Cloning of amplified miRNA target sites into 3’-UTR of luciferase reporter vector.

2006 ◽  
Author(s):  
Irina Naguibneva
Keyword(s):  
Mirna Target ◽  
Luciferase Reporter ◽  
Reporter Vector ◽  
Target Sites ◽  
Luciferase Reporter Vector

Methyl Methacrylate Activates the Gsta1 Promoter

2008 ◽  
Vol 87 (12) ◽  
pp. 1117-1121 ◽  
Author(s):  
N. Hattori ◽  
T. Suzuki ◽  
S. Jinno ◽  
H. Okeya ◽  
A. Ishikawa ◽  
...  
Keyword(s):  
Methyl Methacrylate ◽  
Hepg2 Cells ◽  
Luciferase Reporter ◽  
Promoter Activation ◽  
Glutathione S Transferase ◽  
Cis Acting ◽  
Reporter Vector ◽  
Anti Oxidant ◽  
Luciferase Reporter Vector ◽  
Responsive Element

Residual monomers in resin-based biomaterials cause cytotoxicity. We previously showed that methyl methacrylate (MMA) induced mRNA expression of the glutathione S-transferase alpha 1 gene ( Gsta1) located downstream of the cis-acting anti-oxidant responsive element (ARE). Herein, we tested the hypothesis that MMA activated the Gsta1 promoter through the ARE. HepG2 cells were transfected with a luciferase reporter vector containing the ARE and the Gsta1 promoter (−990 to +46 bp) and cultured for 12 hrs with MMA (initial concentration, 10 mM). Analysis of the expressed luciferase activity indicated that MMA activated the promoter 2.6-fold. MMA (from 1 to 30 mM) dose-dependently increased the promoter activity, which reached a plateau between 6 and 12 hrs. In HepG2 cells transfected with a reporter vector containing 2 AREs and a TATA-like promoter, 10 mM MMA increased the reporter expression 2.8-fold. These results suggest that MMA increases Gsta1 transcription through ARE-mediated promoter activation.

Molecular Cloning and Characterization of Human Homeobox Gene Nkx3.1 Promoter

2004 ◽  
Vol 36 (1) ◽  
pp. 64-67 ◽  
Author(s):  
An-Li Jiang ◽  
Jian-Ye Zhang ◽  
Charles Young ◽  
Xiao-Yan Hu ◽  
Yong-Mei Wang ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Gene Transcription ◽  
Promoter Activity ◽  
Homeobox Gene ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Reporter Vector ◽  
Luciferase Reporter Vector ◽  
Dual Luciferase Reporter Assay

Abstract Nkx3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. To study its regulation of transcription, 1.06 kb 5′ flanking region of Nkx3.1 gene and its 5′ deletion mutants (861, 617, 417 and 238 bp) were obtained by PCR and cloned into pGL3-basic, a promoter-less luciferase reporter vector, to examine their promoter activities driving the reporter gene transcription. pRL-TK, a Renilla luciferase reporter vector was used as internal control, and pGL3-control and pGL3-basic were used as positive and negative control respectively. The promoter activities were determined by dual-luciferase reporter assay 48 h after pGL3 constructs were cotransfected with pRL-TK into prostate cancer cell LNCaP. The results showed that dual-luciferase reporter assay (M1/M2) of pGL3-1.06 kb cotransfection with pRL-TK was 2.7, which was about 1.5-fold higher than that of pGL3-control cotransfection with pRL-TK and 50-fold higher than that of pGL3-basic cotransfection with pRL-TK. The results also showed that the relative activities (M1/M2) were 0.71, 0.84, 0.44 and 2.07 respectively for pGL3-861 bp, pGL3-617 bp, pGL3-417 bp, pGL3-238 bp, the last one still had 80% promoter activity compared with pGL3-1.06 kb, which showed that deletion from 1.06 kb to 238 bp had small effects on promoter activity. The conclusion was that the 238 bp fragment containing a TATA box and two CAAT boxes had strong promoter activity. However, the deletion from 1.06 kb to 861 bp reduced activity 3.8-fold while the deletion from 417 bp to 238 bp enhanced activity 4.7-fold, which indicated that these deleted sequences might contain some important positive or negative regulatory elements. It will be important to identify the elements within the Nkx3.1 promoter that contribute to regulation of the gene transcription in the future studies.

scholarly journals MirSNP, a database of polymorphisms altering miRNA target sites, identifies miRNA-related SNPs in GWAS SNPs and eQTLs

BMC Genomics ◽  
2012 ◽  
Vol 13 (1) ◽  
pp. 661 ◽  
Author(s):  
Chenxing Liu ◽  
Fuquan Zhang ◽  
Tingting Li ◽  
Ming Lu ◽  
Lifang Wang ◽  
...  
Keyword(s):  
Mirna Target ◽  
Gwas Snps ◽  
Target Sites

scholarly journals Global Analysis of the Genetic Variations in miRNA-Targeted Sites and Their Correlations With Agronomic Traits in Rapeseed

2021 ◽  
Vol 12 ◽  
Author(s):  
Pengfei Xu ◽  
Yantao Zhu ◽  
Yanfeng Zhang ◽  
Jianxia Jiang ◽  
Liyong Yang ◽  
...  
Keyword(s):  
Mirna Target ◽  
Target Genes ◽  
Rare Variants ◽  
Agronomic Traits ◽  
Global Analysis ◽  
Variant Calling ◽  
Genetic Variations ◽  
Nucleotide Polymorphisms ◽  
A Genome ◽  
Target Sites

MicroRNAs (miRNAs) and their target genes play vital roles in crops. However, the genetic variations in miRNA-targeted sites that affect miRNA cleavage efficiency and their correlations with agronomic traits in crops remain unexplored. On the basis of a genome-wide DNA re-sequencing of 210 elite rapeseed (Brassica napus) accessions, we identified the single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) in miRNA-targeted sites complementary to miRNAs. Variant calling revealed 7.14 million SNPs and 2.89 million INDELs throughout the genomes of 210 rapeseed accessions. Furthermore, we detected 330 SNPs and 79 INDELs in 357 miRNA target sites, of which 33.50% were rare variants. We also analyzed the correlation between the genetic variations in miRNA target sites and 12 rapeseed agronomic traits. Eleven SNPs in miRNA target sites were significantly correlated with phenotypes in three consecutive years. More specifically, three correlated SNPs within the miRNA-binding regions of BnSPL9-3, BnSPL13-2, and BnCUC1-2 were in the loci associated with the branch angle, seed weight, and silique number, respectively; expression profiling suggested that the variation at these 3 miRNA target sites significantly affected the expression level of the corresponding target genes. Taken together, the results of this study provide researchers and breeders with a global view of the genetic variations in miRNA-targeted sites in rapeseed and reveal the potential effects of these genetic variations on elite agronomic traits.

Bioinformatics Methods for Studying MicroRNA and ARE-Mediated Regulation of Post-Transcriptional Gene Expression

2010 ◽  
Vol 1 (3) ◽  
pp. 97-112 ◽  
Author(s):  
Richipal Singh Bindra ◽  
Jason T. L. Wang ◽  
Paramjeet Singh Bagga
Keyword(s):  
Mirna Target ◽  
Target Prediction ◽  
Untranslated Regions ◽  
Regulatory Rna ◽  
Rna Motifs ◽  
Protein Coding ◽  
Rna Molecules ◽  
Cellular Processes ◽  
Target Sites ◽  
Transcriptional Gene Regulation

MicroRNAs (miRNAs) are short single-stranded RNA molecules with 21-22 nucleotides known to regulate post-transcriptional expression of protein-coding genes involved in most of the cellular processes. Prediction of miRNA targets is a challenging bioinformatics problem. AU-rich elements (AREs) are regulatory RNA motifs found in the 3’ untranslated regions (UTRs) of mRNAs, and they play dominant roles in the regulated decay of short-lived human mRNAs via specific interactions with proteins. In this paper, the authors review several miRNA target prediction tools and data sources, as well as computational methods used for the prediction of AREs. The authors discuss the connection between miRNA and ARE-mediated post-transcriptional gene regulation. Finally, a data mining method for identifying the co-occurrences of miRNA target sites in ARE containing genes is presented.

scholarly journals An Integrating Approach for Genome-Wide Screening of MicroRNA Polymorphisms Mediated Drug Response Alterations

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Xianyue Wang ◽  
Hong Jiang ◽  
Wei Wu ◽  
Rongxin Zhang ◽  
Lingxiang Wu ◽  
...  
Keyword(s):  
Drug Targets ◽  
Large Scale ◽  
Drug Response ◽  
Mirna Target ◽  
Mrna Translation ◽  
Gene Interaction ◽  
Mirna Regulation ◽  
Small Noncoding Rnas ◽  
Target Sites ◽  
Drug Efficiency

MicroRNAs (miRNAs) are a class of evolutionarily conserved small noncoding RNAs, ~22 nt in length, and found in diverse organisms and play important roles in the regulation of mRNA translation and degradation. It was shown that miRNAs were involved in many key biological processes through regulating the expression of targets. Genetic polymorphisms in miRNA target sites may alter miRNA regulation and therefore result in the alterations of the drug targets. Recent studies have demonstrated that SNPs in miRNA target sites can affect drug efficiency. However, there are still a large number of specific genetic variants related to drug efficiency that are yet to be discovered. We integrated large scale of genetic variations, drug targets, gene interaction networks, biological pathways, and seeds region of miRNA to identify miRNA polymorphisms affecting drug response. In addition, harnessing the abundant high quality biological network/pathways, we evaluated the cascade distribution of tarSNP impacts. We showed that the predictions can uncover most of the known experimentally supported cases as well as provide informative candidates complementary to existing methods/tools. Although there are several existing databases predicting the gain or loss of targeting function of miRNA mediated by SNPs, such as PolymiRTS, miRNASNP, MicroSNiPer, and MirSNP, none of them evaluated the influences of tarSNPs on drug response alterations. We developed a user-friendly online database of this approach named Mir2Drug.

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