Polymeric nanoparticle-based delivery of microRNA-199a-3p inhibits proliferation and growth of osteosarcoma cells.
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ABSTRACT:
SUBMITTER: Zhang L
PROVIDER: S-EPMC4404938 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Osteosarcoma (OS) is a common bone tumor with high mortality worldwide. The long-term survival rate of patients with metastatic or recurrent disease is <20%. The present study explored the biological role of microRNA (miRNA/miR)-1236-3p in OS. miRNA and mRNA expression levels were measured via reverse transcription-quantitative PCR. Fluorescence in situ hybridization was performed to determine miR-1236-3p expression levels in clinical specimens. Protein expression was measured via western blotting. Immunohistochemical analysis was used to detect Wnt target gene expression in tumor tissues. The interaction between the Wnt3a 3'untranslated region and miR-1236-3p was assessed via dual-luciferase reporter assays. Cell cycle, Transwell, Cell Counting Kit-8 and wound healing assays were conducted to evaluate the function of the miR-1236-3p/Wnt3a axis. Human OS (HOS) cells stably transfected with vector or miR-1236-3p sponge were injected subcutaneously into nude mice to assess the role of miR-1236-3p in vivo. miR-1236-3p expression was downregulated in OS tissues compared with chondroma tissues, and miR-1236-3p overexpression inhibited OS cell migration and proliferation compared with the negative control group. Furthermore, in vivo xenograft assays displayed enhanced tumour growth rates in the miR-1236-3p sponge group compared with the vector control group. In the present study, the results indicated that miR-1236-3p inhibited OS progression and Wnt3a was identified as a target of miR-1236-3p.
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Project description:microRNAs (miRNA, miR) play an important role in cancer cell growth and migration; however, the potential roles of miRNAs in osteosarcoma remain largely uncharacterized. By applying a miRNA microarray platform and unsupervised hierarchical clustering analysis, we found that several miRNAs have altered expression levels in osteosarcoma cell lines and tumor tissues when compared with normal human osteoblasts. Three miRNAs, miR-199a-3p, miR-127-3p, and miR-376c, were significantly decreased in osteosarcoma cell lines, whereas miR-151-3p and miR-191 were increased in osteosarcoma cell lines in comparison with osteoblasts. Transfection of precursor miR-199a-3p into osteosarcoma cell lines significantly decreased cell growth and migration, thus indicating that the inhibition effect is associated with an increase in the G(1)-phase and a decrease of the S-phase cell population. In addition, we observed decreased mTOR and Stat3 expression in miR-199a-3p transfected cells. This study provides new insights for miRNAs in osteosarcoma and suggests that miR-199a-3p may play a functional role in osteosarcoma cell growth and proliferation. Restoring miR-199a-3p's function may provide therapeutic benefits in osteosarcoma.
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Project description:Hepatic apoptosis and the initiated liver inflammation play the initial roles in inflammation-induced hepatocarcinogenesis. Molecular mechanisms underlying the regulation of hepatocyte apoptosis and their roles in hepatocarcinogenesis have attracted much attention. A set of microRNAs (miRNAs) have been determined to be dysregulated in hepatocellular carcinoma (HCC) and participated in cancer progression, however, the roles of these dysregulated miRNAs in carcinogenesis are still poorly understood. We previously analyzed the dysregulated miRNAs in HCC using high-throughput sequencing, and found that miR-199a/b-3p was abundantly expressed in human normal liver while markedly decreased in HCC, which promotes HCC progression. Whether miR-199a/b-3p participates in HCC carcinogenesis is still unknown up to now. Hence, we focused on the role and mechanism of miR-199a/b-3p in hepatocarcinogenesis in this study. Hepatic miR-199a/b-3p was determined to be expressed by miR-199a-2 gene in mice, and we constructed miR-199a-2 knockout and hepatocyte-specific miR-199a-2 knockout mice. Diethylnitrosamine (DEN)-induced hepatocarcinogenesis were markedly increased by hepatocyte-specific miR-199a-3p knockout, which is mediated by the enhanced hepatocyte apoptosis and hepatic injury by DEN administration. In acetaminophen (APAP)-induced acute hepatic injury model, hepatocyte-specific miR-199a-3p knockout also aggravated hepatic apoptosis. By proteomic screening and reporter gene validation, we identified and verified that hepatic programed cell death 4 (PDCD4), which promotes apoptosis, was directly targeted by miR-199a-3p. Furthermore, we confirmed that miR-199a-3p-suppressed hepatocyte apoptosis and hepatic injury by targeting and suppressing PDCD4. Thus, hepatic miR-199a-3p inhibits hepatocyte apoptosis and hepatocarcinogenesis, and decreased miR-199a-3p in hepatocytes may aggravate hepatic injury and HCC development.
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Project description:Osteosarcoma (OS) is a primary bone tumor with a high incidence and mortality in children and adolescents. Emerging evidence shows that microRNAs (miRNAs) participate in biological tumor mechanisms by targeting downstream messenger RNAs (mRNAs). This article aimed to investigate the potential regulatory targets of microRNA-199a-3p (miR-199a-3p) in OS and to contribute to the understanding of miR-199a-3p-related OS regulatory mechanisms. MicroRNA-related Gene Expression Omnibus (GEO) chips, ArrayExpress chips and literature data were used to determine the expression of miR-199a-3p in OS and pooled to explore its potential clinical value. To investigate the target genes of miR-199a-3p further, we integrated the results from the following three-part gene study: Twelve online prediction tools were used to predict the target genes of miR-199a-3p; the GEO GSE89370 chip transfected with miRSelect pEP-miR-199a-3p was used to analyze the downregulated differentially expressed genes (DEGs) in OS cells; and highly expressed DEGs were derived from an in-house microarray generated from three pairs of clinical OS and normal tissue samples acquired through our department. Then, we analyzed the target genes using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases and the protein-protein interaction (PPI) network to further identify the primary target genes. In addition, we constructed transcription factor (TF)-miRNA-joint gene feed-forward regulatory loops (FFLs) with Circuits DB using miR-199a-3p as the core. A comprehensive meta-analysis of a hub of miR-199a-3p targeted genes was performed to integrate expression level, summary ROC (sROC) curves and survival analysis results from the GEO data for verification and exploration. Finally, the expression levels of the hub genes were verified in OS tissues and U2OS cells by immunohistochemistry (IHC) and immunocytochemistry (ICC). Data on miR-199a-3p expression were obtained from three data sets (GSE65071, GSE69524, and PMID 21666078), which showed low miR-199a-3p expression levels in OS tissues. The combined data indicated the same tendency, with the SMD of the random effect model, as shown in forest plots, being -2.8 (95% CI: -4.49, -1.11). In addition, we determined that miR-199a-3p may serve as a molecular marker useful for distinguishing OS tissues from normal tissues with high sensitivity and specificity, with the measured outcomes being 0.94 (95% CI: 0.80, 0.99) and 0.96 (95% CI: 0.78, 1.00), respectively. In addition, 391 genes were considered targets of miR-199a-3p in OS, and the enrichment analysis indicated that these targets were mainly enriched in proteoglycans in cancer and in spliceosomes. Four genes, CDKI, CCNB1, AURKA and NEK2, were regarded as hub targets based on the PPI data. Subsequently, TF-miRNA-joint genes FFLs were constructed in Circuits DB and included 17 TFs and 82 joint targets. These joint targets were mainly enriched in spliceosomes. UBE2D1 and RBM25 were regarded as hub joint targets based on the enrichment analysis. All selected target genes were further verified to ensure that they were upregulated in OS and to determine their prognostic significance. At the experimental verification level, the CDK1 protein was confirmed to be positively expressed in the cytoplasm of OS tissues and the U2OS cell line. Our study verified that miR-199a-3p was obviously downregulated in OS. CDK1, CCNB1, NEK2, AURKA, UBE2D1 and RBM25 were identified as potential target genes of miR-199a-3p in OS.
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Project description:MicroRNAs (miRNAs) play a crucial role in maintaining the balance between the rapid growth and suppression of tumorigenesis during antler regeneration. This study investigated the role of a novel miRNA, PC-3p-2869 (miR-PC-2869), in antler growth and its therapeutic potential in human osteosarcoma and chondrosarcoma. Stem-loop RT-qPCR showed that miR-PC-2869 was expressed extensively in diverse layers of antler tissues. Overexpression of miR-PC-2869 suppressed the proliferation and migration of antler cartilage cells. Similarly, heterologous expression of miR-PC-2869 reduced the proliferation, colony formation, and migration of osteosarcoma cell line MG63 and U2OS and chondrosarcoma cell line SW1353. Moreover, 18 functional target genes of miR-PC-2869 in humans were identified based on the screening of the reporter library. Among them, 15 target genes, including CDK8, EEF1A1, and NTN1, possess conserved miR-PC-2869-binding sites between humans and red deer (Cervus elaphus). In line with this, miR-PC-2869 overexpression decreased the expression levels of CDK8, EEF1A1, and NTN1 in MG63, SW1353, and antler cartilage cells. As expected, the knockdown of CDK8, EEF1A1, or NTN1 inhibited the proliferation and migration of MG63, SW1353, and antler cartilage cells, demonstrating similar suppressive effects as miR-PC-2869 overexpression. Furthermore, we observed that CDK8, EEF1A1, and NTN1 mediated the regulation of c-myc and cyclin D1 by miR-PC-2869 in MG63, SW1353, and antler cartilage cells. Overall, our work uncovered the cellular functions and underlying molecular mechanism of antler-derived miR-PC-2869, highlighting its potential as a therapeutic candidate for bone cancer.
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Project description:Osteosarcoma (OS) is the most common primary malignant neoplasm of the bone. Recent studies have indicated that the inhibitory effects of microRNA (miR)-324-3p could affect the development of numerous cancers. However, its biological roles and underlying mechanisms in OS progression remain unexplored. In this study, miR-324-3p expression was markedly reduced in OS cell lines and tissues. Functionally, miR-324-3p overexpression suppressed OS progression and was involved in the Warburg effect. Mechanistically, miR-324-3p negatively regulated phosphoglycerate mutase 1 (PGAM1) expression by targeting its 3'-UTR. Moreover, high expression of PGAM1 promoted OS progression and aerobic glycolysis, which were associated with inferior overall survival in patients with OS. Notably, the tumor suppressor functions of miR-324-3p were partially recovered by PGAM1 overexpression. In summary, the miR-324-3p/PGAM1 axis plays an important role in regulating OS progression by controlling the Warburg effect. Our results provide mechanistic insights into the function of miR-324-3p in glucose metabolism and subsequently on the progression of OS. Targeting the miR-324-3p/PGAM1 axis could be a promising molecular strategy for the treatment of OS.
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Project description:Osteosarcoma (OS) is the most common primary bone malignancy and remains a leading cause of cancer-related deaths in adolescents. Emerging evidence indicates that microRNAs (miRNAs) are correlated with clinical and biological characteristics of OS. However, the involvement of miR-199a-5p in OS development remains unclear. In this study, we examined the function of miR-199a-5p in vitro and in vivo. The results showed that miR-199a-5p was significantly up-regulated in OS patient tissues and cells. The inhibition of miR-199a-5p led to a significant decrease in cell proliferation and tumour growth. We further demonstrated that miR-199a-5p could directly bind to the 3'UTRs of the mRNA of both PIAS3 and p27 and mediate a decrease in the protein levels of PIAS3 and p27, thereby stimulating STAT3 activation and cell cycle progression in OS cells. Rescue experiments of PIAS3 and p27 further revealed that PIAS3 and p27 were functional targets of miR-199a-5p. Moreover, enhancing the expressions of both PIAS3 and p27 using miR-199a-5p-targeted inhibitors in an OS xenograft model was shown to be a promising approach for OS clinical therapy. Our findings indicate that the pathway of miR-199a-5p targeting both PIAS3 and p27 is a possible mechanism that contributes to tumour growth in OS.
| S-EPMC5264164 | biostudies-literature
Project description:BackgroundGastric cancer (GC) is one of the most frequent malignant tumors and the molecular mechanism underlying its proliferation remains far from completely understood. Although accumulating evidence shows that abnormal expression of microRNA (miRNA) is involved in tumorigenesis, the role of specific miRNAs involved in GC remains elusive. MiR-199a/b-3p functions as a tumor suppressor in diverse cancers, but its expression, function, and mechanism in GC remain unclear. Our aim is to explore miR-199a/b-3p expression and its role in regulating GC cell proliferation.MethodsReal-time PCR was performed to determine miR-199a/b-3p expression in GC tissues and normal adjacent tissues as well as normal gastric mucosal cell line GES-1 and GC cell lines MGC-803 and SGC-7901. MTT assay and Western blot were performed to determine cell proliferation and expression of PAK4, p-MEK and p-ERK, respectively. MiR-199a/b-3p mimics-transfected assay and PAK-specific siRNA assay were performed to determine their function in cell proliferation, respectively. GC xenograft nude mice were used to determine miR-199a/b-3p function in cell proliferation.ResultsMiR-199a/b-3p expression was significantly decreased in GC tissues and GC cell lines MGC-803 and SGC-7901. MiR-199a/b-3p over-expression and PAK4 silencing inhibited cell proliferation and diminished the activation of p-MEK and p-ERK in MGC-803 and SGC-7901 cells, and miR-199a/b-3p over-expression reduced PAK4 expression. MiR-199a/b-3p over-expression suppressed MGC-803 cell growth and PAK4 expression in nude mice.ConclusionsmiR-199a/b-3p inhibits GC cell proliferation via down-regulating PAK4/MEK/ERK signaling pathway and may be a novel prognostic biomarker and a potential therapeutic target for GC patients.
| S-EPMC5756398 | biostudies-literature
Project description:BackgroundMicroRNAs (miRNAs) play vital roles in regulating various biological processes. The dysregulations of miRNAs may result in severe human diseases, including cancer.MethodsWe performed the qRT-PCR, western blot and the luciferase reporter assays to test whether Adenylate Kinase 4 (AK4) is the target of miR-199a-3p. Up- or down-regulation of miR-199a-3p and/or the AK4 gene was done to detect their roles in OS multi-drug resistance using drug resistance profiling assays. We further predicted the putative signal pathway involved in the miR-199a-3p-mediated OS drug-resistance.ResultsThe AK4 gene is one of the targets of miR-199a-3p and negatively correlates with the effect of miR-199a-3p on OS drug-resistance. In addition, the activity of the NF-кB signaling pathway was drastically altered by the forced changes of the miR-199a-3p level in OS cells.ConclusionsOur data revealed that both miR-199a-3p and its target gene AK4 are reversely correlated with the OS drug resistance.
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