Abstract
MicroRNA-22 (miR-22) was suggested to be important for type 2 diabetes but its functions for this disease remained unclear. Recombinant adeno-associated virus (rAAV)-mediated miR delivery is a powerful approach to study miR functions in vivo, however, the overexpression of miR-22 by rAAV remains challenging because it is one of the most abundant miRs in the liver. In this study, a series of expression cassettes were designed and compared. It was shown that different lengths of primary miR-22 were overexpressed in HEK293 and HeLa cells but the longer ones were more efficiently expressed. miR-22 may be placed in either introns or the 3′ UTR of a transgene for efficient overexpression. RNA polymerase III or II promoters were successfully utilized for miR expression but the latter showed higher expression levels in cell lines. Specifically, miR-22 was expressed efficiently together with an EGFP gene. After screening, a liver-specific TTR promoter was chosen to overexpress miR-22 in diabetic mice fed a high-fat diet. It was shown that miR-22 was overexpressed 2-3 folds which improved the insulin sensitivity significantly. The approach utilized in this study to optimize miR overexpression is a powerful tool for the creation of efficient rAAV vectors for the other miRs.
Highlights
MicroRNAs are single-stranded non-coding RNAs, 20- to 24-nt long, which regulate gene expression post-transcriptionally by binding to 3′ untranslated region (3′ Untranslated region (UTR)) of their target mRNAs [1]
A series of Recombinant adeno-associated virus (rAAV) plasmid vectors containing different lengths of pri-miR-22 were designed, all of which were under the control of human H1 promoter (Fig. 1a)
We investigated whether miR-22 overexpression affected bodyweight and insulin sensitivity in obese and diabetic mice induced by a high-fat diet (HFD)
Summary
MicroRNAs (miRs) are single-stranded non-coding RNAs, 20- to 24-nt long, which regulate gene expression post-transcriptionally by binding to 3′ untranslated region (3′ UTR) of their target mRNAs [1]. The targeted mRNAs are degraded or destabilized, resulting in the reduction of targeted proteins. Single-strand primary microRNAs (pri-miRs) are generally transcribed by RNA polymerase II. Pri-miRs form a hairpin loop structure, which is cleaved by Drosha, resulting in short hairpin precursor microRNAs (pre-miRs). Pre-miRs are released into the cytoplasm and further cleaved into double-stranded ~ 22-nt long mature miRs by Dicer. A matured single-stranded miR is incorporated into the silencing complex miRISC
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