Differential expression of miR-499 and validation of predicted target genes in the testicular tissue of swine at different developmental stages

DNA and Cell Biology
Xiaojun ZhangBoxing Sun

Abstract

microRNAs (miRNAs) represent a newly identified class of nonprotein-coding ∼ 22 nt small RNA that plays important roles in multiple biological processes by degrading targeted mRNA or repressing mRNA translation. This study observed the morphology of swine testicular tissue at different developmental stages (including 1-day old, 1-7 month old) by Hematoxylin-eosin staining. We also examined the expression of miR-499 and its target genes (CYLC1, DMRT1, QKI, XRN2, ZNF313) in samples of tissue slices using quantitative reverse-transcription polymerase chain reaction, which showed that miR-499 had a significant negative correlation with QKI gene. Then, the proteins of QKI gene expression were determined by western blot, which were consistent with results of quantitative polymerase chain reaction (qPCR) detection. Therefore, the luciferase reporter gene system was used to verify correlation between miR-499 and QKI gene. Activity of luciferase was significantly lower in miR-499 co-transfected with pmiR-RB-REPORT-QKI-WT group than the miR-499 co-transfected with pmiR-RB-REPORT-QKI-mut/si groups, indicating that target sequence of miR-499 existed in 3'UTR of QKI gene. Furthermore, the expressions of miR-499 and QKI were detected in test...Continue Reading

References

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Methods Mentioned

BETA
transfection
PCR
Protein Assay
chip

Software Mentioned

SPSS
miRGen
TargetScan
miRDB

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