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

Abstract

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.

Keywords: AdEasy system; lncRNA H19; mesenchymal stem cells (MSCs); osteogenic differentiation.

Figure 1

Schematic depiction of the construction of recombinant adenovirus vector. Whole mouse genomic DNA were extracted from mouse embryonic fibroblasts (MEFs), which were isolated from new born mice. Then, H19 fragment 1 and H19 fragment 2 were amplified from mouse genome separately and then connected for full-length H19. Full-length H19 was firstly subcloned to pMOK plasmid, and then full-length H19 was cut off from pMOK-H19 and subcloned to recombinant adenovirus vector. After recombinant in AdEasier cells (BJ5183 cell), packing in RAPA cells and amplification in 293pTP cells, high titre AdH19 were generated.

Figure 2

AdH19 mediated overexpression of H19 in MSCs. A: IMAD cells were infected with high titre AdH19 or AdH19. Two days later, bright light and fluorescence microscope examination showed the transduction efficiency of recombinant adenoviruses in iMAD cells, AdFRP as controls (100×). B, C: AdH19 mediated overexpression of H19 (with 2 different pair of primers) and H19 encoded microRNA miR-675 at day 2 and day 5. IMAD cells were infected with AdH19 or AdRFP, at indicated time points, Total RNA was isolated and subjected to TqPCR analysis, relative expressions of H19 or miR675 were normalized with the reference gene GAPDH, **P<0.01.

Figure 3

Functional identification of AdH19 mediated overexpression of H19. Subconfluent iMADs were infected with AdH19 or AdRFP for 2 days, total RNA was isolated from the infected iMADs and subjected to TqPCR analysis of the selected 14 miRNAs which may be associated with H19. The relative normalized expression of these miRNAs was subjected to clustering analysis, 3 miRs are upregulated and 11 miRs are downregulated.

Figure 4

Expression levels of H19 and osteogenic differentiation markers during osteogenic differentiation of MSCs. IMAD cells were cultured in osteogenic medium, at the indicated time points, total RNA was isolated and subjected to TqPCR analysis. (A) Expression level of H19 (A) and osteogenic differentiation markers (B-E) at different time point. All samples were normalized with the reference gene GAPDH. Each assay condition was done in triplicate. Relative expression was calculated by dividing the relative expression values (i.e., gene/GAPDH). **P<0.01, *P<0.05, Runx2, runt-related transcription factor 2; BSP, bone sialoprotein; OPN, osteopontin; OCN, osteocalcin.

Figure 5

AdH19 mediated overexpression of H19 in MSCs is in a dosage dependent manner. A: Bright light and fluorescence microscope examination showed the transduction efficiency of recombinant adenoviruses in iMAD cells two days after infection, AdFRP as controls (100×). The infection rate of low dosage was about 20%, 40% for medium dosage and 80% for high dosage. B: Expression levels of H19 in different infection rate at day 2. All samples were normalized with the reference gene GAPDH. Each assay condition was done in triplicate, **P<0.01.

Figure 6

AdH19 mediated overexpression of H19 biphasic regulating osteogenic differentiation markers expression during osteogenic differentiation of MSCs. IMAD cells were cultured in osteogenic medium, at the indicated time points, total RNA was isolated and subjected to TqPCR analysis. A: Expression level of Runx2 at day 7. B: Expression level of OPN at day 9. C: Expression level of OCN at day 9. Each assay condition was done in triplicate. **P<0.01, Runx2, runt-related transcription factor 2; OPN, osteopontin; OCN, osteocalcin.

Figure 7

AdH19 mediated overexpression of H19 biphasic regulating early and late osteogenic differentiation of MSCs. (A, B) AdH19 mediated overexpression of H19 biphasic regulating ALP activity in MSCs. Subconfluent iMAD cells were infected with AdH19 or AdRFP with different dosage. At day 7 after infection, the infected cells were subjected to ALP activity assays by either histochemical staining (100×) (A) or quantitative bioluminescence assay (B). Each assay conditions were done in triplicate. Representative staining is shown, **P<0.01. (C) AdH19 mediated overexpression of H19 biphasic regulating matrix mineralization of MSCs. Subconfluent iMAD cells were infected with AdH19 or AdRFP with different dosage. At day 15 after infection, the infected cells were subjected to Alizarin Red S staining. Each assay condition was done in triplicate. Representative microscopic images are shown (100×).