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

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.

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

10.21203/rs.3.rs-57994/v1 ◽

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.

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Long Noncoding RNA KCNQ1OT1 Induces Resistance of HCC Cells To Cisplatin Through Regulating The miR-26a/CCND2 Molecular Axis

10.21203/rs.3.rs-858904/v1 ◽

2021 ◽

Author(s):

Cai LI ◽

Qi-Fa YE

Keyword(s):

Cell Proliferation ◽

Reporter Gene Assay ◽

Molecular Axis ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Luciferase Reporter Gene Assay ◽

Qrt Pcr ◽

Gene Assay ◽

Proliferation And Invasion ◽

Hcc Cells

Abstract Objective: To explore the molecular mechanism by which LncRNA KCNQ1OT1 regulated the miR-26a/CCND2 molecular axis to participate in the resistance of Hepatocellular carcinoma(HCC) cells to cisplatin.Methods: Cancer tissue and corresponding para-carcinoma tissue specimens were collected from 25 HCC patients with complete data admitted from January 2018 to December 2018 at The Transplantation Center of the Third Xiangya Hospital. Then, the expression levels of KCNQ1OT1, miR-26a and CCND2 in HCCtissues and cell lines were detected through qRT-PCR. Meanwhile, the sensitivity of HCC cells to cisplatin was examined through Transwell and Annexin V-FITC/PI double staining flow cytometry. Further, the targeted relationships among KCNQ1OT1, miR-26a and CCND were verified through dual-luciferase reporter gene assay, and the regulatory relationships were detected through Western blotting and qRT-PCR.Results: KCNQ1OT1 was highly expressed in HCC tissues and cisplatin-resistant cell lines; meanwhile, over-expression of KCNQ1OT1 promoted the resistance of Huh7/CDDP cells to cisplatin. Dual-luciferase reporter gene assay verified that, KCNQ1OT1 targeted miR-26a and down-regulated its expression level. miR-26a suppressed Huh7/CDDP cell proliferation and invasion, while promoting their apoptosis, thus down-regulating the promoting effect of KCNQ1OT1 on the cisplatin resistance of HCC cells. miR-26a negatively regulated CCND2 expression, while KCNQ1OT1 down-regulated the suppression of miR-26a on CCND2 to promote Huh7/CDDP cell proliferation and invasion and to suppress apoptosis, thereby up-regulating the resistance of HCCcells to cisplatin. Conclusions: LncRNA KCNQ1OT1 regulates the miR-26a/CCND2 molecular axis to induce the resistance of HCC cells to cisplatin.

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

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v20i9.10 ◽

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.

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[34] Luciferase reporter gene assay in mammalian cells

Methods in Enzymology - Recombinant DNA Part G ◽

10.1016/0076-6879(92)16036-j ◽

1992 ◽

pp. 386-397 ◽

Cited By ~ 26

Author(s):

Allan R. Brasier ◽

David Ron

Keyword(s):

Reporter Gene ◽

Mammalian Cells ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Luciferase Reporter Gene Assay ◽

Gene Assay

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

Molecules ◽

10.3390/molecules23102585 ◽

2018 ◽

Vol 23 (10) ◽

pp. 2585 ◽

Cited By ~ 7

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.

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Use of a Dual Firefly and Renilla Luciferase Reporter Gene Assay to Simultaneously Determine Drug Selectivity at Human Corticotrophin Releasing Hormone 1 and 2 Receptors

Analytical Biochemistry ◽

10.1006/abio.2000.4570 ◽

2000 ◽

Vol 281 (2) ◽

pp. 187-192 ◽

Cited By ~ 20

Author(s):

S.J.W. Parsons ◽

S.A. Rhodes ◽

H.E. Connor ◽

S. Rees ◽

J. Brown ◽

...

Keyword(s):

Reporter Gene ◽

Reporter Gene Assay ◽

Renilla Luciferase ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Luciferase Reporter Gene Assay ◽

Releasing Hormone ◽

Gene Assay ◽

Drug Selectivity

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Screening of 19 traditional Mexican medicinal plants for effects on the metabolic syndrome related targets PPARα, β/δ, and γ in an in vitro luciferase reporter gene assay

Planta Medica ◽

10.1055/s-0035-1565656 ◽

2015 ◽

Vol 81 (16) ◽

Author(s):

K Kuchta ◽

N Matsuura ◽

HW Rauwald ◽

R Quintanilla-Licea ◽

M Iinuma

Keyword(s):

Metabolic Syndrome ◽

Medicinal Plants ◽

Reporter Gene ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Luciferase Reporter Gene Assay ◽

The Metabolic Syndrome ◽

Gene Assay

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

Gerontology ◽

10.1159/000520325 ◽

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

Objective: 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. Methods: 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. Results: 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. Conclusion: 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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