scholarly journals miR-199a Targeting PNRC1 to Promote Keratinocyte Proliferation and Invasion in Cholesteatoma

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Lihui Yao ◽  
Wenjing Zhang ◽  
Jian Zheng ◽  
Xing Lu ◽  
Fan Zhang
Keyword(s):  
Target Gene ◽  
Target Genes ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Keratinocyte Proliferation ◽  
Qrt Pcr ◽  
Candidate Target ◽  
Candidate Target Gene ◽  
Proliferation And Invasion ◽  
Dual Luciferase Reporter Assay

Introduction. miR-199a has been reported as an oncogene of various cancers. However, the biological function and regulatory mechanism of miR-199a in keratinocytes of cholesteatoma are still unclear. Methods. Detection by qRT-PCR was conducted on miR-199a’s expression in both thirty pairs of cholesteatoma tissues and normal skins. For characterizing the function of miR-199a, this research adopted transwell assay, wound healing assay, and CCK8 assays. Under the support of qRT-PCR, efforts were made to investigate the relative expression of candidate target genes. Moreover, the evaluation of the targeting relationship between miR-199a and the candidate target gene was conducted with the dual-luciferase reporter assay. Results. The upregulation of miR-199a was found in cholesteatoma tissues, which facilitated the proliferation, migration, and invasion of HaCaT cells, while its downregulation caused opposite results. Conclusions. The findings of the present research offer more insights into the molecular mechanism of cholesteatoma progression.

scholarly journals MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4

2021 ◽  
Vol 20 ◽  
pp. 153303382098586
Author(s):  
Xuhui Wu ◽  
Gongzhi Wu ◽  
Huaizhong Zhang ◽  
Xuyang Peng ◽  
Bin Huang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Invasion ◽  
Target Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Invasion And Migration ◽  
And Migration ◽  
Dual Luciferase Reporter Assay

Objective: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. Methods: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. Results: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. Conclusion: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

scholarly journals lncRNA CRNDE promotes the proliferation and metastasis by acting as sponge miR-539-p to regulate POU2F1 expression in HCC

2020 ◽  
Author(s):  
Zhixi Li ◽  
Gang Wu ◽  
Jie Li ◽  
Youyu Wang ◽  
Xueming Ju ◽  
...  
Keyword(s):  
Western Blot ◽  
Luciferase Reporter ◽  
Akt Pathway ◽  
Migration And Invasion ◽  
Reporter Assay ◽  
Normal Cells ◽  
Normal Tissues ◽  
Qrt Pcr ◽  
Hcc Cells ◽  
Dual Luciferase Reporter Assay

Abstract Background This article focuses on the roles and mechanism of lncRNA CRNDE on the progression of HCC. Methods We used qRT-PCR to detect the expression of lncRNA CRNDE in HCC cells, normal cells and clinical tissues. MTT assay, FCM analysis, Transwell migration and invasion assay were used to detect the effects of lncRNA CRNDE on cell viability, apoptosis, migration and invasion of HCC cells. The expression of apoptosis-related proteins Bcl-2, Bax, Cleaved Caspase 3, Cleaved Caspase 9, EMT epithelial marker E-cadherin and mesothelial marker Vimentin were analyzed by Western blot. Online prediction software was used to predict the binding sites between lncRNA CRNDE and miR-539-5p, or miR-539-5p and POU2F1 3’UTR. Dual luciferase reporter assay, qRT-PCR and RNA pulldown were used to detect target-relationship between lncRNA CRNDE and miR-539-5p. Dual luciferase reporter assay, qRT-PCR, Western blot and Immunofluorescence were used to detect target-relationship between miR-539-5p and POU2F1. qRT-PCR was used to detect the expression of miR-539-5p and POU2F1 in clinical tissues. Rescue experiments was used to evaluate the association among lncRNA CRNDE, miR-539-5p and POU2F1. Finally, we used Western blot to detect the effects of lncRNA CRNDE, miR-539-5p and POU2F1 on NF-κB and AKT pathway. Results lncRNA CRNDE was highly expressed in HCC cells and HCC tissues compared with normal cells and the corresponding adjacent normal tissues. lncRNA CRNDE promoted the cell viability, migration and invasion of HCC cells, while inhibited the apoptosis and promoted the EMT process of HCC cells. lncRNA CRNDE adsorbed miR-539-5p acts as a competitive endogenous RNA to regulate POU2F1 expression indirectly. In HCC clinical tissues, miR-539-5p expression decreased and POU2F1 increased compared with the corresponding adjacent normal tissues. lncRNA CRNDE/miR-539-5p/POU2- F1 participated the NF-κB and AKT pathway in HCC. Conclusion lncRNA CRNDE promotes the expression of POU2F1 by adsorbing miR-539-5p, thus promoting the progression of HCC. Keywords: HCC, lncRNA CRNDE, miR-539-5p, POU2F1, ceRNA

scholarly journals Circular RNA CircITGA7 Promotes Tumorigenesis of Osteosarcoma via miR-370/PIM1 Axis

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Chuanwu Fang ◽  
Xiaohong Wang ◽  
Dongliang Guo ◽  
Run Fang ◽  
Ting Zhu
Keyword(s):  
Circular Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Functional Assay ◽  
Transwell Assay ◽  
Qrt Pcr ◽  
Proliferation And Metastasis ◽  
And Migration ◽  
Dual Luciferase Reporter Assay ◽  
Proliferation And Migration

Many studies have shown that there are many circular RNA (circRNA) expression abnormalities in osteosarcoma (OS), and this abnormality is related to the development of osteosarcoma. But at present, it is unclear as to what circITGA7 has in the OS and what it does. In this study, qRT-PCR was used to detect the expression of circITGA7, miR-370, and PIM1 mRNA in OS tissues and cells. The CCK-8 assay was used to detect the effect of circITGA7 on cell proliferation. Later, the transwell assay was used to detect cell migration and invasion. The dual-luciferase reporter assay confirmed the existence of the targeting relationship between circITGA7 and miR-370, and miR-370 and PIM1. We found that circITGA7 was upregulated in OS tissues and cell lines. Knockdown of circITGA7 weakened the cell’s ability to proliferate and metastasize. Furthermore, we observed that miR-370 was negatively regulated by circITGA7, while PIM1 was positively regulated by it. A functional assay validated that circITGA7 promoted OS progression via suppressing miR-370 and miR-370 affected OS proliferation and migration via PIM6 in OS. In summary, this study shows that circITGA7 promotes OS proliferation and metastasis via miR-370/PIM1.

scholarly journals LncRNA ZEB1-AS1 regulates hepatocellular carcinoma progression by targeting miR-23c

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Shuai Xue ◽  
Fengqin Lu ◽  
Chunhui Sun ◽  
Jingjing Zhao ◽  
Honghua Zhen ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Correlation Analysis ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Transwell Assay ◽  
Qrt Pcr ◽  
Proliferation And Invasion ◽  
Hcc Cells ◽  
Dual Luciferase Reporter Assay

Abstract Background It has been reported that long-chain non-coding RNA (lncRNA) zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) is an oncogene in various cancers, including hepatocellular carcinoma (HCC). We investigated the role and mechanism of ZEB1-AS1 as a competitive endogenous RNA (ceRNA) combined with miR-23c in HCC cell proliferation and invasion. Methods QRT-PCR was used to detect ZEB1-AS1 and miR-23c expressions in HCC tissues and cells. The dual luciferase reporter assay detected the targeted regulation of miR-23c and ZEB1-AS1. We also performed the correlation analysis of their expression in HCC tissues by the Spearman’s correlation analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of hepatoma cells. Cell invasion was assessed by the Transwell assay. Results QRT-PCR results indicated ZEB1-AS1 was upregulated and miR-23c was downregulated in HCC tissues and cell lines. ZEB1-AS1 knockdown hampered the proliferation and invasion of HCC cells. Dual luciferase reporter assay showed that miR-23c is a target of ZEB1-AS1, and ZEB1-AS1 was significantly negatively correlated with the miR-23c expression in HCC tissues. The results of MTT and Transwell assay showed that miR-23c inhibition restored the inhibitory effect of ZEB1-AS1 knockdown on HCC cells proliferation and invasion. Conclusions As a ceRNA, lncRNA ZEB1-AS1 may play a vital role in inhibiting HCC progression through miR-23c, which will provide new clues and theoretical basis for the HCC diagnosis and treatment.

scholarly journals LncRNA SNHG16 Contributes to Tumor Progression via miR-302b-3p/SLC2A4 in Pancreatic Cancer

2020 ◽  
Author(s):  
Hao Xu ◽  
Xin Miao ◽  
Xin Li ◽  
Haofei Chen ◽  
Bo Zhang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Caspase 3 ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Caspase 9 ◽  
Transwell Assay ◽  
Qrt Pcr ◽  
Rna Immunoprecipitation ◽  
Related Proteins ◽  
Dual Luciferase Reporter Assay

Abstract Background: It is reported that lncRNA SNHG16 is significantly highly expressed in pancreatic cancer (PC). However, the functions and mechanisms of SNHG16 are not clear. The aim of this study was to explore the effects of SNHG16 in PC.Methods: qRT-PCR analyze was applied to detect the expression levels of SNHG16, miR-302b-3p and SLC2A4 in PC tissues and cells. CCK8 and EdU assays were used to determine the proliferation of PC cells. Transwell assay were used to measure the capacities of PC cells migration and invasion. Apoptosis were evaluated by flow cytometry, and the expression of apoptosis related proteins (including Bax, Bcl-2, cleaved caspase-3 and cleaved caspase-9), which were tested by western blot. The interactions between miR-302b-3p and SNHG16 or miR-302b-3p and SLC2A4 mRNA 3’UTR were clarified by Dual luciferase reporter assay and RNA immunoprecipitation.Results: SNHG16 was significantly elevated in PC tissues and cell lines, which was associated with poor prognosis of PC patients. Knockdown of SNHG16 reduced PC cells proliferation, migration and invasion. SNHG16 acted as a sponge to regulate miR-302b-3p expression in PC cells. And miR-302b-3p targeted SLC2A4 directly. Conclusions: SNHG16 promoted the progression of PC via miR-302b-3p/SLC2A4 axis and was expected to be a potential target for early diagnosis and treatment of PC.

scholarly journals miR-1270 enhances the proliferation, migration, and invasion of osteosarcoma via targeting cingulin

2021 ◽  
Vol 65 (4) ◽  
Author(s):  
Yang Liu ◽  
Weichun Guo ◽  
Shuo Fang ◽  
Bin He ◽  
Xiaohai Li ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Target Genes ◽  
Bioinformatics Analysis ◽  
Tumor Xenograft ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
High Morbidity ◽  
Proliferation And Apoptosis ◽  
Dual Luciferase Reporter Assay

Osteosarcoma (OS), characterized by high morbidity and mortality, is the most common bone malignancy worldwide. MicroRNAs (miRNAs) play a crucial role in the initiation and development of OS. The purpose of this study was to investigate the roles of miR-1270 in OS. RT-qPCR and Western blot were applied to detect the mRNA and protein level, respectively. CCK-8, colony formation, and TUNEL assays were conducted to determine the cell viability, proliferation, and apoptosis of OS cells. Wound healing and transwell assay were performed to detect the migration and invasion ability of OS cells. Bioinformatics analysis and dual-luciferase reporter assay were used to predict the target genes of miR-1270. Tumor xenograft in vivo assay was carried out to determine miR-1270 effect on the tumor size, volume, and weight. In this study, miR-1270 was overexpressed in OS tissues and cells. However, miR-1270 knockdown inhibited the proliferation, migration and invasion, and promoted the OS cells’ apoptosis. Mechanistically, cingulin (CGN) was predicted and proved to be a target of miR-1270 and partially alleviated the effects of miR-1270 on the proliferation, migration and invasion ability of OS cells. Taken together, knockdown of miR-1270 may inhibit the development of OS via targeting CGN. This finding may provide a novel therapeutic strategy for OS.

scholarly journals MicroRNA-145 Aggravates Hypoxia-Induced Injury by Targeting Rac1 in H9c2 Cells

2017 ◽  
Vol 43 (5) ◽  
pp. 1974-1986 ◽  
Author(s):  
Ximing Wang ◽  
Yanxia Zhang ◽  
Hongshan Wang ◽  
Genshang Zhao ◽  
Xianen Fa
Keyword(s):  
Target Gene ◽  
Target Genes ◽  
Underlying Mechanism ◽  
Mitogen Activated Protein Kinase ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
H9c2 Cells ◽  
Down Regulation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Background/Aims: Myocardial infarction (MI) is a leading cause of morbidity and mortality. Here, we sought to explore the potential role and underlying mechanism of miR-145 in MI. Methods: H9c2 cells were cultured under persistent hypoxia to simulate MI. The hypoxia-induced injury was assessed on the basis of cell viability, migration, invasion and apoptosis. The expression of miR-145 was evaluated by qRT-PCR and the influence of aberrantly expressed miR-145 on H9c2 cells under hypoxia was also estimated. Utilizing bioinformatics methods, the target genes of miR-145 were verified by luciferase reporter assay. Then, effects of abnormally expressed target gene on miR-145 silenced H9c2 cells were assessed. Finally, the phosphorylation levels of key kinases in the phosphatidylinositol-3-kinase (PI3K)/AKT and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways were detected by Western blot analysis. Results: Hypoxia remarkably lowered viability, migration and invasion but promoted cell apoptosis. Meantime, the miR-145 level was up-regulated in H9c2 cells under hypoxia. Following experiments suggested that hypoxia-induced injury was exacerbated by miR-145 overexpression while was alleviated by miR-145 silence. Rac1 was predicted and further validated to be a target gene of miR-145. The influence of miR-145 silencing on H9c2 cells under hypoxia could be reversed by down-regulation of Rac1. Additionally, the phosphorylation levels of PI3K, AKT, MAPK and ERK were all elevated in miR-145 silenced cells and these alterations were reversed by down-regulation of Rac1. Conclusion: miR-145 silencing could protect H9c2 cells against hypoxia-induced injury by targeting Rac1, in which PI3K/AKT and MAPK/ERK pathways might be involved.

scholarly journals Bta-miR-124a Affects Lipid Metabolism by Regulating PECR Gene

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Binglei Shen ◽  
Zhuonina Yang ◽  
Shuo Han ◽  
Ziwen Zou ◽  
Juan Liu ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Metabolism ◽  
Dairy Cows ◽  
Signaling Pathway ◽  
Target Gene ◽  
Target Genes ◽  
Mammary Epithelial ◽  
Luciferase Reporter ◽  
Milk Lipid ◽  
Candidate Target

According to our previous studies, bta-miR-124a was differentially expressed in breast tissue between high-fat and low-fat dairy cows. However, the function of bta-miR-124a in lipid metabolism of dairy cows and the identification of its target genes have not been reported. Therefore, this study will identify the target gene of bta-miR-124a and explore its role in the regulation of milk lipid metabolism. First, preliminary bioinformatics prediction of bta-miR-124a candidate target genes was performed, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze relative expression changes of bta-miR-124a and its candidate target genes and the expression level of the downstream gene of the target gene in the lipid metabolism signaling pathway in dairy mammary epithelial cell lines (Mac-T), using the dual luciferase reporter system for the identification of the targeting relationship between bta-miR-124a and the candidate target gene. Then, the effect of transfection of bta-miR-124a mimics and inhibitors on triglyceride (TG) and free fatty acid (FFA) levels was analyzed. The results indicate that bta-miR-124a directly interacts with the 3′-untranslated region of peroxisomal trans-2-enoyl-CoA reductase (PECR) to downregulate its expression in Mac-T cells. Further, bta-mir-124a regulates the expression of PECR and the downstream gene extension of very long chain fatty acid protein 2 (ELOVL2) through an unsaturated fatty acid biosynthesis signaling pathway. In conclusion, bta-miR-124a is involved in lipid metabolism by directly downregulating the PECR gene and affecting the expression of the downstream gene ELOVL2 and regulates the content of some key secretory elements such as TG and FFA. The function of bta-miR-124a has a certain effect on the synthesis and secretion of milk fat in the mammary epithelial cells of dairy cows.

scholarly journals Overexpression of NELFE contributes to gastric cancer progression via Wnt/β-catenin signaling-mediated activation of CSNK2B expression

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Shijun Yu ◽  
Li Li ◽  
Hui Cai ◽  
Bin He ◽  
Yong Gao ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Proliferation ◽  
Cancer Progression ◽  
Target Genes ◽  
Mrna Level ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Loss Of Function ◽  
Mice Model ◽  
Qrt Pcr

Abstract Background Accumulating evidence has highlighted the importance of negative elongation factor complex member E (NELFE) in tumorigenesis. However, the relationship between NELFE and gastric cancer (GC) remains unclear. This study aimed to explore the expression pattern and specific function of NELFE in GC. Methods NELFE expression was evaluated by immunohistochemistry and qRT-PCR in GC tissues, respectively. Cell proliferation, migration and invasion were measured by CCK-8, colony formation, transwell assays, and nude mice model. Bioinformatics analysis was performed to search potential target genes of NELFE, and a Cignal Finder 10-Pathway Reporter Array was used to explore potential signaling pathways regulated by NELFE. Dual-luciferase reporter assays, qRT-PCR and western blotting were conducted to verify their regulatory relationship. The expression correlations among NELFE, β-catenin and CSNK2B were further explored by immunohistochemistry on consecutive resections. Results NELFE was significantly overexpressed in GC tissues both in protein and mRNA level and negatively correlated with the prognosis of GC patients. Gain- and loss-of-function experiments showed that NELFE potentiated GC cell proliferation and metastasis in vitro and in vivo. CSNK2B was identified as a downstream effector of NELFE. Wnt/β-catenin signaling may mediate the regulation of CSNK2B by NELFE. In addition, NELFE, β-catenin and CSNK2B were all remarkably upregulated in tumor tissues compared with adjacent normal tissues, and their expression levels in GC were positively correlated with each other. Conclusion Our findings reveal a new NELFE-Wnt/β-catenin-CSNK2B axis to promote GC progression and provide new candidate targets against this disease.

scholarly journals miR-17~92 Promotes Progression of ABC-DLBCL Lymphoma via Regulation of Canonical NF-kB Signaling

2021 ◽  
Author(s):  
Xianhuo Wang ◽  
Huaqing Wang ◽  
Xiaoyan Zhang ◽  
Chengfeng Bi ◽  
Timothy W. McKeithan ◽  
...  
Keyword(s):  
B Cell ◽  
Therapeutic Strategy ◽  
Target Gene ◽  
Target Genes ◽  
B Cell Lymphoma ◽  
Aggressive Lymphoma ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Over Expression ◽  
Dual Luciferase Reporter Assay

Abstract Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive lymphoma characterized by constitutive NF-κB activation. Nevertheless, the role and mechanisms of miR-17~92 in contributing to the NF-κB activation in ABC-DLBCL are still elusive. Methods: The expression of miR-17~92 primary transcript (MIR17HG) and NF-κB target genes was determined using RNA-sequencing. The expression of miR-17~92 was performed using microarray analysis. Plasmids carrying conditional over-expression and loss-of-function of miR-17~92 were respectively constructed and dual-luciferase reporter assay was used to validate the target gene of miR-17~92. Immunoprecipitation and polyubiquitination were further used to the study of potential mechanisms.Results: Expression of MIR17HG was positively correlated with NF-κB activity, miR-17~92 activated the NF-κB signaling in ABC-DLBCL, and its over-expression promoted ABC-DLBCL cell growth, accelerated cell G1 to S phase transition and enhanced cell resistance to NF-κB inhibitor. Importantly, miR-17~92 promoted NF-κB activation through directly targeting multiple ubiquitin-editing regulators to lead to increase the K63-linked polyubiquitination and decrease the K48-linked polyubiquitination of RIP1 complex in ABC-DLBCL. We further found that miR-17~92 selectively activated IκB-α and NF-κB p65 but not NF-κB p52/p100, and high miR-17~92 expression was also associated with poorer outcome in ABC-DLBCL patients. Conclusions: Taken together, miR-17~92 selectively activate the canonical NF-κB signaling via targeting ubiquitin-editing regulators to lead to constitutively NF-κB activation and poorer outcome, which is an innovative function of miR-17~92 and previously unappreciated regulatory mechanism of NF-κB activation in ABC-DLBCL. Targeting miR-17~92 may thus provide a novel bio-therapeutic strategy for ABC-DLBCL patients.

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