Recombinant adenovirus (AdEasy system) mediated exogenous expression of long non-coding RNA H19 (lncRNA H19) biphasic regulating osteogenic differentiation of mesenchymal stem cells (MSCs).

BACKGROUND

We previously constructed AdEasy system for rapid generation of recombinant adenovirus expressing coding genes. However, it is unclear if AdEasy system could be used for exogenously expression of long noncoding RNAs (lncRNAs). Here we investigated how to overexpress lncRNA H19 with AdEasy system and identified the effect of overexpression H19 on mesenchymal stem cells (MSCs) osteogenic differentiation.

METHODS

H19 fragment 1 and H19 fragment 2 were amplified from mouse genomic DNA separately and then connected for full-length H19. H19 was firstly subcloned to homemade pMOK plasmid, then H19 was cut off from pMOK-H19 and subcloned to recombinant adenovirus plasmid. After homologous recombination in AdEasier cells (BJ5183 cell), packing in mammalian packaging cell line and amplification in 293pTP cells, high titer AdH19 was obtained. Immortalized mouse adipose-derived progenitors (iMADs) were infected by AdH19 with different infection rate, the expression of H19, H19 related microRNAs (miRs) and osteogenic differentiation markers were determined by TqPCR. Alkaline phosphatase (ALP) activities and matrix mineralization were determined by ALP assays and Alizarin red S staining respectively.

RESULTS

AdEasy system was suitable for rapid generation and production of H19, AdH19 can effectively overexpress H19 and serve as functional lncRNA in mesenchymal stem cells (MSCs). Higher dosage of AdH19 inhibited osteogenic differentiation of MSCs, however, lower dosage of AdH19 promoted osteogenic differentiation of MSCs.

CONCLUSIONS

We firstly reported the method for the generation of functional lncRNA with AdEasy system, and identified the biphasic effect of H19 on MSCs osteogenic differentiation.