scholarly journals RBM15 facilitates laryngeal squamous cell carcinoma progression by regulating TMBIM6 stability through IGF2BP3 dependent

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Xin Wang ◽  
Linli Tian ◽  
Yushan Li ◽  
Jingting Wang ◽  
Bingrui Yan ◽  
...  
Keyword(s):  
Regulatory Mechanism ◽  
Biological Effects ◽  
Tissue Samples ◽  
Downstream Target ◽  
Transcriptomic Sequencing ◽  
Rna Immunoprecipitation ◽  
M6a Rna Methylation ◽  
Hoechst Staining

Abstract Background Laryngeal cancer has the highest mortality rate among head and neck tumours. RNA N6-methyladenosine (m6A) is the most plentiful and variable in mammalian mRNA. Yet, the m6A regulatory mechanism underlying the carcinogenesis or progression of LSCC remains poorly understood. Methods The m6A RNA methylation quantification kit was used to detect tissue methylation levels. m6A microarray analysis, mRNA transcriptomic sequencing (mRNA-seq), and proteomics were used to determine RBM15, TMBIM6, and IGF2BP3. Immunohistochemical (IHC), quantitative real-time PCR (qRT-PCR) and Western blot were used to investigate RBM15, TMBIM6, and IGF2BP3 expression in tissue samples and cell lines. The biological effects of RBM15 were detected both in vitro and in vivo. The combination relationship between RBM15/IGF2BP3 and TMBIM6 was verified by RNA immunoprecipitation (RIP) assay, Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNase Mazf, and luciferase report assay. RNase Mazf was used to determine the methylation site on TMBIM6 mRNA. Hoechst staining assay was used to confirm the apoptotic changes. The actinomycin D verified TMBIM6 stability. Results The global mRNA m6A methylation level significantly increased in LSCC patients. RBM15, as a “writer” of methyltransferase, was significantly increased in LSCC and was associated with unfavorable prognosis. The knockdown of RBM15 reduced the proliferation, invasion, migration, and apoptosis of LSCC both in vitro and in vivo. The results were reversed after overexpressing RBM15. Mechanically, TMBIM6 acted as a downstream target of RBM15-mediated m6A modification. Furthermore, RBM15-mediated m6A modification of TMBIM6 mRNA enhanced TMBIM6 stability through IGF2BP3-dependent. Conclusion Our results revealed the essential roles of RBM15 and IGF2BP3 in m6A methylation modification in LSCC, thus identifying a novel RNA regulatory mechanism.

scholarly journals RBM15 Facilitates Laryngeal Squamous Cell Carcinoma Progression by Regulating TMBIM6 Stability Through IGF2BP3 Dependent

2020 ◽  
Author(s):  
Xin Wang ◽  
Linli Tian ◽  
Yushan Li ◽  
Jingting Wang ◽  
Bingrui Yan ◽  
...  
Keyword(s):  
Regulatory Mechanism ◽  
Biological Effects ◽  
Tissue Samples ◽  
Downstream Target ◽  
Transcriptomic Sequencing ◽  
Rna Immunoprecipitation ◽  
M6a Rna Methylation ◽  
Hoechst Staining

Abstract Background: Laryngeal cancer has the highest mortality rate among head and neck tumours. RNA N6-methyladenosine (m6A) is the most plentiful and variable in mammalian mRNA. Yet, the m6A regulatory mechanism underlying the carcinogenesis or progression of LSCC remains poorly understood. Methods: The m6A RNA methylation quantification kit was used to detect tissue methylation levels. m6A microarray analysis, mRNA transcriptomic sequencing (mRNA-seq), and proteomics were used to determine RBM15, TMBIM6, and IGF2BP3. Immunohistochemical (IHC), quantitative real-time PCR (qRT-PCR) and Western blot were used to investigate RBM15, TMBIM6, and IGF2BP3 expression in tissue samples and cell lines. The biological effects of RBM15 were detected both in vitro and in vivo. The combination relationship between RBM15/IGF2BP3 and TMBIM6 was verified by RNA immunoprecipitation (RIP) assay, Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNase Mazf, and luciferase report assay. RNase Mazf was used to determine the methylation site on TMBIM6 mRNA. Hoechst staining assay was used to confirm the apoptotic changes. The actinomycin D verified TMBIM6 stability. Results: The global mRNA m6A methylation level significantly increased in LSCC patients. RBM15, as a “writer” of methyltransferase, was significantly increased in LSCC and was associated with unfavorable prognosis. The knockdown of RBM15 reduced the invasion, migration, and apoptosis of LSCC both in vitro and in vivo. The results were reversed after overexpressing RBM15. Mechanically, TMBIM6 acted as a downstream target of RBM15-mediated m6A modification. Furthermore, RBM15-mediated m6A modification of TMBIM6 mRNA enhanced TMBIM6 stability through IGF2BP3-dependent. Conclusion: Our results revealed the essential roles of RBM15 and IGF2BP3 in m6A methylation modification in LSCC, thus identifying a novel RNA regulatory mechanism.

scholarly journals RBM15 facilitates laryngeal squamous cell carcinoma progression by regulating TMBIM6 stability through IGF2BP3 dependent

2021 ◽  
Author(s):  
Xin Wang ◽  
Linli Tian ◽  
Yushan Li ◽  
Jingting Wang ◽  
Bingrui Yan ◽  
...  
Keyword(s):  
Regulatory Mechanism ◽  
Biological Effects ◽  
Tissue Samples ◽  
Downstream Target ◽  
Transcriptomic Sequencing ◽  
Rna Immunoprecipitation ◽  
M6a Rna Methylation ◽  
Hoechst Staining

Abstract Background: Laryngeal cancer has the highest mortality rate among head and neck tumours. RNA N6-methyladenosine (m6A) is the most plentiful and variable in mammalian mRNA. Yet, the m6A regulatory mechanism underlying the carcinogenesis or progression of LSCC remains poorly understood. Methods: The m6A RNA methylation quantification kit was used to detect tissue methylation levels. m6A microarray analysis, mRNA transcriptomic sequencing (mRNA-seq), and proteomics were used to determine RBM15, TMBIM6, and IGF2BP3. Immunohistochemical (IHC), quantitative real-time PCR (qRT-PCR) and Western blot were used to investigate RBM15, TMBIM6, and IGF2BP3 expression in tissue samples and cell lines. The biological effects of RBM15 were detected both in vitro and in vivo. The combination relationship between RBM15/IGF2BP3 and TMBIM6 was verified by RNA immunoprecipitation (RIP) assay, Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNase Mazf, and luciferase report assay. RNase Mazf was used to determine the methylation site on TMBIM6 mRNA. Hoechst staining assay was used to confirm the apoptotic changes. The actinomycin D verified TMBIM6 stability. Results: The global mRNA m6A methylation level significantly increased in LSCC patients. RBM15, as a “writer” of methyltransferase, was significantly increased in LSCC and was associated with unfavorable prognosis. The knockdown of RBM15 reduced the proliferation, invasion, migration, and apoptosis of LSCC both in vitro and in vivo. The results were reversed after overexpressing RBM15. Mechanically, TMBIM6 acted as a downstream target of RBM15-mediated m6A modification. Furthermore, RBM15-mediated m6A modification of TMBIM6 mRNA enhanced TMBIM6 stability through IGF2BP3-dependent. Conclusion: Our results revealed the essential roles of RBM15 and IGF2BP3 in m6A methylation modification in LSCC, thus identifying a novel RNA regulatory mechanism.

scholarly journals Long noncoding RNA SNHG14 promotes hepatocellular carcinoma progression by regulating miR-876-5p/SSR2 axis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Zhibin Liao ◽  
Hongwei Zhang ◽  
Chen Su ◽  
Furong Liu ◽  
Yachong Liu ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Animal Experiments ◽  
Luciferase Reporter ◽  
Western Blot Assay ◽  
Downstream Target ◽  
Protein Levels ◽  
Elevated Expression ◽  
Rna Immunoprecipitation

Abstract Background Aberrant expressions of long noncoding RNAs (lncRNAs) have been demonstrated to be related to the progress of HCC. The mechanisms that SNHG14 has participated in the development of HCC are obscure. Methods Quantitative real-time PCR (qRT-PCR) was used to measure the lncRNA, microRNA and mRNA expression level. Cell migration, invasion and proliferation ability were evaluated by transwell and CCK8 assays. The ceRNA regulatory mechanism of SNHG14 was evaluated by RNA immunoprecipitation (RIP) and dual luciferase reporter assay. Tumorigenesis mouse model was used to explore the roles of miR-876-5p in vivo. The protein levels of SSR2 were measured by western blot assay. Results In this study, we demonstrated that SNHG14 was highly expressed in HCC tissues, meanwhile, the elevated expression of SNHG14 predicted poor prognosis in patients with HCC. SNHG14 promoted proliferation and metastasis of HCC cells. We further revealed that SNHG14 functioned as a competing endogenous RNA (ceRNA) for miR-876-5p and that SSR2 was a downstream target of miR-876-5p in HCC. Transwell, CCK8 and animal experiments exhibited miR-876-5p inhibited HCC progression in vitro and in vivo. By conducting rescue experiments, we found the overexpression of SSR2 or knocking down the level of miR-876-5p could reverse the suppressive roles of SNHG14 depletion in HCC. Conclusion SNHG14 promotes HCC progress by acting as a sponge of miR-876-5p to regulate the expression of SSR2 in HCC.

scholarly journals TCF4 and HuR Mediated-METTL14 Suppresses Dissemination of Colorectal Cancer via N6-Methyladenosine-Dependent Silencing of ARRDC4

2021 ◽  
Author(s):  
Hao Wang ◽  
Wei Wei ◽  
Zhong-Yuan Zhang ◽  
Yao Liu ◽  
Bin Shi ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Chromatin Immunoprecipitation ◽  
Dependent Manner ◽  
Dot Blot ◽  
Downstream Target ◽  
In Vivo Assays ◽  
Rna Immunoprecipitation ◽  
Underlying Mechanisms

Abstract Background: Metastasis remains the major obstacle to improved survival for colorectal cancer (CRC) patients. Dysregulation of N6-methyladenosine (m6A) is causally associated with the development of metastasis through poorly understood mechanisms. Methods: The expression of METTL14 and its correlation with clinicopathological features were evaluated by western blot and immunohistochemistry. The roles of METTL14 in CRC metastasis were determined through in vitro and in vivo assays. The underlying mechanisms of METTL14 regulation were explored using transcriptome-sequencing, m6A-seguencing, methylated RNA immunoprecipitation (MeRIP), m6A dot blot, RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP) assay.Results: METTL14 is functionally related to the inhibition of ARRDC4/ZEB1 signaling and to the consequent suppression of CRC metastasis. We unveil METTL14-mediated m6A modification profile and identify ARRDC4 as a direct downstream target of METTL14. Knockdown of METTL14 significantly enhances ARRDC4 mRNA stability relying on the “reader” protein YHTDF2 dependent manner. Moreover, TCF4 can induce METTL14 protein expression, and HuR suppresses METTL14 expression by directly binding to its promoter. Clinically, decreased METTL14 is correlated with poor prognosis and acts as an independent predictor of CRC survival. Conclusion: Our data suggest that TCF4 and HuR mediated-METTL14 can trigger the metastasis of CRC during cancer development via YHTDF2/ARRDC4/ZEB1 axis, which imposes great challenge that inhibition of METTL14 is a potential approach for cancer treatment.

scholarly journals HOTAIR contributes to cell proliferation and metastasis of cervical cancer via targetting miR-23b/MAPK1 axis

2018 ◽  
Vol 38 (1) ◽  
Author(s):  
Qin Li ◽  
Yanhong Feng ◽  
Xu Chao ◽  
Shuai Shi ◽  
Man Liang ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Mitogen Activated Protein Kinase ◽  
Cancer Tissue ◽  
Tissue Samples ◽  
Downstream Target ◽  
Cervical Cancer Cells

The long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) has been found to be overexpressed in many human malignancies and involved in tumor progression and metastasis. Although the downstream target through which HOTAIR modulates tumor metastasis is not well-known, evidence suggests that miR-23b might be involved in this event. In the present study, the expressions of HOTAIR and miR-23b were detected by real-time PCR in 33 paired cervical cancer tissue samples and cervical cell lines. The effects of HOTAIR on the expressions of miR-23b and mitogen-activated protein kinase 1 (MAPK1) were studied by overexpression and RNAi approaches. We found that HOTAIR expression was significantly increased in cervical cancer cells and tissues. In contrast, the expression of miR-23b was obviously decreased. We further demonstrated that HOTAIR knockdown promoted apoptosis and inhibited cell proliferation and invasion in vitro and in vivo. Moreover, our data indicated that HOTAIR may competitively bind miR-23b and modulate the expression of MAPK1 indirectly in cervical cancer cells. Taken together, our study has identified a novel pathway through which HOTAIR exerts its oncogenic role, and provided a molecular basis for potential applications of HOTAIR in the prognosis and treatment of cervical cancer.

scholarly journals circEVI5 acts as a miR-4793-3p sponge to suppress the proliferation of gastric cancer

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Meinan Yan ◽  
Liling Niu ◽  
Jing Liu ◽  
Yuan Yao ◽  
Hui Li
Keyword(s):  
Gastric Cancer ◽  
Cell Cycle ◽  
Circular Rnas ◽  
Tissue Samples ◽  
Expression Levels ◽  
Rna Immunoprecipitation ◽  
Incorporation Assay ◽  
Underlying Mechanisms

AbstractCircular RNAs (circRNAs) are a novel class of endogenous noncoding RNAs (ncRNAs) with a covalently closed loop structure. Accumulating evidence shows that circRNAs play vital roles in the growth, metastasis, treatment and prognosis of various cancers. However, the detailed functions and underlying mechanisms of circEVI5 (hsa_circ_0013162) in gastric cancer (GC) remain undocumented. In this study, the expression levels and prognostic value of circEVI5 were validated in GC tissue samples by using qRT-PCR. circEVI5 was significantly downregulated in GC tissues and cells, and low circEVI5 expression was correlated with poor prognosis. Next, in vitro CCK-8 assay, EdU incorporation assay, PI staining cell cycle assay, and in vivo xenograft mouse models were conducted to assess the functions of circEVI5. Gain of function experiments indicated that circEVI5 could inhibit GC cell proliferation and retard the cell cycle. Moreover, bioinformatics prediction showed that circEVI5 binds to miR-4793-3p, while FOXO1 may be a target of miR-4793-3p. Pull-down assays, RNA immunoprecipitation (RIP) assays, luciferase assays, and western blot were used to confirm the interactions between circEVI5, miR-4793-3p, and FOXO1. Functional assays demonstrated that circEVI5 suppressed the proliferation of GC by sponging miR-4793-3p and increasing FOXO1 expression levels. In conclusion, our study demonstrated that circEVI5 can bind miR-4793-3p as a ceRNA to eliminate the negative regulation of FOXO1, therefore suppressing GC proliferation.

scholarly journals Circular RNA circDLC1 Inhibits MMP1-Mediated Liver Cancer Progression via Interaction with HuR

2020 ◽  
Author(s):  
Hailing Liu ◽  
Tian Lan ◽  
Hui Li ◽  
Lin Xu ◽  
Xing Chen ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Rna Binding ◽  
Circular Rna ◽  
Hepatoma Cells ◽  
Circular Rnas ◽  
Rna Seq ◽  
Downstream Target ◽  
Rna Immunoprecipitation

Abstract Background: N6-methyladenosine (m6A) modification has been demonstrated to be closely related to cancer progression. KIAA1429, a key component of the m6A methyltransferase complex, has recently been reported to promote hepatocellular carcinoma (HCC) progression by regulating the m6A methylation. However, the involvement of circular RNAs (circRNAs) in KIAA1429-mediated HCC progression is still unknown.Methods: RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (m6A-seq) were utilized to identify KIAA1429-regulated circRNAs. The effects of circDLC1 on proliferation and metastasis of hepatoma cells were examined in vitro and in vivo. RT-qPCR was used to measure the expression of circDLC1 in HCC tissues and hepatoma cells. RNA FISH, RIP assays and biotin-labeled RNA pull-down were used to investigate the downstream effector of circDLC1. The downstream targets of circDLC1 were identified using RNA-seq.Results: Our data demonstrated that circDLC1 was downregulated in HCC tissues and closely relevant to favorable prognosis. Overexpression of circDLC1 inhibited the proliferation and motility of hepatoma cells in vitro and in vivo, while silencing of circDLC1 played the opposite role. Mechanistic investigations revealed that circDLC1 could bind to RNA-binding protein HuR, which subsequently reduced the interaction between HuR and MMP1 mRNAs, thus inhibited the expression of MMP1, finally contributed to inhibition of HCC progression.Conclusion: Our work suggests that circDLC1, a downstream target of KIAA1429, is a promising prognostic marker for HCC patients, and the circDLC1-HuR-MMP1 axis may serve as a potential therapeutic target for HCC treatment.

scholarly journals METTL14 Suppresses Pyroptosis and Diabetic Cardiomyopathy by Down-Regulating TINCR lncRNA

2021 ◽  
Author(s):  
Liping Meng ◽  
Hui Lin ◽  
Xingxiao Huang ◽  
Jingfan Wen ◽  
Shengjie Wu
Keyword(s):  
Diabetic Cardiomyopathy ◽  
Epigenetic Regulation ◽  
Regulatory Mechanism ◽  
Rat Tissues ◽  
The Novel ◽  
Qrt Pcr ◽  
Rna Immunoprecipitation

Abstract Background: N6-methyladenosine (m6A) is one of the most important epigenetic regulation of RNAs, such as lncRNAs. However, the underlying regulatory mechanism of m6A in diabetic cardiomyopathy (DCM) is very limited. In this study, we sought to define the role of METTL14-mediated m6A modification in pyroptosis and DCM progression.Methods: DCM rat model was established and qRT-PCR, western blot and immunohistochemistry (IHC) were used to detect the expression of METTL14 and TINCR. Gain-and-loss functional experiments were performed to define the role of METTL14-TINCR-NLRP3 axis in pyroptosis and DCM. RNA pulldown and RNA immunoprecipitation (RIP) assays were carried out to verify the underlying interaction.Results: In vivo and in vitro studies showed that pyroptosis was tightly involved in DCM progression. METTL14 was downregulated in cardiomyocytes and hear tissues of DCM rat tissues. Functionally, METTL14 suppressed pyroptosis and DCM via downregulating lncRNA TINCR, which further decreased the expression of key pyroptosis-related protein, NLRP3. Mechanistically, METTL14 increased m6A methylation level of TINCR gene, resulting in its downregulation. Moreover, the m6A reader protein YTHDF2 was essential for m6A methylation and mediated the degradation of TINCR. Finally, TINCR positively regulated NLRP3 through increasing its mRNA stability.Conclusions: Our work revealed the novel role of METTL14-mediated m6A methylation and lncRNA regulation in pyroptosis and DCM, which could help extend our understanding the epigenetic regulation of pyroptosis in DCM progression.

scholarly journals LncRNA-MIAT promotes thyroid cancer progression and function as ceRNA to target EZH2 by sponging miR-150-5p

2021 ◽  
Vol 12 (12) ◽  
Author(s):  
Kai Guo ◽  
Kai Qian ◽  
Yuan Shi ◽  
Tuanqi Sun ◽  
Zhuoying Wang
Keyword(s):  
Cancer Progression ◽  
Human Cancer ◽  
Biological Effects ◽  
Loss Of Function ◽  
Downstream Target ◽  
Cerna Network ◽  
Ezh2 Expression ◽  
And Migration

AbstractWhile long noncoding RNAs (lncRNAs) have been reported to play an important role in human cancer types, they remain poorly understood in papillary thyroid carcinoma (PTC). The aim of this study was to use genome-wide expression profiling to identify lncRNAs acting as competing endogenous RNAs (ceRNAs) in PTC. We constructed a ceRNA network based on our lncRNA microarray data and validated the correlation between myocardial infarction-associated transcript lncRNA (MIAT), miRNA-150-5p, and EZH2 in vitro and in vivo. We found 15 lncRNAs, 28 miRNAs, and hundreds of mRNAs involved in this ceRNA network. Splendid positive correlations were found between the MIAT and EZH2 expression in types of cancer in TCGA data. Besides, significant differences in MIAT/EZH2 expression were found among various clinicopathological features. Gain- and loss-of-function experiments revealed that MIAT inhibited cell proliferation and migration in vitro. Moreover, EZH2 was identified as a direct downstream target of miR-150-5p in PTC cells. Restoration of EZH2 expression partially abolished the biological effects of miR-150-5p. Furthermore, overexpression of MIAT was inversely correlated with miR-150-5p expression. Knockdown of MIAT produced significant behavioral alter maybe partly due to the function of the MIAT-150-5p-EZH2 network. Our findings suggest MIAT may inhibit EZH2 expression and promote PTC cell invasion via the miR-150/EZH2 pathway. Therefore, MIAT may serve as a valuable prognostic biomarker and therapeutic target for PTC.

scholarly journals The m6A methyltransferase METTL3 promotes hypoxic pulmonary arterial hypertension via targeting PTEN

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
Q I N Yuhan ◽  
T A N G Chengchun
Keyword(s):  
Pulmonary Arteries ◽  
Rna Modification ◽  
Funding Sources ◽  
Pten Mrna ◽  
Rna Immunoprecipitation ◽  
M6a Rna Methylation ◽  
And Migration

Abstract Background N6-methyladenosine (m6A) is the most prevalent internal RNA modification in mammal mRNAs. Accumulating evidence has indicated the crucial role of m6A modification in cardiovascular diseases including cardiac hypertrophy, heart failure, ischemic heart disease, vascular calcification, restenosis, and aortic aneurysm. However, the role of m6A methylation in the occurrence and development of hypoxic pulmonary hypertension (HPH) remains largely unknown. Purpose The present study aims to explore the role of key transferase METTL3, in the development of HPH. Methods Hypoxic rat models and pulmonary artery smooth muscle cells (PASMCs) and were used to research the METTL3-mediated m6A in HPH in vivo and in vitro. CCK-8, EdU, PCNA, transwell and TUNEL assay were performed to evaluate the proliferation, migration and apoptosis rates of PASMCs. m6A RNA Methylation Quantification Kit and m6A-qPCR were utilized to measure the total m6A level and m6A-PTEN mRNA expression. RNA immunoprecipitation and RNA pull down were used to detect the interaction between METTL3 and PTEN mRNA. The half-life of mRNA was detected through actinomycin D assay. Results Both METTL3 mRNA and protein were found abnormally upregulated in pulmonary arteries of HPH rats and hypoxia induced PASMCs. Furthermore, downregulation of METTL3 attenuated PASMCs proliferation and migration exposed to hypoxia. In addition, m6A binding protein YTHDF2 was found significantly increased in HPH group in vivo and in vitro. Mechanistically, YTHDF2 recognized METTL3 mediated m6A-PTEN mRNA and promoted the degradation of PTEN. Decreased PTEN led to over-proliferation of PASMCs through activation of PI3K/Akt signaling pathway. Conclusion METTL3/YTHDF2/PTEN axis exerts a significant role in hypoxia induced PASMCs proliferation, providing a novel therapeutic target for HPH. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Natural Science Foundation of China Figure 1

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