Knockdown of METTL14 suppresses the malignant progression of non-small cell lung cancer by reducing Twist expression
ABSTRACT: Non-small cell lung cancer (NSCLC) is one of the most malignant cancer types. N6-methyladenosine (m6A), an abundant eukaryotic mRNA modification, has been observed in multiple diseases, particularly cancer. Methyltransferase-like 14 (METTL14) is a central component of the m6A methyltransferase complex and has been reported to promote tumor development in several cancer types. The present study aimed to investigate the role of METTL14 in NSCLC. Relevant clinical and mRNA sequencing data for m6A-related genes were downloaded from The Cancer Genome Atlas database. R software was used to evaluate the expression of m6A regulators in NSCLC. The biological functions of METTL14 were evaluated using Cell Counting Kit-8, colony formation, Transwell migration and western blot analyses. The results demonstrated that METTL14 expression was upregulated in NSCLC tissues and cell lines, and its expression was high in cancer tissues from patients with NSCLC with all four stages (I, II, III and IV) of disease. METTL14 downregulation inhibited cell proliferation and migration in A549 and SK-MES-1 lung cancer cell lines. Knockdown of METTL14 in lung cancer cell lines increased E-cadherin expression and suppressed N-cadherin expression. Furthermore, METTL14 downregulation reduced the expression levels of the transcription factor Twist and the p-AKT/AKT ratio. In conclusion, the present findings revealed that silencing of METTL14 suppressed NSCLC malignancy by inhibiting Twist-mediated activation of AKT signaling. These data suggest that METTL14 may be a potential therapeutic target for NSCLC.
Project description:N6-methyladenosine (m6A) messenger RNA methylation is a gene regulatory mechanism affecting cell differentiation and proliferation in development and cancer. To study the roles of m6A mRNA methylation in cell proliferation and tumorigenicity, we investigated human endometrial cancer in which a hotspot R298P mutation is present in a key component of the methyltransferase complex (METTL14). We found that about 70% of endometrial tumours exhibit reductions in m6A methylation that are probably due to either this METTL14 mutation or reduced expression of METTL3, another component of the methyltransferase complex. These changes lead to increased proliferation and tumorigenicity of endometrial cancer cells, likely through activation of the AKT pathway. Reductions in m6A methylation lead to decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2. Together, these results reveal reduced m6A mRNA methylation as an oncogenic mechanism in endometrial cancer and identify m6A methylation as a regulator of AKT signalling.
Project description:Results from various studies reveal that the role of G protein-coupled oestrogen receptor (GPER) is cancer-context dependent, and the function of GPER in non-small-cell lung cancer (NSCLC) is still unclear. The present study demonstrated that neoplasm lung tissues expressed higher level of GPER compared with the normal lung tissues. The clinical data also showed that GPER expression level was positively correlated with the tumour stage of NSCLC. Our experimental data confirmed that GPER played an oncogenic role to promote cell growth of NSCLC cells. Mechanistic dissection revealed that GPER could modulate the NOTCH1 pathway to regulate cell growth in NSCLC cells. Further exploration of the mechanism demonstrated that GPER could up-regulate circNOTCH1, which could compete with NOTCH1 mRNA for METTL14 binding. Because of the lack of m6A modification by METTL14 on the NOTCH1 mRNA, NOTCH1 mRNA was more stable and much easier to undergo protein translation. Subsequently, we found that GPER could prevent YAP1 phosphorylation and promote YAP1-TEAD's transcriptional regulation on QKI, a transacting RNA-binding factor involved in circRNA biogenesis, to facilitate circNOTCH1 generation. Supportively, data from preclinical mice model with implantation of H1299 cells also demonstrated that knock-down of circNOTCH1 could block GPER-induced NOTCH1 to suppress NSCLC tumour growth. Together, our data showed that GPER could promote NSCLC cell growth via regulating the YAP1/QKI/circNOTCH1/m6A methylated NOTCH1 pathway, and targeting our identified molecules may be a potentially therapeutic approach to suppress NSCLC development.
Project description:N6-methyladenosine (m6A) is a common transcriptomic modification in cancer. Recently, it has been found to be involved in the regulation of non-small cell lung cancer (NSCLC) formation and metastasis. Interleukin 37 (IL-37) plays a crucial protective role in lung cancer. In our previous studies, we found that IL-37 is a potential novel tumor suppressor by inhibiting IL-6 expression to suppress STAT3 activation and decreasing epithelial-to-mesenchymal transition. Moreover, we found that treatment of IL-37 in lung cancer cells induced widespread and dynamic RNA m6A methylation. The effects of RNA m6A methylation of IL-37 treatment require further study. However, the functions of RNA m6A methylation of IL-37 treatment still await elucidation. Using MeRIP-seq and RNA-seq, we uncovered a unique m6A methylation profile in the treatment of IL-37 on the A549 cell line. We also showed the expression of m6A writers METTL3, METTL14, and WTAP and erasers ALKBH5 and FTO in A549 cells and lung cancer tissues after the treatment of IL-37. This study showed that IL-37 could lead to changes in m6A methylation level and related molecule expression level in A546 cells and may downregulate the proliferation by inhibiting Wnt5a/5b pathway in A549 cells. We conclude that IL-37 suppresses tumor growth through regulation of RNA m6A methylation in lung cancer cells.
Project description:<h4>Background</h4>TP53 is an important tumor suppressor gene on human 17th chromosome with its mutations more than 60% in tumor cells. Lung cancer is the highest incidence malignancy in men around the world. N-6 methylase (m6A) is an enzyme that plays an important role in mRNA splicing, translation, and stabilization. However, its role in TP53-mutant non-small-cell lung cancer (NSCLC) remains unknown.<h4>Method</h4>First, we investigated 17 common m6A regulators' prognostic values in NSCLC. Then, after the establishment of risk signature, we explored the diagnostic value of m6A in TP53-mutant NSCLC. Finally, gene set enrichment analysis (GSEA), gene ontology (GO) enrichment analysis, and differential expression analysis were used to reveal the possible mechanism of m6A regulators affecting TP53-mutant NSCLC patients.<h4>Results</h4>Study showed that nine m6A regulators (YTHDC2, METTL14, FTO, METTL16, YTHDF1, HNRNPA2B1, RBM15, KIAA1429, and WTAP) were expressed differently between TP53-mutant and wild-type NSCLC (p < 0.05); and ALKBH5 and HNRNPA2B1 were associated with the prognostic of TP53-mutant patients. After construction of the risk signature combined ALKBH5 and HNRNPA2B1, we divided patients with TP53 mutations into high- and low-risk groups, and there was a significant survival difference between two groups. Finally, 338 differentially expression genes (DEGs) were found between high- and low-risk groups. GO enrichment analysis, PPI network, and GSEA enrichment analysis showed that m6A may affect the immune environment in extracellular and change the stability of mRNA.<h4>Conclusion</h4>In conclusion, m6A regulators can be used as prognostic predictors in TP53-mutant patients.
Project description:<h4>Objective</h4>Non-small-cell lung cancer (NSCLC) is one of the most common fatal cancers in the world. Although the treatment of NSCLC has been significantly improved, there is still an unmet need to identify novel targets for developing therapeutic agents and diagnostic/prognostic markers. The aim of this study is explore the role and underlying mechanism of the epithelial splicing regulatory protein (ESRP1) in the development and progression of NSCLC.<h4>Methods</h4>A total of 115 participants, 65 cases of NSCLC, 20 cases of precancerous lesions, and 30 cases of benign lung nodules, were included in this study. The expressions of ESRP1 and related transcription factor Twist in enrolled lung tissues were evaluated by histochemistry and immunohistochemistry assay. The survival analysis and related prognosis factors were evaluated by the Kaplan-Meier curve and Cox regression. In addition, the expression of ESRP1 and epithelial-mesenchymal transition (EMT)related transcription factor Twist and EMT markers E-cadherin and N-cadherin were ascertained by immunohistochemical and immunoblotting assay on A549 lung adenocarcinoma cell lines that were exposed to transforming growth factor β1 (TGFβ1).<h4>Results</h4>Compared with normal lung tissues, the abundance of ESRP1 protein was significantly increased in precancerous lesions and lung cancer. Correlation analysis demonstrated that ESRP1 was an independent prognostic factor in NSCLC. The expression of ESRP1 and Twist was positively correlated in lung tissues (r = 0.285, p < 0.001). In vitro analysis further showed that TGFβ1 could upregulate the expression of EMT transcription factor Twist while downregulating ESRP1.<h4>Conclusions</h4>Our data suggest that the aberrant expression of ESRP1 is an early event in the development of NSCLC. The ESRP1 could serve as a prognostic biomarker for NSCLC, particularly when combined with Twist. The Twist negatively regulated the expression of ESRP1, emphasizing the role of the TGFβ/ESRP1 pathway in the development of NSCLC, which warrants further investigation.
Project description:Histone deacetylase 5 (HDAC5) belongs to class II HDAC subfamily and is reported to be increased in the kidneys of diabetic patients and animals. However, little is known about its function and the exact mechanism in diabetic kidney disease (DKD). Here, we found that HDAC5 was located in renal glomeruli and tubular cells, and significantly upregulated in diabetic mice and UUO mice, especially in renal tubular cells and interstitium. Knockdown of HDAC5 ameliorated high glucose-induced epithelial-mesenchymal transition (EMT) of HK2 cells, indicated in the increased E-cadherin and decreased ?-SMA, via the downregulation of TGF-?1. Furthermore, HDAC5 expression was regulated by PI3K/Akt signaling pathway and inhibition of PI3K/Akt pathway by LY294002 treatment or Akt phosphorylation mutation reduced HDAC5 and TGF-?1 expression in vitro high glucose-cultured HK2 cells. Again, high glucose stimulation downregulated total m6A RNA methylation level of HK2 cells. Then, m6A demethylase inhibitor MA2 treatment decreased Akt phosphorylation, HDAC5, and TGF-?1 expression in high glucose-cultured HK2 cells. In addition, m6A modification-associated methylase METTL3 and METTL14 were decreased by high glucose at the levels of mRNA and protein. METTL14 not METTL3 overexpression led to PI3K/Akt pathway inactivation in high glucose-treated HK2 cells by enhancing PTEN, followed by HDAC5 and TGF-?1 expression downregulation. Finally, in vivo HDACs inhibitor TSA treatment alleviated extracellular matrix accumulation in kidneys of diabetic mice, accompanied with HDAC5, TGF-?1, and ?-SMA expression downregulation. These above data suggest that METTL14-regulated PI3K/Akt signaling pathway via PTEN affected HDAC5-mediated EMT of renal tubular cells in diabetic kidney disease.
Project description:Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-associated mortality in the United States. AXL, which is a member of the receptor tyrosine kinases, has been established as a strong candidate for the targeted therapy of cancer. Therefore, the present study aimed to investigate the role of AXL in NSCLC; in particular the molecular mechanisms underlying the involvement of AXL in the epithelial-to-mesenchymal transition (EMT). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis demonstrated that AXL, EMT-inducing Twist and the mesenchymal marker N-cadherin were upregulated, and the epithelial markers E-cadherin and ?-cadherin were downregulated, in the PC9 NSCLC cell line. Furthermore, downregulation of AXL expression by RNA interference was shown to inhibit cell growth by inducing the apoptosis of PC9 cells, as demonstrated by MTT and flow cytometry analyses. Notably, inhibition of AXL attenuated the regulation of EMT-associated genes, specifically downregulating Twist and N-cadherin, and upregulating E-cadherin and ?-cadherin. Conversely, downregulation of Twist did not affect the expression levels of AXL. These results suggested that AXL may inhibit the EMT by the regulation of EMT-associated genes in the PC9 cell line. The results of the present study indicated that AXL may have a role in the regulation of EMT and the cell cycle of the PC9 cells; thus suggesting that AXL may have clinical significance in the design of therapeutic strategies targeting NSCLC and EMT signaling pathways.
Project description: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.
Project description:In the United States, lung cancer is the second most common cancer in men and women. In 2017, 222,500 new cases and 155,870 deaths from lung cancer are estimated to have occurred. A tyrosine kinase receptor, epidermal growth factor receptor (EGFR), is over expressed or mutated in non-small cell lung cancer (NSCLC) resulting in increased cell proliferation and survival. Tyrosine kinase inhibitors (TKIs) are currently being used as therapy for NSCLC patients, however, they have limited efficacy in NSCLC patients due to acquisition of resistance. This study investigates the role of epithelial-mesenchymal transition (EMT) in the development of resistance against TKIs in NSCLC. Currently, the role of p120-catenin, Kaiso factor and PRMT-1 in reversal of EMT in T790M mutated and TKI-resistant NSCLC cells is a new line of study. In this investigation we found upregulation of cytoplasmic p120-catenin, which was co-localized with Kaiso factor. In the nucleus, binding of p120-catenin to Kaiso factor initiates transcription by activating EMT-transcription factors such as Snail, Slug, Twist, and ZEB1. PRMT-1 was also found to be upregulated, which induces methylation of Twist and repression of E-cadherin activity, thus promoting EMT. We confirmed that TKI-resistant cells have mesenchymal cell type characteristics based on their cell morphology and gene or protein expression of EMT related proteins. EMT proteins, Vimentin and N-cadherin, displayed increased expression, whereas E-cadherin expression was downregulated. Finally, we found that the knockdown of p120-catenin and PRMT-1 by siRNA or use of a PRMT-1 inhibitor Furamidine increased Erlotinib sensitivity and could reverse EMT to overcome TKI resistance.
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.