Increasing the specific expression of Cdk9 gene by single-stranded adult microRNA-1 in fibroblast cells

MicroRNAs (miRNAs) are endogenous, non-coding short RNAs (~22 nt) that can downregulate gene expression by translational repression, mRNA degradation, or transcriptional repression. miRNA misregulation has been implicated in pathogenic alterations such as cancer. In order to investigate microRNA functions in gene regulation and/or to modulate their expression in pathogenic conditions, microRNAs are overexpressed by using plasmid or synthetic RNA duplexes designed to mimic the miRNA of interest.
Mouse fibroblast cells were cultured in DMEM containing 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Lipofectamine 2000 reagent (Invitrogen, 11668-027) was used for transfection according to the manufacturer’s instructions. RNA was extracted from cells and tissues using Qiazol (Qiagen, Germany), according to the manufacturer’s protocol. 2 µg RNA samples were reverse transcribed to cDNA using microRNA-1 specific stem loop and random hexamer primers and MLV reverse transcriptase.q-PCR was performed with the ‘SYBR Green I Master Mix’ kit.
miR-1-expressing plasmid (pPRO-miR-1) was transfected into fibroblast cells. Quantitative real time PCR determination showed that Cdk9 expression was inhibited in transfected cells compared to control. Similarly, downregulation of Cdk9 was also observed following transfection of 22 nucleotide mature double stranded miR-1. Interestingly, transfection of synthetic 22 nucleotide mature single stranded miR-1 (miR-1-3p) induced Cdk9 expression in the fibroblast cells.
Unlike miR-expressing vector or synthetic RNA duplexes designed to mimic the miRNA of interest that can downregulate gene expression, synthetic 22 nucleotide mature single stranded microRNA probably induces gene expression in a locus-specific manner.