The m6A methyltransferase METTL3 affects autophagy and progression of nasopharyngeal carcinoma by regulating the stability of lncRNA ZFAS1

Abstract Background Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the epithelial cells of the nasopharyngeal mucosa of the head and neck. The role of long non-coding RNA and RNA methylation in NPC has received increasing attention. Therefore, this study aims to investigate the mechanism of lncRNA ZFAS1 in NPC and its relationship with RNA methylation, providing evidence for targeted therapy of NPC. Methods Microarray arrays were used to screen the differentially expressed miRNAs in normal tissues and tumor tissues. QRT-PCR was used to quantify ZFAS1, miR-100-3p, ATG10, autophagy and epithelial-mesenchymal transition related genes. The interactive relationship between ZFAS1 and miR-100-3p was verified using dual-luciferase reporter gene assay and RIP assay. CCK-8, transwell and apoptosis were used to detect the occurrence of tumor cells after different treatments. The m6A modification test is used to verify the effect of METTL3 on ZFAS1. BALB/c mice and BALB/c nude mice are used to detect the effects of different treatments on tumor growth and immune escape in vivo. Results ZFAS1 is upregulated in tumor tissues and NPC cells. N (6)-methyladenosine (m6A) is highly enriched in ZFAS1 and enhances its RNA stability. ZFAS1 is used as an oncogenic lncRNA, which can promote NPC cell proliferation, migration and tumor growth. In terms of mechanism, ZFAS1 up-regulates the expression of ATG10 by competitively adsorbing miR-100-3p and regulates the level of autophagy by inhibiting the PI3K/Akt signaling pathway to promote the proliferation and migration of NPC cells. Conclusion In short, our study verified the cancer-promoting effect of ZFAS1 in NPC and explained part of the reason for its upregulation. In addition, we confirmed that ZFAS1 can regulate the autophagy level of NPC cells through the PI3K/AKT pathway through miR-100-3p/ATG10 to affect tumor progression.

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LncRNA KCNQ1OT1 Secreted by Tumor Cell-Derived Exosomes Mediates Immune Escape in Colorectal Cancer by Regulating PD-L1 Ubiquitination via MiR-30a-5p/USP22

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.653808 ◽

2021 ◽

Vol 9 ◽

Author(s):

Di Xian ◽

Liangbo Niu ◽

Jie Zeng ◽

Lei Wang

Keyword(s):

Colorectal Cancer ◽

Tumor Cell ◽

Immune Escape ◽

T Cell Response ◽

Cell Counting ◽

Luciferase Reporter ◽

Promoting Effect ◽

Tumor Immune Escape ◽

Tumor Tissues ◽

Gene Assay

Background: This study tried to explore the mechanism of long non-coding RNA (lncRNA) KCNQ1OT1 in tumor immune escape.Methods: Gene Expression Omnibus (GEO) and microarray analysis were used to screen the differentially expressed lncRNA and microRNA (miRNA) in normal tissues and tumor tissues. Quantitative reverse transcription PCR (RT-qPCR) was used to quantify KCNQ1OT1, miR-30a-5p, ubiquitin-specific peptidase 22 (USP22), and programmed death-ligand 1 (PD-L1). The interactive relationship between KCNQ1OT1 and miR-30a-5p was verified using dual-luciferase reporter gene assay and ribonucleoprotein immunoprecipitation (RIP) assay. Cell Counting Kit (CCK)-8, clone formation, wound healing, and apoptosis are used to detect the occurrence of tumor cells after different treatments. Protein half-life and ubiquitination detection are used to study the influence of USP22 on PD-L1 ubiquitination. BALB/c mice and BALB/c nude mice are used to detect the effects of different treatments on tumor growth and immune escape in vivo.Results: The expression of lncRNA KCNQ1OT1 in tumor tissues and tumor cell-derived exosomes was significantly increased. The tumor-promoting effect of lncRNA KCNQ1OT1 was through the autocrine effect of tumor cell-derived exosomes, which mediates the miR-30a-5p/USP22 pathway to regulate the ubiquitination of PD-L1 and inhibits CD8+ T-cell response, thereby promoting colorectal cancer development.Conclusion: Tumor cell-derived exosomes’ KCNQ1OT1 could regulate PD-L1 ubiquitination through miR-30a-5p/USP22 to promote colorectal cancer immune escape.

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MiR-32-5p knockdown inhibits epithelial to mesenchymal transition and renal fibrosis by targeting SMAD7 in diabetic nephropathy

Human & Experimental Toxicology ◽

10.1177/0960327120952157 ◽

2020 ◽

pp. 096032712095215

Author(s):

H-J Wang ◽

H Liu ◽

Y-H Lin ◽

S-J Zhang

Keyword(s):

Diabetic Nephropathy ◽

Renal Fibrosis ◽

Epithelial Mesenchymal Transition ◽

Interstitial Fibrosis ◽

Kidney Tissue ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Tubular Atrophy ◽

Mesenchymal Transition ◽

Gene Assay

Diabetic nephropathy (DN) is primary cause of end-stage renal disease. A previous study has shown that miR-32-5p (miR-32) is highly expressed in kidney tissue during chronic allograft dysfunction with interstitial fibrosis and tubular atrophy. However, the role of miR-32-5p (miR-32) in DN is still unclear. In this study, streptozotocin-induced DN rat models and high glucose (HG)-incubated human kidney proximal tubular epithelial (HK-2) cells were established to investigate the role and underlying mechanisms of miR-32 in DN. Results of real-time PCR revealed that miR-32 levels were greatly increased in DN rats and HG-incubated HK-2 cells. Downregulation of miR-32 effectively relieved HG-induced autophagy suppression, fibrosis, epithelial-mesenchymal transition (EMT) and inflammation in HK-2 cells. Besides, miR-32 overexpression significantly down-regulated the expression of mothers against decapentaplegic homolog 7 (SMAD7), whereas knockdown of miR-32 markedly up-regulated the level of SMAD7. Dual-luciferase reporter gene assay confirmed that SMAD7 was a target of miR-32. Reintroduction of SMAD7 expression rescued miR-32-induced HK-2 cells autophagy suppression, EMT and renal fibrosis. Our findings indicate that miR-32 may play roles in the progression of EMT and fibrosis in DN.

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Tumor-Suppressive Function of lncRNA-MEG3 in Glioma Cells by Regulating miR-6088/SMARCB1 Axis

BioMed Research International ◽

10.1155/2020/4309161 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15 ◽

Cited By ~ 1

Author(s):

Xin Gong ◽

Meng-Yi Huang

Keyword(s):

Cell Proliferation ◽

Epithelial Mesenchymal Transition ◽

Glioma Cells ◽

Luciferase Reporter ◽

Favorable Effect ◽

Mesenchymal Transition ◽

Gene Assay ◽

And Migration ◽

Proliferation And Migration ◽

Glioma Progression

Objective. Mounting evidence has elaborated the implication of long noncoding RNAs (lncRNAs) in tumorigenesis of several cancers, including glioma. However, little was known about the mechanism of lncRNA maternally expressed gene 3 (MEG3) in the development and progression of glioma. This work is designed to explore the effect of MEG3 on glioma progression and its possible mechanism. Methods. Expressions of lncRNA-MEG3 and SMARCB1 were detected in human glioblastoma U87 and U251 cell lines. Gain and loss of function of MEG3 or/and miR-6088 was performed in U87 and U251 cells to observe its effect on cell proliferation and migration as well as on epithelial-mesenchymal transition (EMT) related markers. Luciferase reporter gene assay was employed to inspect the interactions among MEG3, miR-6088, and SMARCB1. Results. MEG3 and SMARCB1 expressions were downregulated in glioma cells. Transfection of pcDNA3.1-MEG3 or pcDNA3.1-SMARCB1 plasmids could clearly block cell proliferation, migration, and EMT progression. MEG3 functions as a sponge for miR-6088, while SMARCB1 is a downstream protein of miR-6088. Transfection of miR-6088 mimic or si-SMARCB1 could obviously reverse the favorable effect of pcDNA3.1-MEG3 on glioma progression. Conclusion. Collectively, the evidence in this study indicated that MEG3 was downregulated in glioma cells and inhibited proliferation and migration of glioma cells via regulating miR-6088/SMARCB1 axis.

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INFLUENCE OF CHRONIC NEUROGENIC PAIN ON DYNAMICS OF ENDOTHELIN-1 LEVEL AND COMPONENTS OF NO-SYSTEM DURING MELANOMA B16/F10 GROWTH

Russian Journal of Oncology ◽

10.18821/1028-9984-2018-23-3-6-180-188 ◽

2018 ◽

Vol 23 (3-6) ◽

pp. 180-188

Author(s):

O. I Kit ◽

I. M Kotieva ◽

E. M Frantsiyants ◽

Ekaterina I. Surikova ◽

I. V Kaplieva ◽

...

Keyword(s):

Chronic Pain ◽

Tumor Growth ◽

Tumor Antigens ◽

Epithelial Mesenchymal Transition ◽

Neurogenic Pain ◽

Mesenchymal Transition ◽

Endothelin 1 ◽

Tumor Tissues ◽

Group 2 ◽

Skin Conditions

Chronic neurogenic pain is a pathogenic factor triggering mechanisms of homeostasis disfunction. As chronic neurogenic pain has been found to affect the biological features of B16/F10 melanoma, the purpose of the study was to determine the levels of endothelin-1 and components of the NO-system in mice during the growth of transplantable B16/F10 melanoma with chronic pain. Methods. The study included 64 female mice. B16/F10 melanoma was transplanted under the skin of the back to animals of the main group 2 weeks after the sciatic nerve ligation. Levels of endothelin-1, NOS-2, NOS-3, L-arginine, citrulline, total nitrite, nitrotyrosine and ADMA were determined by ELISA in the intact skin and in tumor tissues. Results. The dynamics of the studied parameters in tumor growth with and without chronic pain was different. Increased levels of endothelin-1 in the skin and in tumor tissues, stably elevated levels of NO-synthases in the tumor and stably elevated ADMA levels with their decrease by week 3 of the growth were observed in the tumor growth with pain. Conclusions. Chronic pain can contribute to the development of the immune tolerance to tumor antigens in the skin. Conditions are formed that both facilitate the survival of tumor cells and contribute to the further development of melanoma. The dynamics of activity of endothelin-1 and NO systems can promote stimulation of the epithelial-mesenchymal transition, enhance tumor invasion and hemangio- and lymphangiogenesis. Changes in the ADMA inhibitor levels in the tumor growth with chronic pain may indicate a more subtle control of the NO level providing increased melanoma invasiveness.

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MiR-196a promoted cell migration, invasion and the epithelial-mesenchymal transition by targeting HOXA5 in osteosarcoma

Cancer Biomarkers ◽

10.3233/cbm-201674 ◽

2020 ◽

Vol 29 (2) ◽

pp. 291-298

Author(s):

Xiaoli Wang ◽

Lili Zhang ◽

Xingfeng Zhang ◽

Cuihong Xing ◽

Ruidong Liu ◽

...

Keyword(s):

Cell Migration ◽

Epithelial Mesenchymal Transition ◽

Bone Cancer ◽

Luciferase Reporter ◽

Mesenchymal Transition ◽

Tumor Tissues ◽

Cell Metastasis ◽

Primary Bone ◽

Polymerase Chain ◽

Dual Luciferase Reporter Assay

INTRODUCTION: Osteosarcoma (OS), aggressive neoplasms of the bone, is the most common primary bone cancer in children. MiR-196a usually low expressed in several tumors and its functions in osteosarcoma still unclear. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the expression of miR-196a and the HOXA5. Cell metastasis and epithelial-mesenchymal transition (EMT) abilities were assessed using Transwell and western blot. The dual luciferase reporter assay was carried out to verify whether miR-196a directly targeted the 3’-untranslated region (UTR) of HOXA5 mRNA. RESULTS: MiR-196a was overexpressed and HOXA5 was low expressed in osteosarcoma versus the non-tumor tissues and normal cell lines. Upregulation of miR-196a or downregulation of HOXA5 was associated with worse outcome of osteosarcoma patients. MiR-196a enhanced cell migration, invasion and EMT by regulating the expression of HOXA5 through directly targeting the 3’-UTR of its mRNA in osteosarcoma. HOXA5 partially reversed roles of miR-196a on metastasis and EMT in osteosarcoma. CONCLUSIONS: MiR-196a promoted cell metastasis and EMT by targeting the 3’-UTR of HOXA5 mRNA in osteosarcoma. The newly identified miR-196a/HOXA5 axis provides novel insight into the pathogenesis of osteosarcoma.

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miR-4458 inhibits the epithelial–mesenchymal transition of hepatocellular carcinoma cells by suppressing the TGF-β signaling pathway via targeting TGFBR1

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa029 ◽

2020 ◽

Vol 52 (5) ◽

pp. 554-562

Author(s):

Yuke Zhang ◽

Kun Shi ◽

Hang Liu ◽

Wei Chen ◽

Yunhai Luo ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Signaling Pathway ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Epithelial Mesenchymal Transition ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Mesenchymal Transition ◽

Gene Assay ◽

Hcc Cells

Abstract Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world. MicroRNAs play a pivotal role in the progression of various cancers. To date, very little attention has been paid to miR-4458. Therefore, the aim of our study was to explore the function and underlying molecular mechanism of miR-4458 in HCC. We found that the expression of miR-4458 was reduced in HCC tissues and cell lines. Forced overexpression of miR-4458 inhibited the migration, invasion, and epithelial–mesenchymal transition (EMT) of HCC cells, while downregulation of miR-4458 promoted the aggressive phenotype. Furthermore, transforming growth factor beta receptor 1 (TGFBR1), the modulator of the TGF-β signaling pathway, was verified to be a novel target gene of miR-4458 by dual-luciferase reporter gene assay. Upregulated miR-4458 dramatically abolished TGFBR1 and p-Smad2/3 expression, thus blocking the TGF-β signaling pathway. Moreover, restoration of TGFBR1 partially rescued the miR-4458-mediated suppressive effect on the migration, invasion, and EMT and reactivated the TGF-β signaling pathway in HCC cells. In summary, our findings first demonstrated a mechanism of miR-4458 in HCC cell migration, invasion, and EMT by regulating the TGF-β signaling pathway via directly targeting TGFBR1.

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Long Noncoding RNA PTTG3P Promotes the Development of Childhood Asthma by Targeting the miR-192-3p/CCNB1 Axis

10.21203/rs.3.rs-676965/v1 ◽

2021 ◽

Author(s):

Bing Dai ◽

Feifei Sun ◽

Xuxu Cai ◽

Chunlu Li ◽

Fen Liu ◽

...

Keyword(s):

Childhood Asthma ◽

Noncoding Rna ◽

Epithelial Mesenchymal Transition ◽

Cell Counting ◽

Luciferase Reporter ◽

Mesenchymal Transition ◽

Gene And Protein Expression ◽

Gene Assay ◽

And Migration ◽

Proliferation And Migration

Abstract Background: Increasing studies have suggested that long non-coding RNAs (lncRNAs) affect the regulation of immune responses, airway inflammation, and other pathological processes of asthma. In this study, we investigated the functions of the lncRNA PTTG3P in the progression of childhood asthma.Methods: A competitive endogenous RNA network PTTG3P/miR-192-3p/CCNB1 was identified via bioinformatics analyses. Real-time qPCR and western blot were used to quantify gene and protein expression levels, respectively. Cell counting kit‑8 and transwell assays were used to evaluate the proliferation and migration abilities of bronchial epithelial cells (16HBE). Double luciferase reporter gene assay was used to validate the predictive targets in PTTG3P, miR-192-3p, and CCNB1.Results: PTTG3P was highly expressed in the peripheral blood of children with asthma. Knocking down PTTG3P could inhibit the epithelial-mesenchymal transition (EMT), proliferation, and migration of 16HBE cells. Mechanistically, PTTG3P promoted childhood asthma progression by targeting the miR-192-3p/CCNB1 axis.Conclusions: Childhood asthma development can be stemmed by targeting the PTTG3P/miR-192-3p/CCNB1 axis. This study provides potential diagnosis and treatment biomarkers for childhood asthma.

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Exosomes overexpressing miR-34c inhibit malignant behavior and reverse the radioresistance of nasopharyngeal carcinoma

Journal of Translational Medicine ◽

10.1186/s12967-019-02203-z ◽

2020 ◽

Vol 18 (1) ◽

Cited By ~ 10

Author(s):

Fang-Zhu Wan ◽

Kai-Hua Chen ◽

Yong-Chu Sun ◽

Xi-Chan Chen ◽

Ren-Ba Liang ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Tumor Suppressor ◽

Tumor Progression ◽

Epithelial Mesenchymal Transition ◽

Luciferase Reporter ◽

Serum Samples ◽

Mesenchymal Transition ◽

Malignant Behavior

Abstract Background Malignant behavior and radioresistance, which severely limits the efficacy of radiation therapy (RT) in nasopharyngeal carcinoma (NPC), are associated with tumor progression and poor prognosis. Mesenchymal stem cells (MSCs) are used as a therapeutic tool in a variety of tumors. The aim of this study was to reveal the effect of tumor suppressor microRNA-34c-5p (miR-34c) on NPC development and radioresistance, as well as to confirm that exosomes derived from MSCs overexpressing miR-34c restore the sensitivity to radiotherapy in NPCs. Methods Potentially active microRNAs were screened by cell sequencing, Gene Expression Omnibus (GEO) database analysis, and analysis of clinical serum samples from 70 patients. The expression of genes and proteins was detected by Western blotting, quantitative reverse transcription PCR (qRT-PCR), and immunohistochemistry (IHC). Proliferation, apoptosis, invasion, migration and radioresistance of NPC were detected. Luciferase reporter assays were used to verify the interactions of microRNAs with their downstream targets. MSCs exosomes were isolated by ultrafiltration and verified by electron microscopy and nanoparticle tracking technology. Results The expression of miR-34c was associated with the occurrence and radiation resistance of NPC. In vitro and in vivo experiments indicated that overexpression of miR-34c inhibit malignant behavior such as invasion, migration, proliferation and epithelial-mesenchymal transition (EMT) in NPCs by targeting β-Catenin. In addition, we found alleviated radioresistance upon miR-34c overexpression or β-catenin knockdown in NPCs. Exosomes derived from miR-34c-transfected MSCs attenuated NPC invasion, migration, proliferation and EMT. Moreover, miR-34c-overexpressing exosomes drastically increased radiation-induced apoptosis in NPC cells. Conclusion miR-34c is a tumor suppressor miR in NPC, which inhibits malignant behavior as well as radioresistance of tumor. Therefore, exogenous delivery of miR-34c to NPCs via MSC exosomes inhibits tumor progression and increases the efficiency of RT. Combination IR with miR-34c-overexpressing exosomes may be effective treatment for radioresistant NPCs.

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MicroRNA-296-5p inhibits cell metastasis and invasion in nasopharyngeal carcinoma by reversing transforming growth factor-β-induced epithelial–mesenchymal transition

Cellular & Molecular Biology Letters ◽

10.1186/s11658-020-00240-x ◽

2020 ◽

Vol 25 (1) ◽

Author(s):

Meihui Chen ◽

Chen Chen ◽

Haiqing Luo ◽

Jing Ren ◽

Qiuqin Dai ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Real Time ◽

Real Time Pcr ◽

Transforming Growth Factor ◽

Epithelial Mesenchymal Transition ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Cell Metastasis

Abstract Aim To explore the effect of miR-296-5p on the metastasis of nasopharyngeal carcinoma (NPC) cells and investigate the underlying mechanism. Methods The expressions of miR-296-5p in NPC tissues and cells were determined using GSE32920 database analysis and real-time PCR and miRNA microarray assays. An miR-296-5p mimic and inhibitor were transfected into NPC cells. Then, immunofluorescence imaging, scratch wound-healing, transwell migration and invasion assays were used to observe the effects of miR-296-5p on cell metastasis and invasion. Real-time PCR and western blotting were carried out to detect the expressions of genes and proteins related to epithelial–mesenchymal transition (EMT). A dual luciferase reporter assay was used to identify whether TGF-β is the target gene of miR-296-5p. Finally, TGF-β expression plasmids were transfected into NPC cells to verify the role of TGF-β in the miR-296-5p-mediated inhibition of nasopharyngeal carcinoma cell metastasis. Results Our results show that miR-296-5p inhibits the migratory and invasive capacities of NPC cells by targeting TGF-β, which suppresses EMT. Importantly, the miR-296-5p level was significantly lower in human NPC tissues than in adjacent normal tissues. It also negatively correlated with TGF-β and was significantly associated with the lymph node metastasis of patients with NPC. Conclusions Our findings show that miR-296-5p represses the EMT-related metastasis of NPC by targeting TGF-β. This provides new insight into the role of miR-296-5p in regulating NPC metastasis and invasiveness.

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MiR-205 suppresses epithelial–mesenchymal transition and inhibits tumor growth of human glioma through down-regulation of HOXD9

Bioscience Reports ◽

10.1042/bsr20181989 ◽

2019 ◽

Vol 39 (5) ◽

Cited By ~ 3

Author(s):

Bin Dai ◽

Guanghua Zhou ◽

Zhiqiang Hu ◽

Guangtong Zhu ◽

Beibei Mao ◽

...

Keyword(s):

Tumor Growth ◽

Cancer Progression ◽

Epithelial Mesenchymal Transition ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Pcr Analysis ◽

Human Glioma ◽

Mesenchymal Transition

AbstractEpithelial–mesenchymal transition (EMT) plays a pivotal role in cancer progression. Hsa-miR-205 is considered one of the fundamental regulators of EMT. In the present study, we found that miR-205 was down-regulated in glioma tissues and human glioma cells U87 and U251. Meanwhile, miR-205 overexpression enhanced E-cadherin, reduced mesenchymal markers, and decreased cell proliferation, migration, and invasion in vitro. In vivo, miR-205 suppressed tumor growth. Additionally, HOXD9 was confirmed as a direct target of miR-205. Suppression of HOXD9 by miR-205 was demonstrated by luciferase reporter assay, quantitative real time-PCR analysis, and western blot. Moreover, we observed a negative correlation between miR-205 and HOXD9 in human glioma tissues. In summary, our findings demonstrated that miR-205 suppresses glioma tumor growth, invasion, and reverses EMT through down-regulating its target HOXD9.

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