Construction of a miR-23a-27a cluster expression plasmid: a preliminary study of its function / 中华病理学杂志
Chinese Journal of Pathology
;
(12): 470-474, 2012.
Article
in Chinese
| WPRIM
| ID: wpr-303545
ABSTRACT
<p><b>OBJECTIVE</b>To construct a miR-23a-27a cluster expression plasmid and to explore the target genes and function of the cluster.</p><p><b>METHODS</b>The pre-miR-23a-27a-pcDNA3.1, pre-miR-23a and pre-miR-27a plasmids were cloned by molecular biology method, and their expression efficiency was tested by dual luciferase reporter gene assay and real-time PCR. Several possible target genes of miR-23a and miR-27a were chosen using softwares and further tested by dual luciferase reporter gene assay. Finally, the function of miR-27a was analyzed in MCF-7 cell by Western blot and real-time PCR.</p><p><b>RESULTS</b>miR-23a and miR-27a were transcribed from pre-miR-23a-27a-pcDNA3.1, pre-miR-23a and pre-miR-27a plasmids in HEK293T cells, and both influenced the MRE of Sprouty2 gene in pRL-TK vector, and only miR-27a influenced the 3'-untranslated regions (UTR) full length of Sprouty2 gene while miR-27a did not influence the 3'-UTR of Sprouty2 gene with the sited-mutation in the MRE. The protein expression level of Sprouty2 gene was altered after transfection of pre-miR-27a-pcDNA3.1 plasmid while the RNA level remained unchanged.</p><p><b>CONCLUSION</b>Sprouty2 may be the functional target gene of miR-27a, and the construction of plasmids in the study may provide a fundamental basis for the further functional investigation of miR-23a and miR-27a.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Plasmids
/
Transfection
/
Gene Expression Regulation, Neoplastic
/
Genes, Reporter
/
3' Untranslated Regions
/
MicroRNAs
/
Intracellular Signaling Peptides and Proteins
/
HEK293 Cells
/
MCF-7 Cells
/
Genetics
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Pathology
Year:
2012
Type:
Article
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