scholarly journals Circ_0000005 Facilitates Proliferation, Apoptosis, Migration and Invasion of Acute Myeloid Leukemia Cells via Modulating miR-139-5p/Tspan3 Axis

2020 ◽  
Author(s):  
Juan Tong ◽  
Huilan Liu ◽  
Changcheng Zheng ◽  
Xiaoyu Zhu ◽  
Xiang Wan ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Qrt Pcr ◽  
Gene Assay

Abstract Background: Accumulating circular RNAs (circRNAs) are reported to be abnormally expressed in diverse cancers, hematologic malignancies included. This study aimed to investigate the biological function and underlying mechanisms of circ_0000005 in acute myeloid leukemia (AML).Materials and methods: Bone marrow samples were enrolled from AML patients with normal samples as controls. Circ_0000005, miR-139-5p and tetraspanin 3 (Tspan3) were detected by qRT-PCR and Western blot, respectively. AML cell lines (KG1 and HL60) were used as cell models. CCK-8, Transwell and flow cytometry assays were adopted to study the biological functions of circ_0000005 on AML cells in vitro. The interrelation between circ_0000005 and miR-139-5p was detected by bioinformatics, qRT-PCR, luciferase reporter gene assay, RNA pull-down assay, and RNA-binding protein immunoprecipitation (RIP) assays. Ultimately, Western blot, qRT-PCR, luciferase reporter gene assay were adopted to corroborate the interrelation between miR-139-5p and its target Tspan3. Results: Circ_0000005 was demonstrably elevated in both AML clinical samples and cell lines. Circ_0000005 overexpression promoted the viability, migration and invasion of AML cells, and repressed the apoptosis of AML cells, while silencing circ_0000005 showed opposite biological effects. Circ_0000005 interacted with miR-139-5p and repressed its expression, and Tspan3 was proved to be negatively regulated by miR-139-5p. Circ_0000005 could promote the expression of Tspan3 via repressing miR-139-5p, and the oncogenic functions of circ_0000005 were dependent on its regulatory function on miR-139-5p/Tspan3 axis.Conclusion: Circ_0000005 facilitates the malignant phenotypes of AML cells via miR-139-5p/Tspan3 axis. Circ_0000005 may serve as a potential therapeutic target in AML.

scholarly journals Hsa_circ_0060927 Is a Novel Tumor Biomarker by Sponging miR-195-5p in the Malignant Transformation of OLK to OSCC

2022 ◽  
Vol 11 ◽  
Author(s):  
Siming Xu ◽  
Yuhan Song ◽  
Yanxiong Shao ◽  
Haiwen Zhou
Keyword(s):  
Cell Proliferation ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Qrt Pcr ◽  
Cerna Network ◽  
Gene Assay ◽  
And Migration

ObjectiveTo investigate the clinical significance of differentially expressed circRNAs and candidate circRNAs in the transformation of oral leukoplakia (OLK) to oral squamous cell carcinoma (OSCC).MethodsWe performed high-throughput circRNA sequencing in six cases of normal oral mucosal (NOM) tissues, six cases of OLK tissues, and six cases of OSCC tissues. Ten circRNAs with significant differential expression were verified by qRT-PCR. Enzyme tolerance assay and Sanger sequencing were performed on the screened target circRNA hsa_circ_0060927, and a qRT-PCR assay of hsa_circ_0060927 was performed in three tissues (24 cases in each group); this was followed by an ROC analysis. The ceRNA network was predicted using TargetScan and miRanda. MiR-195-5p and TRIM14 were selected as the downstream research objects of hsa_circ_0060927. The sponge mechanism of hsa_circ_0060927 was detected by AGO2 RIP. The interaction between hsa_circ_0060927 and miR-195-5p was verified by RNA pull-down assay and dual luciferase reporter gene assay. The expressions of hsa_circ_0060927, miR-195-5p, and TRIM14 were verified by normal oral epithelial primary cells and cell lines of LEUK1, SCC9, and SCC25. The hsa_circ_0060927 overexpressed plasmid and miR-195-5p mimics were constructed to transfection LEUK1 to detect the changes in cell proliferation, apoptosis, and migration.ResultsThe results of qRT-PCR validation were consistent with the sequencing results. Hsa_circ_0060927 is a true circRNA with trans-splicing sites. The expression of hsa_circ_0060927 increased in NOM, OLK, and OSCC. Overexpression of hsa_circ_0060927 enhanced the ability of cell proliferation and migration, and decreased cell apoptosis capacity. The prediction of ceRNA network suggested that hsa_circ_0060927 could regulate the target gene TRIM14 through sponging miR-195-5p. AGO2 RIP indicated that hsa_circ_0060927 had a sponge mechanism. RNA pull-down and dual luciferase reporter gene assay suggested that hsa_circ_0060927 interacted with miR-195-5p. Hsa_circ_0060927 was positively correlated with the expression of TRIM14, and could relieve the inhibition of miR-195-5p on TRIM14 to regulate cell proliferation, apoptosis, and migration of LEUK1 cells.ConclusionHsa_circ_0060927 acted as a potential key ceRNA to sponge downstream miR-195-5p and promote OLK carcinogenesis by upregulating TRIM14. Hsa_circ_0060927 was expected to be a molecular marker for the prevention and treatment of OLK carcinogenesis through the hsa_circ_0060927/miR-195-5p/TRIM14 axis.

scholarly journals The Effect of LncRNA H19/miR-194-5p Axis on the Epithelial-Mesenchymal Transition of Colorectal Adenocarcinoma

2018 ◽  
Vol 50 (1) ◽  
pp. 196-213 ◽  
Author(s):  
Chang-feng Li ◽  
Yong-chao  Li ◽  
Yun Wang ◽  
Li-bo Sun
Keyword(s):  
Cell Lines ◽  
Reporter Gene ◽  
Colorectal Adenocarcinoma ◽  
Epithelial Mesenchymal Transition ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Mesenchymal Transition ◽  
Gene Assay

Background/Aims: Since the combined actions of lncRNAs and miRNAs have been considered to be involved in the occurrence and development of various neoplasms, the main purpose of this study was to discover whether and how lncRNA H19 and miR-194 influenced the epithelial-mesenchymal transition (EMT) process of colorectal adenocarcinoma (CRA). Methods: Totally 214 pairs of CRA and adjacent normal tissues were collected, and 5 human CRA cell lines (i.e. HCT116, HT-29, RKO SW280 and Lovo) were purchased. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was adopted to quantify the H19 and miR-194-5p expressions in cells and tissues. The expressions of FoxM1, E-cadherin, vimentin, N-cadherin were determined using western blot. On the side, si-H19, si-NC, miR-194-5p mimic, miR-194-5p inhibitor and negative control (NC) were transfected into CRA cell lines. Meanwhile, the invasive, migratory and proliferative conditions of the cells were assessed through transwell, wound healing and colony-forming experiments, with final verification of the relationship between H19 and miR-194-5p employing dual-luciferase reporter gene assay. Results: Highly-expressed H19, lowly-expressed miR-194-5p, low-grade differentiation and lymph node metastasis appeared as the independent predictors of unfavorable prognosis in CRA patients’ (all P< 0.05). It indicated that FoxM1 expression displayed positive correlations with H19 expression, yet negative associations with miR-194-5p expression within CRA tissues (P< 0.05). In addition, transfection of H19-siRNA and miR-145-5p mimic triggered a conspicuous increase in E-cadherin expression, as well as an evidently down-regulation in vimentin and N-cadherin expressions within HT29 and RKO cells (P< 0.05). On the other hand, the invasive and migratory capacities of CRA cells were significantly hindered (P< 0.05). Moreover, the luciferase reporter gene assay confirmed that H19 modified miR-194-5p expression through directly targeting at it (P< 0.05). Ultimately, FoxM1 could reverse the role of miR-194-5p in inhibiting invasion, migration and EMT of CRA cells (P< 0.05). Conclusion: LncRNA H19/miR-194/FoxM1 axis could serve as a profound target for the diagnosis and treatment of CRA.

scholarly journals miR-138-5p inhibits the metastasis of prostate cancer by targeting FOXC1

2020 ◽  
Author(s):  
Dapeng Zhang ◽  
Xiaodong Liu ◽  
Qingwei Zhang ◽  
Xin Chen
Keyword(s):  
Cell Lines ◽  
Poor Prognosis ◽  
Bioinformatics Analysis ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Over Expression ◽  
Qrt Pcr ◽  
Gene Assay

Abstract Background: This study aimed to uncover the regulatory effect of miR-138-5p on the metastasis of PCa cells, and further explore the potential regulatory mechanisms via regulating FOXC1. Methods: 60 pairs tumor specimens from PCa patients were collected to determine the expression level of miR-138-5p by qRT-PCR. Subsequently, over-expression of miR-138-5p were established to explore the proliferation and metastasis of miR-138-5p in PCa cell lines was analyzed by CCK-8, Tranwell assay and Wounding healing assay, respectively. Bioinformatics analysis and luciferase reporter gene assay were performed to search for the target genes of miR-138-5p, and FOXC1 was selected. Finally, the biological role of miR-138-5p and FOXC1 in the progression of PCa was clarified by a series of rescue experiments. Results: The results of qRT-PCR revealed that miR-138-5p was lowly expressed in PCa tissues and cell lines. Besdies, these PCa patients with low-miR-138-5p had a higher Gleason score, lymph node metastasis, bone metastasis and poor prognosis of PCa, compared with the patients with high-miR-138-5p. Over-expression of miR-138-5p inhibited the viability, migratory and invasive capacities of PC-3 and DU-145 cells. Bioinformatics analysis and luciferase reporter gene assay suggested that FOXC1 was predicted to be the target of miR-138-5p. Moreover, FOXC1 level was negatively correlated to that of miR-138-5p in PCa tissues. Importantly, FOXC1 could reverse miR-138-5p mimic induced-inhibition of PCa malignant progression. Conclusions: Downregulated miR-138-5p was closely associated with Gleason score, distant metastasis and poor prognosis of PCa patients. In addition, miR-138-5p alleviated the malignant progression of PCa by targeting and downregulating FOXC1.

scholarly journals miR-138-5p inhibits the metastasis of prostate cancer by targeting FOXC1

2020 ◽  
Author(s):  
Dapeng Zhang ◽  
Xiaodong Liu ◽  
Qingwei Zhang ◽  
Xin Chen
Keyword(s):  
Cell Lines ◽  
Bioinformatics Analysis ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Over Expression ◽  
Qrt Pcr ◽  
Gene Assay ◽  
Proliferation And Metastasis

Abstract Background: This study aimed to uncover the effect of miR-138-5p on the proliferation and metastasis of PCa cell lines, and further explore the potential regulatory mechanisms via regulating FOXC1.Methods: 60 pairs tumor tissues and corresponding paracancerous tissues from PCa patients were collected to determine the expression level of miR-138-5p by qRT-PCR. Subsequently, over-expression of miR-138-5p were established to explore the proliferation and metastasis of miR-138-5p in PCa cell lines was analyzed by CCK-8, Tranwell assay and Wounding healing assay, respectively. Bioinformatics analysis and luciferase reporter gene assay were performed to search for the target genes of miR-138-5p, and FOXC1 was selected. Finally, the biological role of miR-138-5p and FOXC1 in the progression of PCa was clarified by a series of rescue experiments. Results: The results of qRT-PCR revealed that miR-138-5p was lowly expressed in PCa tissues and cell lines. Besdies, these PCa patients with low-miR-138-5p had a higher Gleason score, lymph node metastasis and poor prognosis of PCa, compared with the patients with high-miR-138-5p. Over-expression of miR-138-5p inhibited the viability, migratory and invasive capacities of PC-3 and DU-145 cells. Bioinformatics analysis and luciferase reporter gene assay suggested that FOXC1 was predicted to be the target of miR-138-5p. Moreover, FOXC1 level was negatively correlated to that of miR-138-5p in PCa tissues. Importantly, FOXC1 could reverse miR-138-5p mimic induced-inhibition of PCa malignant progression.Conclusions: Downregulated miR-138-5p was closely associated with Gleason score, distant metastasis and poor prognosis of PCa patients. In addition, miR-138-5p alleviated the malignant progression of PCa by targeting and downregulating FOXC1.

scholarly journals MiR-486 protects against acute myocardial infarction via regulation of PTEN

2021 ◽  
Vol 20 (9) ◽  
pp. 1845-1853
Author(s):  
Qinfeng Han ◽  
Zhong Xu ◽  
Xiaolei Zhang ◽  
Kun Yang ◽  
Zhifei Sun ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Akt Signaling ◽  
Reporter Gene Assay ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Akt Signaling Pathway ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Qrt Pcr ◽  
Gene Assay

Purpose: To investigate the effect of miR-486 on rats with acute myocardial infarction (AMI), and its mechanism of action.Methods: A rat model of AMI was established. They were randomly divided into 4 groups, namely, sham, model, agomiR-486 and antagomiR-486 groups, respectively. Rats in these different groups were treated with agomiR-21 (5 μL, 40 nmol/mL), antagomiR-21 (5 μL, 40 nmol/mL) or agomiR-NC (5 μL, 40 nmol/mL), respectively. Then, key miRNAs were sorted out using gene-chip assay and verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. Luciferase reporter gene assay was conducted to determine the interaction between miR-486 and gene of PTEN. After intraperitoneal injection of agomiR-486 and antagomiR-486, hemodynamics was measured to determine the effect of miR-486 on myocardial function of the rats. The effect of miR-486 expression level on the expression of myocardial enzymes in serum, the morphology of myocardial tissues, and the apoptosis of myocardial tissues in rats, were investigated. Additionally, the effect of miR-486 expression level on PTEN/AKT signaling pathway in the rats was determined by Western blotting.Results: The results of gene-chip and qRT-PCR assays revealed that there were 8 differentially expressed genes in rat myocardial tissues in the model group when compared with the sham group. MiR-486 improved the cardiac function of rats and the morphology of myocardial tissues, but reduced AMI-induced apoptosis of myocardial cells and the expression of myocardial enzymes (markers of myocardial injury) in a dose-dependent manner (p < 0.05). The results of luciferase reporter gene assay showed that PTEN was a direct target of miR-486. In rat models of AMI, a raised expression of miR-486 remarkably suppressed the protein expression level of PTEN and up-regulated that of p-AKT/AKT (p < 0.05).Conclusion: MiR-486 protects against AMI in rats probably by targeting PTEN and activating the AKT signaling pathway. The results of the current study may provide new insights for the treatment of AMI.

scholarly journals Identification of Alkaloids from Corydalis yanhusuo W. T. Wang as Dopamine D1 Receptor Antagonists by Using CRE-Luciferase Reporter Gene Assay

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2585 ◽  
Author(s):  
Lehao Wu ◽  
Weiyue Zhang ◽  
Xin Qiu ◽  
Chaoran Wang ◽  
Yanfang Liu ◽  
...  
Keyword(s):  
Dopamine Receptors ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
D1 Receptor ◽  
Assay System ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Antagonistic Effects ◽  
Gene Assay

Corydalis yanhusuo W. T. Wang (C. yanhusuo) has been traditionally used for drug addiction and pain relief in China. In our previous study, we showed that the extract of C. yanhusuo blocks dopamine receptors, demonstrating that its pharmacological activities are mostly due to the antagonistic effects of some of its components at dopamine receptors. As part of our ongoing project on C. yanhusuo, the aim of the present study is to establish a high-throughput and low-cost screening assay system and test the abilities of the isolated alkaloids from C. yanhusuo to inhibit dopamine-induced dopamine D1 receptor activity. By using our established cyclic adenosine monophosphate (cAMP)-response element (CRE)-luciferase reporter gene assay system, we identified eight alkaloids from C. yanhusuo with D1 receptor antagonistic activities. We next validated the activities of these compounds using fluorometric imaging plate reader (FLIPR) assay by measuring the intracellular Ca2+ change. Six out of eight compounds, including tetrahydropalmatine, corydaline, 13-methyldehydrocorydalmine, dehydrocorybubine, dehydrocorydaline, and columbamine, can be confirmed for their inhibitory activities. The dopamine-receptor-antagonistic effects of four compounds, including 13-methyldehydrocorydalmine, dehydrocorydaline, columbamine, and corydaline, are reported for the first time. The present study provides an important pharmacological basis to support the traditional use of C. yanhusuo in China.

Circular RNA CCDC66 Regulates Osteoarthritis Progression by Targeting miR-3622b-5p

Gerontology ◽  
2022 ◽  
pp. 1-11
Author(s):  
Chengyuan Zhang ◽  
Ye Lu ◽  
Feng Yuan ◽  
Shilin Jiang
Keyword(s):  
Reporter Gene ◽  
Bioinformatics Analysis ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Chondrocyte Apoptosis ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Tnf Α ◽  
Gene Assay ◽  
Sirt3 Expression

<b><i>Objective:</i></b> CircCCDC66 is involved in cancer progression, but its role in osteoarthritis (OA) remains unknown. This study was carried out to explore the biological role of circCCDC66 in OA and its underlying mechanism. <b><i>Methods:</i></b> The expression levels of miR-3622b-5p and circCCDC66 in OA cartilage tissues were detected by qRT-PCR. Cell Counting Kit-8 (CCK8) and flow cytometry were used to detect the chondrocyte viability and apoptosis. The expression of chondrocyte inflammatory factors (IL-6 and TNF-α) was measured by ELISA. The target genes of circCCDC66 and miR-3622b-5p were analyzed by bioinformatics analysis and luciferase reporter gene assay. The relationship between circCCDC66 and miR-3622b-5p was analyzed by bioinformatics analysis and luciferase reporter gene assay. <b><i>Results:</i></b> It was found that circCCDC66 expression in OA cartilage tissues was upregulated. CircCCDC66 overexpression inhibited proliferation and promoted apoptosis of chondrocytes and increased IL-6 and TNF-α levels in chondrocytes. miR-3622b-5p was predicted to be a downstream target gene of circCCDC66, and circCCDC66 overexpression inhibited miR-3622b-5p expression in chondrocytes. Moreover, miR-3622b-5p expression was downregulated in OA cartilage tissues. miR-3622b-5p overexpression increased chondrocyte proliferation, inhibited chondrocyte apoptosis, and enhanced the expression of IL-6 and TNF-α in chondrocytes. In addition, circCCDC66 overexpression enhanced SIRT3 expression in chondrocytes, while miR-3622b-5p overexpression inhibited SIRT3 expression in chondrocytes. <b><i>Conclusion:</i></b> CircCCDC66 promoted OA chondrocyte apoptosis by regulating the miR-3622b-5p/SIRT3 axis. CircCCDC66 may be a new therapeutic target of OA.

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