scholarly journals Construction of Novel lncRNA–miRNA–mRNA Network Associated With Recurrence and Identification of Immune-Related Potential Regulatory Axis in Hepatocellular Carcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Tian Zhan ◽  
Xiang Gao ◽  
Guoguang Wang ◽  
Fan Li ◽  
Jian Shen ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Correlation Analysis ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Immune Infiltration ◽  
Gene Assay

Hepatocellular carcinoma (HCC) is one of the most common malignant diseases globally. Despite continuous improvement of treatment methods, high postoperative recurrence rate remains an urgent problem. In order to determine the mechanism underlying recurrence of liver cancer and identify prognostic genes, data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were integrated and analyzed. Differentially expressed genes (DEGs) between HCC tissue and normal liver tissue were identified, and a protein–protein interaction network was constructed to find hub genes. Clinical correlation analysis and disease-free survival (DFS) analysis were performed using the R language and GEPIA to identify relapse-related genes. Correlation analysis was used to identify a potential regulatory axis. Dual-luciferase reporter gene assay was used to confirm the reliability of the long non-coding RNA (lncRNA)–microRNA (miRNA)–mRNA regulatory axis. Immune infiltration analysis was performed using the TIMER database. Correlations between immune gene markers and ASF1B were verified using quantitative real-time polymerase chain reaction (RT-qPCR). In this work, we found that nine lncRNAs and five mRNAs were significantly overexpressed in HCC tissues from patients with recurrence. SNHG3, LINC00205, ASF1B, AURKB, CCNB1, CDKN3, and DTL were also closely related to HCC grade and stage. Survival analysis showed that these seven DEGs were significantly correlated with poor DFS. Correlation analysis identified SNHG3–miR-214-3p–ASF1B as a potential regulatory axis. Dual-luciferase reporter gene assay showed that SNHG3 and ASF1B directly bound to miR-214-3p. ASF1B was negatively regulated by miRNA-214-3p, and overexpression of SNHG3 could inhibit the expression of miRNA-214-3p. In addition, ASF1B was positively correlated with immune infiltration. A reduction in ASF1B could markedly inhibit the expression of CD86, CD8, STAT1, STAT4, CD68, and PD1 in HCC cells. Flow cytometry showed that SNHG3 promoted the PD-1 expression by regulating ASF1B. Meanwhile, elevated ASF1B predicted poor prognosis of HCC patients in subgroups with decreased B cells, CD8+ T cells, or neutrophils, and those with enriched CD4+ T cells. In conclusion, we found that a novel lncRNA SNHG3/miR-214-3p/ASF1B axis could promote the recurrence of HCC by regulating immune infiltration.

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.

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.

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.

scholarly journals Comparative oxidative stress elicited by nanosilver in stable HSPA1A promoter-driven luciferase reporter HepG2 and A549 cells

2016 ◽  
Vol 5 (5) ◽  
pp. 1298-1305 ◽  
Author(s):  
Lili Xin ◽  
Jianshu Wang ◽  
Guoqiang Fan ◽  
Bizhong Che ◽  
Kaiming Cheng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reporter Gene ◽  
A549 Cells ◽  
Reporter Gene Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay

HSPA1A promoter-driven luciferase reporter gene assay provides a novel tool for predictive screening of the oxidative stress elicited by nanosilver.

Quenching the firefly bioluminescence by various ions

2016 ◽  
Vol 15 (2) ◽  
pp. 244-249 ◽  
Author(s):  
Huateng Zhang ◽  
Haixiu Bai ◽  
Tianyu Jiang ◽  
Zhao Ma ◽  
Yanna Cheng ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Firefly Luciferase ◽  
Reporter Gene Assay ◽  
Negligible Effect ◽  
Renilla Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Luciferase Reporter Gene Assay ◽  
Gene Assay ◽  
Firefly Bioluminescence

Some specific ions could selectively inhibit firefly luciferase while having a negligible effect on renilla luciferase, which may be used in the improved dual luciferase reporter gene assay.

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