionMIR4435-2HG knockdown suppresses the proliferation, migration and invasion of CC cells through regulating the miR-128-3p/MSI2 axis, providing a possible therapeutic strategy for CC.

Abstract 4140: Dissecting the structural basis for rational inhibitor design: Crystal and solution structures of human oncoprotein Musashi-2 N-terminal RNA recognition motif 1

Abstract RNA-binding proteins (RBPs) are key regulators of cellular functions, especially in post-transcriptional regulations. Dysregulation of RBPs is implicated in many diseases including cancer. One of the RBPs that is overexpressed in a variety of human cancer is Musashi-2 (MSI2). Elevated MSI2 expression is associated with ectopic oncogenic pathways including but not limited to NUMB/Notch, PTEN/mTOR, TGF-β/SMAD3, making MSI2 a promising therapeutic target for cancer. Protein structure is critical for drug discovery and structure-based rational design. Both MSI1 and MSI2 proteins contain two N-terminal RNA recognition motifs and play roles in post-transcriptional regulation of target mRNAs. We have identified several inhibitors of MSI1, all of which bind to MSI2 as well. In order to design MSI2 specific inhibitors and compare the differences of binding mode of the inhibitors, we set out to solve the structure of MSI2-RRM1, the key motif that is responsible for the binding. Here we report the crystal structure and the first NMR solution structure of MSI2-RRM1, and compare these to the structures of MSI1-RBD1 and other RNA binding proteins. A high degree of structural similarity was observed between the crystal and solution NMR structures. MSI2-RRM1 shows a highly similar overall folding topology to MSI1-RBD1 and other RNA binding proteins. The structural information of MSI2-RRM1 will be helpful for understanding MSI2-RNA interaction and for guiding rational design of MSI2 specific inhibitors. Citation Format: Lan Lan, Minli Xing, Maithri Kashipathy, Justin Douglas, Philip Gao, Kevin Battaile, Robert Hanzlik, Scott Lovell, Liang Xu. Dissecting the structural basis for rational inhibitor design: Crystal and solution structures of human oncoprotein Musashi-2 N-terminal RNA recognition motif 1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4140.

Long noncoding RNA LEF1‐AS1 acts as a microRNA‐10a‐5p regulator to enhance MSI1 expression and promote chemoresistance in hepatocellular carcinoma cells through activating AKT signaling pathway

Long noncoding RNAs (lncRNAs) contribute to the development of hepatocellular carcinoma (HCC), which could regulate various HCC biological characteristics. Here, the study seeks to investigate the role of lncRNA LEF1-AS1 in HCC cell chemoresistance by regulating microRNA (miR)-10a-5p and Musashi1 (MSI1). The microarray-based analysis was employed to identify the HCC-related lncRNA-miRNA-gene regulatory network. Expression patterns of LEF1-AS1, miR-10a-5p, and MSI1 in the HCC cell lines, tissues were accessed by means of reverse transcription-quantitative polymerase chain reaction. Next, the interaction among LEF1-AS1, miR-10a-5p, and MSI1 in HCC was accessed by bioinformatics and dual-luciferase reporter gene assay. Then, the cell line resistant to cisplatin was established, which was then treated with sh/oe-lncRNA LEF1-AS1, miR-10a-5p-mimic, and oe/sh-MSI1 vectors alone or in combination. Afterward, the effect of LEF1-AS1, miR-10a-5p, and MSI1 on HCC cell chemoresistance, proliferation, and apoptosis was assessed. At last, in vivo experiments confirmed the role of MSI1 in tumor growth and chemoresistance in HCC. LEF1-AS1 might potentially affect the growth and chemoresistance of HCC cells by regulating miR-10a-5p and MSI1. LEF1-AS1 and MSI1 expression patterns were elevated, while miR-10a-5p was repressed in HCC tissues and cell lines. LEF1-AS1 combined to miR-10a-5p and regulated MSI1, thereby activating the protein kinase B (AKT) signaling pathway. Knockdown of LEF1-AS1 and MSI1 or elevation of miR-10a-5p compromised the proliferation of Huh7 cell line resistant to DDP and promoted its chemosensitivity and apoptosis. At last, these in vitro findings were also confirmed in vivo. Our results unraveled LEF1-AS1 acts as a miR-10a-5p modulator to promote chemoresistance of HCC cells by stimulating MSI1 and activating the AKT signaling pathway, which might provide a novel therapeutic target for HCC.

Musashi2 promotes the progression of pancreatic cancer through a novel ISYNA1-p21/ZEB-1 pathway.

Our previous studies found overexpression of Musashi2 (MSI2) conduced to the progression and chemoresistance of pancreatic cancer (PC) by negative regulation of Numb and wild type p53 (wtp53). Now, we further investigated the novel signalling involved with MSI2 in PC. We identified inositol‐3‐phosphate synthase 1 (ISYNA1) as a novel tumour suppressor regulated by MSI2. High MSI2 and low ISYNA1 expression were prevalently observed in 91 PC tissues. ISYNA1 expression was negatively correlated with MSI2 expression, T stage, vascular permeation and poor prognosis in PC patients. What's more, patients expressed high MSI2 and low ISYNA1 level had a significant worse prognosis. And in wtp53 Capan‐2 and SW1990 cells, ISYNA1 was downregulated by p53 silencing. ISYNA1 silencing promoted cell proliferation and cell cycle by inhibiting p21 and enhanced cell migration and invasion by upregulating ZEB‐1. However, MSI2 silencing upregulated ISYNA1 and p21 but downregulated ZEB‐1, which can be rescued by ISYNA1 silencing. Moreover, reduction of cell migration and invasion resulting from MSI2 silencing was significantly reversed by ISYNA1 silencing. In summary, MSI2 facilitates the development of PC through a novel ISYNA1‐p21/ZEB‐1 pathway, which provides new gene target therapy for PC.

RNA-binding protein Musashi2 regulates Hippo signaling via SAV1 and MOB1 in pancreatic cancer.

Musashi 2 (MSI2), a member of the Musashi RNA-binding family, is reported to be an oncoprotein in pancreatic ductal adenocarcinoma (PDAC), but the mechanisms of MSI2 in the development and progression of PDAC have not been fully demonstrated. In this research, we studied the clinical significance, biologic effects and the underlying mechanism of MSI2 in the progression of PDAC. The expression of MSI2, Mps-binding protein 1 (MOB1) and Salvador family WW domain-containing protein 1 (SAV1) in PDAC tissues were analyzed immunohistochemically. The biologic effects of MSI2 regarding PDAC cell proliferation, migration and invasion were studied using gain- and loss-of-function assays. MSI2 regulated Hippo signaling pathway via SAV1 and MOB1 was tested in several PDAC cell lines, and the mechanisms were studied using molecular biologic methods. The expression of MSI2 was significantly increased in PDAC cell lines and tissues, and positively associated with tumor poorer differentiation, lymph nodes metastasis and TNM stages. Overexpression of MSI2 promoted PDAC cells proliferation, migration and invasion. Further studies demonstrated that MSI2 regulated the Hippo signaling pathway via directly binding to the mRNAs of SAV1 and MOB1, and controlled the translation and stability of SAV1 and the translation of MOB1. This study demonstrated that MSI2 regulated the Hippo signaling pathway via suppressing SAV1 and MOB1 at post-transcriptional level and promoted PDAC progression.

Identification and Validation of an Aspergillus nidulans Secondary Metabolite Derivative as an Inhibitor of the Musashi-RNA Interaction.

RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. MSI1 is involved in tumor proliferation and maintenance, and it regulates target mRNAs at the translational level. The known mRNA targets of MSI1 include Numb, APC, and P21WAF-1, key regulators of Notch/Wnt signaling and cell cycle progression, respectively. In this study, we aim to identify small molecule inhibitors of MSI1–mRNA interactions, which could block the growth of cancer cells with high levels of MSI1. Using a fluorescence polarization (FP) assay, we screened small molecules from several chemical libraries for those that disrupt the binding of MSI1 to its consensus RNA. One cluster of hit compounds is the derivatives of secondary metabolites from Aspergillus nidulans. One of the top hits, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance spectroscopy, which demonstrated that Aza-9 binds directly to MSI1, and the binding is at the RNA binding pocket. We also show that Aza-9 binds to Musashi-2 (MSI2) as well. To test whether Aza-9 has anti-cancer potential, we used liposomes to facilitate Aza-9 cellular uptake. Aza-9-liposome inhibits proliferation, induces apoptosis and autophagy, and down-regulates Notch and Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1/2 function, while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology.

LncRNA DANCR regulates osteosarcoma migration and invasion by targeting miR-149/MSI2 axis.

As a new LncRNA, anti-differentiated non-coding RNA (DANCR) plays an important role in tumorigenesis and development, and its molecular mechanism in osteosarcoma is unclear. In this study, by investigating osteosarcoma tissue and cells, we explored the molecular mechanism by which lncRNA DANCR regulates the occurrence and development of osteosarcoma by targeting the miR-149 / MSI2 axis. In this study, osteosarcoma tissues and adjacent tissues in 109 patients were collected, and the relative expression of DANCR was detected by qPCR. The correlation between DANCR expression and clinical classification was statistically analyzed. In order to explore the potential molecular mechanism of DANCR related to tumor migration and invasion, an overexpression and silencing test was performed on the osteosarcoma cell line Saos-2, and then qPCR method was used to test the expression of miR149, and cell scratch test was used to detect invasion after DANCR silencing and miR149 overexpression. Transwell assay was used to detect the invasion after DANCR silencing and miR149 overexpression. Finally, Western blot was used to verify the expression of MSI2 protein after overexpression and silencing of miR-149. DANCR was significantly up-regulated in both osteosarcoma tissue and cells. The high expression of DANCR was significantly positively correlated with tissue typing and advanced TNM stage. DANCR can significantly reduce the migration and invasion of osteosarcoma cells. miRNA overexpression significantly reduced osteosarcoma cell migration and invasion. When miR-149 was overexpressed, MSI2 protein expression was significantly down-regulated. When miR-149 was silenced, MSI2 protein was significantly up-regulated. LncRNA DANCR plays an important regulatory role in the occurrence and development of osteosarcoma. It may be used as a potential target in the treatment of osteosarcoma in the future, by targeting the miR-149/MSI2 axis to regulate the occurrence and development of osteosarcoma.