Analyzing the whole-transcriptome profiles of ncRNAs and predicting the competing endogenous RNA networks in cervical cancer cell lines with cisplatin resistance

Abstract Background Cervical cancer (CC) is one of the most common malignant tumors in women. In order to identify the functional roles and the interaction between mRNA and non-coding RNA (ncRNA, including lncRNA, circRNA and miRNA) in CC cisplatin (DDP) resistance, the transcription profile analysis was performed and a RNA regulatory model of CC DDP resistance was proposed. Methods In this study, whole-transcriptome sequencing analysis was conducted to study the ncRNA and mRNA profiles of parental SiHa cells and DDP resistant SiHa/DDP cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed for pathway analysis based on the selected genes with significant differences in expression. Subsequently, ceRNA network analyses were conducted using the drug resistance-related genes and signal-transduction pathways by Cytoscape software. Furthermore, a ceRNA regulatory pathway, namely lncRNA-AC010198.2/hsa-miR-34b-3p/STC2, was selected by RT-qPCR validation and literature searching. Further validation was done by both dual-luciferase reporter gene assays and RNA pull-down assays. Besides that, the changes in gene expression and biological function were further studied by performing si-AC010198.2 transfection and DDP resistance analyses in the SiHa and SiHa/DDP cells, respectively. Results Using bioinformatics and dual-luciferase reporter gene analyses, we found that AC010198.2/miR-34b-3p/STC2 may be a key pathway for DDP resistance in CC cells. Significant differences in both downstream gene expression and the biological function assays including colony formation, migration efficiency and cell apoptosis were identified in AC010198.2 knockdown cells. Conclusions Our study will not only provide new markers and potential mechanism models for CC DDP resistance, but also discover novel targets for attenuating it.

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Analyzing the Whole Transcriptome Profiles of ncRNAs and Predicting the Competing Endogenous RNA Networks in Cervical Cancer Cell Lines With Cisplatin Resistance

10.21203/rs.3.rs-651847/v1 ◽

2021 ◽

Author(s):

Huimin Lv ◽

Shanshan Jin ◽

Binbin Zou ◽

Yuxiang Liang ◽

Jun Xie ◽

...

Keyword(s):

Gene Expression ◽

Cervical Cancer ◽

Reporter Gene ◽

High Throughput Sequencing ◽

Biological Function ◽

Luciferase Reporter ◽

Sequencing Analysis ◽

Luciferase Reporter Gene ◽

Siha Cells ◽

Whole Transcriptome

Abstract Objective:: Cervical cancer (CC) is one of the most common malignant tumors in women. In order to identify the function between mRNA and non-coding RNA (ncRNA, including lncRNA,circRNA, miRNA) in CC DDP resistance, we analyzed its expression related to transcription profile and the RNA regulatory network between ncRNA and mRNA.Methods: In this study, whole transcriptome high-throughput sequencing (RNA-sequencing) analysis was used to study the ncRNA profiles of parental SiHA cells and DDP-resistant SiHA/DDP cells. Conducted gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and analyzed mRNAs (DE) with significant differences in expression. Then, based on the authoritative cytoscape software v.3.7.2, drug resistance-related genes and signal transduction pathways, a ceRNA network combining lncRNA and mRNA was predicted and constructed. In addition, a constructed ceRNA regulatory pathway was randomly selected, namely lnc-AC010198.2 / hsa-miR-34b-3p / STC2, and verified by real-time qPCR, dual luciferase reporter gene system, and RNA pull-down assay. After transfection with si-lnc-AC010198.2 and DDP resistance, the changes in gene expression and biological function in SiHA and SiHA/DDP cells were further analyzed.Results: Through bioinformatics and dual-luciferase reporter gene analysis, we found that lnc-AC010198.2 / miR-34b-3p / STC2 may be the mechanism by which SiHA / DDP cells are resistant to DDP compared to parent SiHA cells. After si-lnc-AC010198.2 is resistant to DDP after transfection, there are significant differences between SiHA/DDP and SiHA cells' downstream gene expression, the biological function of colony formation, invasion efficiency, and cell apoptosis.Conclusions: Our study may provide new markers and potential mechanisms for CC DDP resistance, and discover some novel targets for reversing it.

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Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya

Toxicology in Vitro ◽

10.1016/j.tiv.2009.02.005 ◽

2009 ◽

Vol 23 (4) ◽

pp. 617-621 ◽

Cited By ~ 8

Author(s):

Martijn Vermeulen ◽

Anne-Marie M.J.F. Boerboom ◽

Barry M.G. Blankvoort ◽

Jac M.M.J.G. Aarts ◽

Ivonne M.C.M. Rietjens ◽

...

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Reporter Gene Expression ◽

Luciferase Reporter ◽

Glutathione S Transferase ◽

Luciferase Reporter Gene ◽

Responsive Element

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Non-invasive somatotransgenic bioimaging in living animals

F1000Research ◽

10.12688/f1000research.25274.1 ◽

2020 ◽

Vol 9 ◽

pp. 1216 ◽

Cited By ~ 1

Author(s):

Juliette M. Delhove ◽

Rajvinder Karda ◽

Lorna M. FitzPatrick ◽

Suzanne M.K. Buckley ◽

Simon N. Waddington ◽

...

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Viral Vectors ◽

Es Cells ◽

Embryonic Stem ◽

Whole Body ◽

Luciferase Reporter ◽

Gene Promoters ◽

Luciferase Reporter Gene ◽

Endogenous Gene

Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.

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Interfering with Long Non-Coding RNA FBXL19-AS1 Inhibits the Proliferation, Invasion and Migration of Hepatoma Carcinoma Cells via Targeting miR-541-5p

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2777 ◽

2021 ◽

Vol 11 (11) ◽

pp. 2120-2127

Author(s):

Weijun Lu ◽

Qun Wang ◽

Changbo Fu

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Cell Migration ◽

Cell Lines ◽

Reporter Gene ◽

Malignant Tumors ◽

Human Life ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Luciferase Reporter Gene

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and the morbidity and mortality of HCC rate in the first few malignant tumors, seriously threatening the safety of human life. LncRNA is a hot topic in tumor research in recent years. The abnormal expression of LncRNA FBXL19-AS1 and its potential target as a tumor diagnostic marker have been confirmed in colon cancer, breast cancer and lung cancer, etc. However, the study on LncRNA FBXL19-AS1 in HCC has not been reported. Rt-qPCR was used to detect the expression of FBXL19-AS1 and miR-541-5p in HCC cell lines, and luciferase reporter gene was used to detect whether there were binding sites between LncRNA FBXL19-AS1 and miR-541-5p. Interfered with FBXL19-AS1 and overexpressed miR-541-5p were detected by cell transfection. Then CCK-8 and colony formation assay were used to detect cell viability and cell proliferation. Wound healing detected the rate of cell migration and Transwell detected the rate of cell invasion. Western blot was used to detect the expression of proteins related to cell migration and invasion. The expression of FBXL19-AS1 in HCC cell lines was significantly higher than that in normal liver cells (LO2). Moreover, FBXL19-AS1 can promote HCC cell proliferation, migration and invasion. Luciferase reporter gene confirmed the binding site between LncRNA FBXL19-AS1 and miR-541-5p. After interfering with the expression of FBXL19-AS1, miR-541-5p was significantly increased. Subsequently, overexpression of miR-541-5p can inhibit the expression of lncRNA FBXL19-AS11 and promote proliferation, migration and invasion of hepatocellular carcinoma. So we can conclude that lncRNA FBXL19-AS1 promoted the proliferation, migration and invasion of HCC cells through targeting miR-541-5p.

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In vivo regulation of β-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.4.c883 ◽

1999 ◽

Vol 276 (4) ◽

pp. C883-C891 ◽

Cited By ~ 46

Author(s):

Carola E. Wright ◽

F. Haddad ◽

A. X. Qin ◽

P. W. Bodell ◽

K. M. Baldwin

Keyword(s):

Gene Expression ◽

Thyroid Hormone ◽

Reporter Gene ◽

Normal Control ◽

Transcriptional Activity ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Promoter Construct ◽

Hormone Responsiveness

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the −408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the −3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

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MKL-1 regulates the stem cell marker. CD44 in breast cancer cells

E3S Web of Conferences ◽

10.1051/e3sconf/20197801002 ◽

2019 ◽

Vol 78 ◽

pp. 01002

Author(s):

Zhou-Tong Dai ◽

Ao Yao ◽

Yuan Xiang ◽

Jia Peng Li ◽

Wei Guo ◽

...

Keyword(s):

Gene Expression ◽

Malignant Tumors ◽

Regulation Of Gene Expression ◽

Gene Promoter ◽

Expression Regulation ◽

Stem Cell Marker ◽

Luciferase Reporter ◽

Regulation Mechanism ◽

Luciferase Reporter Gene ◽

Factors Affecting

CD44, cluster of differentiation 44 is a typical marker of stem cells. At present, it has been found that CD44 is prevalent in various human malignant tumors, but its expression regulation mechanism is still not clear. The initiation of gene expression, the modification of RNA levels, and the regulation of protein levels are the main factors affecting the expression level of genes, and the most critical one is the regulation of gene expression by signaling pathways. Up to now, there has been no report on the role of MKL-1 in the cloning of the cd44 promoter. Therefore, this study intends to clone the cd44 gene promoter, construct its luciferase reporter gene vector, transfect the MKL-1 overexpression vector, and analyze how it affects transcriptional activity, in order to further study the expression regulation of cd44. The mechanism provides a powerful tool in the future.

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