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METTL14 promotes the migration and invasion of breast cancer cells by modulating N6?methyladenosine and hsa?miR?146a?5p expression.

ABSTRACT: Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer?related death among women worldwide. Evidence indicates that posttranscriptional N6?methyladenosine (m6A) modification modulates BC development. In the present study, we assessed BC and normal tissues to investigate this connection. RNA m6A levels were determined by methylation quantification assay. The effects of methyltransferase?like 14 (METTL14) gain?of?expression or co?transfection with an m6A inhibitor on cell migration and invasion abilities were determined by Transwell assays. The levels of differentially expressed (DE) miRNAs were verified by real?time quantitative PCR (RT?qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG) were performed to analyze potential function of target genes of the DE miRNAs. The effects of candidate miRNAs modulated by METTL14 on cell migration and invasion abilities were confirmed by Transwell assays. We demonstrated that m6A methyltransferase METTL14 was significantly upregulated in BC tissues compared with normal tissues. METTL14 gain? and loss?of?expression regulated m6A levels in MCF?7 and MDA?MB?231 cells. The cell function assays revealed that METTL14 overexpression enhanced the migration and invasion capacities of BC cells. Moreover, treatment with the m6A inhibitor suppressed this enhanced cell migration and invasion. Additionally, aberrant expression of METTL14 reshaped the miRNA profile in BC cell lines. The remodeled DE miRNA/mRNA network was found to be most enriched in cancer pathways, and DE miRNAs were enriched in cell adhesion terms. hsa?miR?146a?5p modulated by METTL14 promoted cell migration and invasion. METTL14 modulates m6A modification and hsa?miR?146a?5p expression, thereby affecting the migration and invasion of breast cancer cells.

SUBMITTER: Yi D

PROVIDER: S-EPMC7107905 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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METTL14 promotes the migration and invasion of breast cancer cells by modulating N6‑methyladenosine and hsa‑miR‑146a‑5p expression.

Yi Dandan D Wang Ru R Shi Xianbiao X Xu Lei L Yilihamu Yiminu'er Y Sang Jianfeng J

Oncology reports 20200224 5

Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer‑related death among women worldwide. Evidence indicates that posttranscriptional N6‑methyladenosine (m6A) modification modulates BC development. In the present study, we assessed BC and normal tissues to investigate this connection. RNA m6A levels were determined by methylation quantification assay. The effects of methyltransferase‑like 14 (METTL14) gain‑of‑expression or co‑transfection with an m6A inhibit ...[more]

PMID: 32323801

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Project description:Rheumatoid arthritis (RA) is largely caused by the inflammatory response triggered by macrophage polarization. Through epigenetic reprogramming, the inflammatory state of macrophages can be modified. Macrophage polarization is associated with the RNA epigenetic alteration N6-methyladenosine (m6A) RNA methylation. However, the specific function and underlying mechanisms of m6A methylation in the role of macrophage polarization in RA remain to be elucidated. The mRNA expression levels of m6A methylase genes and signaling pathway components associated with RA macrophages were determined in the present study using reverse-transcription quantitative PCR. Methyltransferase 14 (METTL14) protein expression levels were determined using western blot analysis, and the levels of specific cellular secretion factors were determined using ELISA and flow cytometry. The results of the present study demonstrated that elevated METTL14 expression was associated with joint tenderness, and METTL14 expression was positively correlated with both C-reactive protein and rheumatoid factor expression levels. Moreover, METTL14 exhibited potential in the prediction of visual analogue scale. Pro-inflammatory cytokines (TNF-α) and M1 macrophage markers (CD68+CD86+) were also positively associated with METTL14 expression. The results of the Kyoto Encyclopedia of Genes and Genomes analysis revealed that METTL14 was strongly associated with the MAPK signaling pathway. Notably, JNK and ERK2 exhibited a positive correlation with the M1 macrophage marker, CD68+CD86+, which was positively associated with the pro-inflammatory factor, TNF-α. JNK and ERK2 expression levels were markedly increased in the METTL14 high-expression group, compared with in the low-expression group; however, p38 and ERK1 expression levels were not significantly different between these groups. Collectively, the results of the present study demonstrated that METTL14 expression was significantly increased in the peripheral blood and synovial tissue of patients with RA, highlighting the potential association with both immunoinflammatory markers and clinical symptoms. In addition, it was suggested that METTL14 may exacerbate the downstream inflammatory response, through mediating macrophage polarization via the MAPK pathway.

Project description:BackgroundFetal growth restriction (FGR) is a worldwide problem, and a major cause of perinatal morbidity. The precise molecular mechanisms involved in placental development and function during FGR remain poorly understood. Circular RNAs (circRNAs) are important biological molecules associated with disease pathogenesis. However, the role of circRNAs in FGR has not been well studied.MethodscircRNA expression profiles in placental tissues with and without FGR were identified by circRNA microarray. circRNA expression was verified by quantitative reverse-transcription PCR (RT-qPCR) assay. The effect of hsa_circ_0000848 and hsa-miR-6768-5p on HTR-8 cell apoptosis, migration, and invasion was evaluated. The association between hsa_circ_0000848 and hsa-miR-6768-5p was confirmed by dual luciferase activity and anti-AGO2 RNA immunoprecipitation (RIP) assays. Protein levels were examined via western blotting.ResultsRT-qPCR results showed that hsa_circ_0000848 expression was significantly down-regulated in FGR placenta. Hsa_circ_0000848 overexpression and hsa-miR-6768-5p inhibitor suppressed apoptosis, and promoted cell migration and invasion. In addition, hsa_circ_0000848 overexpression and hsa-miR-6768-5p inhibitor increased the protein abundance of BCL2, MMP2 and MMP9, and decreased the protein abundance of cleaved caspase-3, cleaved caspase-9, and BAX, whereas hsa_circ_0000848 knockdown caused the opposite effect. Moreover, a significant increase in hsa-miR-6768-5p expression and a negative correlation between hsa_circ_0000848 and hsa-miR-6768-5p were identified in the FGR tissues. Luciferase reporter and RIP assay results revealed binding of hsa-miR-6768-5p to hsa_circ_0000848. Furthermore, hsa-miR-6768-5p overexpression eliminated the effect of hsa_circ_0000848 overexpression in HTR-8 cells.Conclusionshsa_circ_0000848 expression is significantly down-regulated in the FGR placenta. hsa_circ_0000848 promotes trophoblast cell migration and invasion, and inhibits cell apoptosis via the sponging of hsa-miR-6768-5p. Our study provided a novel insight into mechanisms underlying the pathogenesis of FGR, as well as into new strategies for the treatment of FGR.

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Project description:BackgroundTwo mature microRNAs (miRNAs), hsa-miR-125a-3p and hsa-miR-125a-5p (collectively referred to as hsa-miR-125a-3p/5p), are derived from 3' and 5' ends of pre-miR-125a, respectively. Although impaired regulation of hsa-miR-125a-5p has been observed in some tumors, the role of this miRNA in invasion and metastasis remains unclear, and few studies have examined the function of hsa-miR-125a-3p. In order to characterize the functions of hsa-miR-125a-3p/5p in invasion and metastasis of non-small cell lung cancer (NSCLC), we investigated the relationships between hsa-miR-125a-3p/5p expression and lymph node metastasis in NSCLC tissues. We also explored the impact of expression of these miRNAs on invasive and migratory capabilities of lung cancer cells.MethodsExpression of hsa-miR-125a-3p/5p in NSCLC tissues was explored using real-time PCR. The relationships between hsa-miR-125a-3p/5p expression and pathological stage or lymph node metastasis were assessed using the Spearman correlation test. For in vitro studies, lung cancer cells were transfected with sense and antisense 2'-O-methyl oligonucleotides for gain-of-function and loss-of-function experiments. Transwell experiments were performed to evaluate cellular migration and invasion.ResultsExpression of hsa-miR-125a-3p/5p was lower in NSCLC tissues than in adjacent normal lung tissues (LAC). Furthermore, the results from the Spearman correlation test showed a negative relationship between hsa-miR-125a-3p expression and pathological stage or lymph node metastasis and an inverse relationship between hsa-miR-125a-5p expression and pathological stage or lymph node metastasis. In vitro gain-of-function experiments indicated that hsa-miR-125a-3p and hsa-miR-125a-5p function in an opposing manner, suppressing or enhancing cell migration and invasion in A549 and SPC-A-1 cell lines, respectively. These opposing functions were further validated by suppression of hsa-miR-125a-3p and hsa-miR-125a-5p expression in loss-of-function experiments.ConclusionHsa-miR-125a-3p and hsa-miR-125a-5p play distinct roles in regulation of invasive and metastatic capabilities of lung cancer cells, consistent with the opposing correlations between the expression of these miRNAs and lymph node metastasis in NSCLC. These results provide new insights into the roles of miR-125a family members in the development of NSCLC.

Project description:BACKGROUND:Colorectal cancer (CRC) is one of the leading causes of tumor-related death worldwide, and its main cause of death is distant metastasis. Methyltransferase-like 14(METTL14), a major RNA N6-adenosine methyltransferase, is involved in tumor progression via regulating RNA function. The goal of the study is to uncover the biological function and molecular mechanism of METTL14 in CRC. METHODS:Quantitative real-time PCR (qRT-PCR), western blot and immunohistochemical (IHC) assays were employed to detect METTL14 and SOX4 in CRC cell lines and tissues. The biological functions of METTL14 were demonstrated using in vitro and in vivo experiments. Chromatin immunoprecipitation (ChIP), Transcrptomic RNA sequencing (RNA-Seq), m6A-RNA immunoprecipitation sequencing (MeRIP-Seq), RNA immunoprecipitation and luciferase reporter assays were used to explore the mechanism of METTL14 action. RESULTS:METTL14 expression was significantly downregulated in CRC and decreased METTL14 was associated with poor overall survival (OS). Both the univariate and multivariate Cox regression analysis indicated that METTL14 was an independent prognostic factor in CRC. Moreover, lysine-specific histone demethylase 5C(KDM5C)-mediated demethylation of histone H3 lysine 4 tri-methylation(H3K4me3) in the promoter of METTL14 inhibited METTL14 transcription. Functionally, we verified that METTL14 inhibited CRC cells migration, invasion and metastasis through in vitro and in vivo assays, respectively. Furthermore, we identified SRY-related high-mobility-group box 4(SOX4) as a target of METTL14-mediated m6A modification. Knockdown of METTL14 markedly abolished SOX4 mRNA m6A modification and elevated SOX4 mRNA expression. We also revealed that METTL14-mediated SOX4 mRNA degradation relied on the YTHDF2-dependent pathway. Lastly, we demonstrated that METTL14 might inhibit CRC malignant process partly through SOX4-mediated EMT process and PI3K/Akt signals. CONCLUSIONS:Decreased METTL14 facilitates tumor metastasis in CRC, suggesting that METTL14 might be a potential prognostic biomarker and effective therapeutic target for CRC.

Dataset Information

METTL14 promotes the migration and invasion of breast cancer cells by modulating N6?methyladenosine and hsa?miR?146a?5p expression.

Publications

METTL14 promotes the migration and invasion of breast cancer cells by modulating N6‑methyladenosine and hsa‑miR‑146a‑5p expression.

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