To investigate the regulatory effects of anti-differetiation noncoding RNA(DANCR) on the ability of human adipose derived mesenchymal stromal stem cells(hASCs) to differentiate into adipocytes and osteoblasts.

hASCs was isolated from abandoned adipose tissue after liposuction. The lentivirus-based DANCR knockdown and over-expression plasmids were constructed and used to knock-down and over-express DANCR in hASCs respectively, the expression of DANCR in hASCs was detected by real time quantitative PCR. Thus, the experiment was divided into five groups: the DANCR knock-down group (shDANCR1, shDANCR2) and the control group (shScrmble); DANCR over-expression group (PCDH-DANCR) and the control group (PCDH-GFP). All the groups were cultured in the growth medium, adipogenic medium and osteogenic medium, Oil red O staining was used to characterize the adipogenic differentiation capabilities of hASCs after 7 days of adipogenic induction, and alizarin red S staining was used to assess the osteogenic differentiation capacities of hASCs after 14 days of osteogenic induction. In addition, real-time PCR analysis was used to quantify the expressions of the transcription factors that play crucial roles in adipogenic induction (PPAR-γ, C/EBPα, FABP4, Adiponectin), lipid synthase(LPL, GPDH, FASN, ACC1, HSL)and osteogenic differentiation(RUNX2, OCN, OPN, BMP2, ALP). All values are expressed as the mean±standard error of the mean. The differences between the groups were assessed using Student’s t test. The result were considered statistically significant at P<0.05.

The expression of DANCR was correspondingly down-regulated and up-regulated in hASCs after infection of DANCR-specific lentivirus. Compared with the control group(shScramble), DANCR expression respectively decreased 86 % (t=84.07, P<0.001)and 74% (t=75.52, P<0.001) in the DANCR knock-down group (shDANCR1, shDANCR2). The constructed DANCR overexpressed plasmids could effectively increase the DANCR expression, which increased 15.067±1.986 times compared with the control group(pCDH-GFP), with a statistically significant difference (t=12.24, P<0.001). To confirm adipogenesis, cells were stained with the lipid dye oil red O. Compared with the control group(shScramble) with a statistically significant difference, more lipid droplets were apparent in the DANCR knockdown group(shDANCR1, shDANCR2) following 7 d adipogenic induction(t= 17.24, P<0.001,t=30.68, P<0.001), whereas the number of lipid droplets in DANCR overexpression group (pCDH-DANCR) less than the control group(pCDH-GFP). The mRNA expression levels of genes that related to adipogenic differentiation(PPAR-γ, C/EBPα, FABP4, Adiponectin) and lipid synthesis (LPL, GPDH, FASN, ACC1, HSL)were elevated in DANCR knockdown cells (shDANCR1, shDANCR2) compared with the control group(shScramble). In contrast, overexpression of DANCR significantly downregulated the expression of these genes. Osteogenesis was confirmed by alizarin red S staining. Overexpression of DANCR reduced the osteogenic differentiation as revealed by the mineralization assay of alizarin red S staining after 14 d osteogenic induction(t=40.81, P<0.001), whereas knockdown of DANCR significantly promoted the mineralization of hASCs(t=15.25, P<0.001,t=24.61, P<0.001). Consistently, DANCR overexpression downregulated several osteogenic differentiation genes(RUNX2, OCN, OPN, BMP2, ALP) at the mRNA level, whereas knockdown of DANCR significantly upregulated the expression of these genes.

The capabilities of hASCs to differentiate into adipocytes and osteoblasts could be regulated by the expression of lncRNA-DANCR.

Stem cells; Adipose derived mesenchymal stromal stem cells; DANCR; Adipogenic differentiation; Osteogenic differentiation