scholarly journals miR-148a-3p inhibits DNMT1-mediated methylation of VEGF and promotes proliferation and invasion in glioma cells via the PI3K/Akt/eNOS signaling pathway

2020 ◽  
Author(s):  
Jun-feng Huo ◽  
Xiao-bing Chen
Keyword(s):  
Cell Lines ◽  
Methylation Level ◽  
Promoter Region ◽  
Dna Methyltransferase ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Protein Levels ◽  
U87 Cells ◽  
Proliferation And Invasion

Abstract Background: Abnormal DNA methylation in the promoter region of vascular endothelial growth factor (VEGF) has been observed in multiple types of cancer. Increasing evidence shows that miR-148a-3p is involved in the regulation of methylation. However, it is unclear how miR-148a-3p regulates VEGF methylation.Methods: Methylation-specific polymerase chain reaction (MSP) and bisulfate-sequencing PCR (BSP) were performed to detect the methylation level of the VEGF promoter region in glioma tissues and cell lines. Then, we used RT-qPCR to detect the expression of miR-148a-3p and VEGF in glioma tissues and cell lines. Human glioma U87 cells were transfected with miR-148a-3p mimic or a pcDNA3.1 overexpression vector of VEGF, and then, the proliferation, invasion and apoptosis of U87 cells were examined with cell counting kit-8 (CCK-8), Transwell assay and Flow cytometry, respectively. We next examined the relationship between DNA methyltransferase 1 (DNMT1) and miR-148a-3p with online prediction and luciferase reporter gene experiments. Furthermore, we also used Western blotting to detect the protein levels of VEGF, DNMT1, PI3K, Akt and eNOS.Results: Both miR-148a-3p and VEGF were significantly up-regulated, and the promoter methylation of VEGF was hypomethylation in glioma. Overexpression miR-148a-3p or VEGF promoted the proliferation and invasion of U87 cells and inhibited the apoptosis. Silencing VEGF inhibited U87 cells proliferation and invasion and induced the apoptosis. Furthermore, miR-148a-3p regulated DNMT1 at the post-transcriptional. Overexpression of DNMT1 mediated an increase in the methylation level of VEGF, which reduced the expression of VEGF in U87 cells. The opposite was exact when DNMT1 was silenced. We also observed that silencing VEGF inhibited the activation of the PI3K/Akt/eNOS pathway and induced U87 cell apoptosis.Conclusion: These results indicated that miR-148a-3p down-regulates VEGF methylation by targeting DNMT1 and promotes the proliferation, invasion of the glioma cell through the PI3K/Akt/eNOS pathway.

scholarly journals MicroRNA-367 directly targets PIK3R3 to inhibit proliferation and invasion of oral carcinoma cells

2020 ◽  
Vol 40 (5) ◽  
Author(s):  
Haitao Sun ◽  
Xiaodong Feng
Keyword(s):  
Quantitative Pcr ◽  
Cell Lines ◽  
Hepatocellular Cancer ◽  
Carcinoma Cells ◽  
Luciferase Reporter ◽  
Pcr Assay ◽  
Protein Levels ◽  
Cancer Types ◽  
Proliferation And Invasion ◽  
Quantitative Pcr Assay

Abstract Recently, microRNA-367 (miR-367) has been reported to function as both tumor suppressor and oncogene in several cancer types, including gastric cancer, hepatocellular cancer and lung cancer. However, the biological function of miR-367 and its precise mechanisms in oral squamous cell carcinoma (OSCC) have not been well clarified. The aim of the present study was to study the roles of miR-367/PIK3R3 axis in OSCC. The levels of PIK3R3 and miR-367 were detected by quantitative PCR assay in OSCC tissues and cell lines. Moreover, the biological roles of miR-367 and PIK3R3 in OSCC cells were assessed by cell proliferation and invasion. The mRNA and protein levels of PIK3R3 were determined by using quantitative PCR and Western blotting assays. Luciferase assays were used to confirm that PIK3R3 was one target of miR-367. In the present study, the miR-367 level was dramatically reduced in OSCC tissues and cell lines, and the PIK3R3 expression was significantly enhanced. What’s more, the PIK3R3 expression was negatively related to the miR-367 level in OSCC tissues. Furthermore, up-regulation of miR-367 obviously restrained OSCC cells proliferation and invasion. We confirmed that miR-367 could directly target PIK3R3 by luciferase reporter assay. Besides, knockdown of PIK3R3 also could markedly inhibit the proliferation and invasion of OSCC cells. Finally, overexpression of miR-367 in OSCC cells partially reversed the promoted effects of PIK3R3 up-regulation. Overexpression of miR-367 restrained OSCC cells proliferation and invasion via regulation of PIK3R3.

scholarly journals Identification and analysis of the promoter region of the human methionine sulphoxide reductase A gene

2005 ◽  
Vol 393 (1) ◽  
pp. 321-329 ◽  
Author(s):  
Antonella De Luca ◽  
Paolo Sacchetta ◽  
Carmine Di Ilio ◽  
Bartolo Favaloro
Keyword(s):  
Cell Lines ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Promoter Activity ◽  
Regulatory Region ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Mcf7 Cells ◽  
Luciferase Reporter Gene ◽  
Hek 293

MsrA (methionine sulphoxide reductase A) is an antioxidant repair enzyme that reduces oxidized methionine to methionine. Moreover, the oxidation of methionine residues in proteins is considered to be an important consequence of oxidative damage to cells. To understand mechanisms of human msrA gene expression and regulation, we cloned and characterized the 5′ promoter region of the human msrA gene. Using 5′-RACE (rapid amplification of cDNA ends) analysis of purified mRNA from human cells, we located the transcription initiation site 59 nt upstream of the reference MsrA mRNA sequence, GenBank® accession number BC 054033. The 1.3 kb of sequence located upstream of the first exon of msrA gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Sequentially smaller fragments of the msrA promoter region were generated by PCR, and expression levels were monitored from these constructs within HEK-293 and MCF7 human cell lines. Analysis of deletion constructs revealed differences in promoter activity in these cell lines. In HEK-293 cells, the promoter activity was constant from the minimal promoter region to the longest fragment obtained. On the other hand, in MCF7 cells we detected a down-regulation in the longest fragment. Mutation of a putative negative regulatory region that is located between −209 and −212 bp (the CCAA box) restored promoter activity in MCF7 cells. The location of the msrA promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts.

The long non-coding RNA cytoskeleton regulator (CYTOR) sponges microRNA-206 (miR-206) to promote proliferation and invasion of HP75 cells

2021 ◽  
Vol 21 ◽  
Author(s):  
Hu Keqi ◽  
Liu Handong
Keyword(s):  
Cell Lines ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Chi Square ◽  
Qrt Pcr ◽  
Non Coding Rna ◽  
Hp75 Cells ◽  
Long Non Coding Rna

Background: The role and mechanism of long non-coding RNA cytoskeleton regulator (CYTOR) in invasive pituitary adenomas (IPA) has not been elucidated previously. Objective: This study aimed to investigate the interaction between CYTOR and miR-206 and their roles in IPA using HP75 cells as the model. Methods: The expression levels of CYTOR and miR-206 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in IPA tissues and cell lines. The Chi-square test was used to analyze the correlation between CYTOR expression and clinical-pathological parameters. HP75 cell proliferation was detected by Cell Counting Kit-8 assay and colony formation assay. Scratch healing experiments and Transwell assay were used to detect migration and invasion of HP75 cells. The relationship between CYTOR and miR-206 was predicted by bioinformatics and verified by qRT-PCR and dual-luciferase reporter gene method. Result: CYTOR is up-regulated in IPA tissues and cell lines. The high expression of CYTOR is associated with adenoma invasiveness and adenoma size of the patients. Down-regulation of CYTOR decreases the proliferation, migration and invasion of HP75 cells, while up-regulation of miR-206 can inhibit proliferation, migration and invasion of HP75 cells. MiR-206 is identified as a target of CYTOR and could be negatively regulated by it in IPA. Discussion: CYTOR, as a tumor-promoting factor, facilitates the proliferation, migration and invasion of HP75 cells through sponging miR-206. Conclusion: The CYTOR-miR-206 axis provides new insights into the diagnosis and treatment of IPA.

scholarly journals Decreased miR- 31 Suppress Cell Migration and Proliferation By Targeting Syncytin-1 in Pancreatic Carcinoma

2020 ◽  
Author(s):  
Jing Yang ◽  
Judong Luo ◽  
Feng Wang ◽  
Zhiwen Cheng ◽  
Xia Han ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Prognostic Significance ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Kaplan Meier ◽  
Gene Assay ◽  
Proliferation And Invasion

Abstract Background: Pancreatic cancer(PC) is seriously harmful to human health, and the pathogenesis is not clear. The present study aimed to explore the functional role of syncytin-1 in PC.Methods: Syncytin-1 and miR-31 expression was analyzed by qRT-PCR and Western blot analysis in both human PC cell lines and tissuse. The prognostic significance of syncytin-1 was investigated using the immunohistochemistry(IHC) and Kaplan-Meier survival. The CCK-8 assay and transwell assays were used to determine the role of syncytin-1 and miR-31 in cell proliferation, migration and invasion. Luciferase reporter assays was used to identify possible miRNA targets in tumorigenesis.Results: The results showed that the syncytin-1 level was significantly decreased in PC cell lines and tissues than normal(P < 0.05), while miR-31 was markedly higher than normal(P < 0.05), and low expression of syncytin-1 have a poor prognosis than high expression(P < 0.05). Overexpression of syncytin-1 significantly reduced the PC cell proliferation and invasion ability in PANC-1 and BxPC-3 cells(P < 0.05), and miR-31showed contrary results. The Dual-Luciferase reporter gene assay demonstrated that miR-31 binded directly to 3’UTR of syncytin-1 and resulting in the inhibition of syncytin-1. The overexpression of miR-31 promoted migration and proliferation of PC cells through down-regulating the expression of syncytin-1.Conclusion: We verified that syncytin-1 can inhibit proliferation and invasion of PC cell lines by targeting miR-31.

scholarly journals LncRNA SNHG3 Promotes Gastric Cancer Cells Proliferation, Migration, and Invasion by Targeting miR-326

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Jun Rao ◽  
Jinjin Fu ◽  
Chuchen Meng ◽  
Jin Huang ◽  
Xiangrong Qin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Gastric Cancer Cells ◽  
Invasion And Migration ◽  
Protein Levels ◽  
Rna Immunoprecipitation ◽  
And Migration

The function and possible mechanism of lncRNA Small Nucleolar RNA Host Gene 3 (SNHG3) in GC have not been fully studied. The aim of our study was to investigate the role of SNHG3 in the proliferation, migration, and invasion of GC cell lines. The expressions of SNHG3, miR-326, and TWIST in GC9811-P GC cell lines were detected by RT-qPCR. Western blotting was performed to detect the protein levels of TWIST and EMT-related genes. Luciferase reporter gene analysis and RNA immunoprecipitation (RIP) analysis confirmed the interaction between lncRNA SNHG3, miR-326, and TWIST. CCK-8 and Transwell assays were performed to detect cell proliferation, invasion, and migration abilities. The results showed that lncRNA SNHG3 and TWIST were highly expressed in GC cell lines, while miR-326 was expressed to a low degree. Moreover, lncRNA SNHG3 knockdown or miR-326 overexpression significantly inhibited cell proliferation, migration, and invasion of GC cell lines. In addition, TWIST overexpression can reverse the inhibition of lncRNA SNHG3 knockdown or miR-326 overexpression on cell proliferation, migration, and invasion. In conclusion, lncRNA SNHG3 may promote GC progression through the miR-326/TWIST axis, which may provide a new diagnostic and prognostic biomarker for GC.

scholarly journals Linc00887 suppresses tumorigenesis of cervical cancer through regulating the miR-454-3p/FRMD6-Hippo axis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Pei Li ◽  
Jinsheng Wang ◽  
Lingran Zhi ◽  
Fengmei Cai
Keyword(s):  
Cervical Cancer ◽  
Cell Proliferation ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Hippo Signaling ◽  
Luciferase Reporter Gene ◽  
Hippo Signaling Pathway ◽  
Cancer Tissues ◽  
Proliferation And Invasion

Abstract Background Emerging evidence suggested that long intergenic noncoding RNA (lincRNA) 00887 (NR_024480) reduced the invasion and metastasis of non-small cell lung cancer by sponging miRNAs degradation. However, the role and regulatory mechanism of linc00887 in the progression of cervical cancer remain largely unknown. Methods In vivo or vitro, RT-qPCR assay was used to detect the expression of linc00887 in human normal (N = 30), cervical cancer tissues (N = 30), human normal cervical epithelial cells (Ect1/E6E7) and cervical cancer cell lines (HeLa, C33A). Then, CCK-8 and Transwell assays were used to examine cell proliferation and invasion when linc00887 was overexpressed or knocked down. In addition, bioinformatics, luciferase reporter gene and pull-down assays were used to predict and validate the relationship between linc00887 and miR-454-3p. Moreover, we detected the expression of miR-454-3p in Ect1/E6E7, HeLa and C33A cells when linc00887 was overexpressed or knocked down. Cell proliferation and invasion were also measured when pcDNA-linc00887 and miR-454-3p were transfected alone or together. Next, miR-454-3p target gene was predicted and validated by bioinformatics and luciferase reporter gene assays. Gain- and loss-of-function experiments were performed in HeLa cells to evaluate the effect of miR-454-3p or linc00887 on the expression of FERM domain containing protein 6 (FRMD6) protein and several key proteins in the FRMD6-Hippo signaling pathway. Results Linc00887 was downregulated in cervical cancer tissues or human cervical cancer cell lines (Hela, C33A) compared with normal tissues or cell lines. Overexpression of linc00887 inhibited proliferation and invasion HeLa and C33A cells, while linc00887 knockdown had the opposite effect. Linc00887 bound with miR-454-3p, and overexpression of miR-454-3p rescued linc00887-induced inhibition proliferation and invasion of HeLa cells. MiR-454-3p targeted and suppressed the expression of FRMD6, and linc00887 suppressed tumorigenesis of cervical cancer through activating the FRMD6-Hippo signaling pathway. Conclusions Linc00887, sponging miR-454-3p, inhibited the progression of cervical cancer by activating the FRMD6-Hippo signaling pathway.

scholarly journals MiR-326 mediates malignant biological behaviors of lung adenocarcinoma by targeting ZEB1

2021 ◽  
Vol 104 (2) ◽  
pp. 003685042110093
Author(s):  
Mingxin Liu ◽  
Hong Wu ◽  
Yiqiang Liu ◽  
Yan Tan ◽  
Songtao Wang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Invasion And Migration ◽  
Transwell Invasion Assay ◽  
Enhancer Binding Protein ◽  
Adenocarcinoma Cells ◽  
And Migration ◽  
Related Proteins

MiR-326 functions as an antioncogene in the several types of cancer. However, the underling mechanisms through which miRNA-326 regulates the anti-carcinogenesis of lung adenocarcinoma have remained elusive. The aim of this study was to explore the role and regulatory mechanism of miR-326 in cell proliferation, invasion, migration and apoptosis in lung adenocarcinoma. Quantitative real-time PCR (qRT-PCR) was used to detect the expression pattern of miR-326 in human bronchial epithelial cells (HBES-2B), 4 kinds of lung adenocarcinoma cell lines (H23, H1975, H2228, H2085) and 20 lung adenocarcinoma tissues. Then, H23 cells were infected with miR-326 mimics, miR-326 inhibitors and si-ZEB1 to build up-regulated miR-326 cell lines, down-regulated ZEB1(zinc-finger-enhancer binding protein 1)cell lines, simultaneous down-regulated ZEB1 and miR-326 cell lines. Moreover, CCK-8 assay, transwell invasion assay, wound healing assay and flow cytometry assay were employed to examine the effects of miR-326 and ZEB1 on the proliferation, invasion, migration and apoptosis abilities of H23 cells. Western blot was performed to explore the effects of miR-326 and ZEB1 on the expression of invasion and migration related proteins N-cadherin, E-cadherin, MMP7, MMP13, SLUG and apoptotic proteins PARP, BAX. On the mechanism, a dual-luciferase reporter gene was used to measure the target relationship between miR-326 and ZEB1. MiR-326 expression was significantly downregulated in lung adenocarcinoma tissues and cells. Overexpression of miR-326 significantly inhibited the malignant behaviors of H23 cells. Mechanically, luciferase reporter assay showed that ZEB1 was a direct target of miR-326. MiR-326 mimic downregulated the expression of ZEB1. Furthermore, knocking down ZEB1 strongly inhibited the proliferation, invasion and migration of H23 cells but promoted apoptosis. MiR-326 could target ZEB1 to inhibit the proliferation, invasion and migration of lung adenocarcinoma cells and promote apoptosis, which is a potential therapeutic target for lung adenocarcinoma.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

scholarly journals Circ_0003266 sponges miR-503-5p to suppress colorectal cancer progression via regulating PDCD4 expression

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Caihong Wen ◽  
Xiaoqing Feng ◽  
Honggang Yuan ◽  
Yong Gong ◽  
Guangsheng Wang
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Progression ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Circular Rnas ◽  
Pdcd4 Expression ◽  
Novel Target

Abstract Background Circular RNAs (circRNAs) feature prominently in tumor progression. However, the biological function and molecular mechanism of circ_0003266 in colorectal cancer (CRC) require further investigation. Methods Circ_0003266 expression in 46 pairs CRC tissues / adjacent tissues, and CRC cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR); after circ_0003266 was overexpressed or knocked down in CRC cells, cell proliferation, apoptosis, migration, and invasion were evaluated by the cell counting kit-8 (CCK-8), flow cytometry, and Transwell assays, respectively; the interaction among circ_0003266, miR-503-5p, and programmed cell death 4 (PDCD4) was confirmed using bioinformatics analysis and dual-luciferase reporter assay; PDCD4 protein expression in CRC cells was quantified using Western blot. Results Circ_0003266 was significantly lowly expressed in CRC tissues and cell lines. Circ_0003266 overexpression markedly repressed CRC cell proliferation, migration, and invasion, and accelerated the cell apoptosis, but its overexpression promoted the malignant phenotypes of CRC cells. PDCD4 was a direct target of miR-503-5p and circ_0003266 promoted PDCD4 expression by competitively sponging miR-503-5p. Conclusion Circ_0003266 suppresses the CRC progression via sponging miR-503-5p and regulating PDCD4 expressions, which suggests that circ_0003266 may serve as a novel target for the treatment of CRC.

scholarly journals Blockade of LINC01605-enriched exosome generation in M2 macrophages impairs M2 macrophage-induced proliferation, migration, and invasion of human dermal fibroblasts

2021 ◽  
Vol 35 ◽  
pp. 205873842110167
Author(s):  
Zhensen Zhu ◽  
Bo Chen ◽  
Liang Peng ◽  
Songying Gao ◽  
Jingdong Guo ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
M2 Macrophage ◽  
M2 Macrophages ◽  
Luciferase Reporter Gene ◽  
Gene Assay

Activated M2 macrophages are involved in hypertrophic scar (HS) formation via manipulating the differentiation of fibroblasts to myofibroblasts having the proliferative capacity and biological function. However, the function of exosomes derived from M2 macrophages in HS formation is unclear. Thus, this study aims to investigate the role of exosomes derived by M2 in the formation of HS. To understand the effect of exosomes derived from M2 macrophages on formation of HS, M2 macrophages were co-cultured with human dermal fibroblast (HDF) cells. Cell Counting Kit-8 assay was performed to evaluate HDF proliferation. To evaluate the migration and invasion of HDFs, wound-healing and transwell invasion assays were performed, respectively. To investigate the interaction between LINC01605 and miR-493-3p, a dual-luciferase reporter gene assay was adopted; consequently, an interaction between miR-493-3p and AKT1 was detected. Our results demonstrated that exosomes derived from M2 macrophages promoted the proliferation, migration, and invasion of HDFs. Additionally, we found that long noncoding RNA LINC01605, enriched in exosomes derived from M2 macrophages, promoted fibrosis of HDFs and that GW4869, an inhibitor of exosomes, could revert this effect. Mechanistically, LINC01605 promoted fibrosis of HDFs by directly inhibiting the secretion of miR-493-3p, and miR-493-3p down-regulated the expression of AKT1. Exosomes derived from M2 macrophages promote the proliferation and migration of HDFs by transmitting LINC01605, which may activate the AKT signaling pathway by sponging miR-493-3p. Our results provide a novel approach and basis for further investigation of the function of M2 macrophages in HS formation.

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