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.

Since the function of miR-22 on type 2 diabetes was ambiguous, it needs to be overexpressed specifically in the mouse liver to investigate the resulting effects on glucose metabolism, the hallmark for identifying diabetes. To this end, the promoter and miR-22 sequence needs to be optimized before the overexpression cassette is delivered by an rAAV vector in serotype 8 which has a strong liver tropism after intravenous injection.

miR-22 expression comparisons driven by the Pol II promoter (CB) and Pol III promoters (hU6, mU6, and hH1)

Article Access: https://doi.org/10.1186/s43556-021-00063-y

Website for Molecular Biomedicine: https://www.springer.com/journal/43556

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