Figure 4.

The expression level of CDKN1A is involved in miR-302d-induced proliferation in hADSCs. (a and b) Downregulation of CDKN1A mRNA expression. Forty-eight hours after transfection with indicated miRNA (a) or siRNA (b), cells were harvested for measurement of mRNA levels by real-time PCR. *P<0.05 versus miR-control. ^##P<0.01 versus siR-control. (c) The increased expression of CDKN1A in miR-302d inhibitor (IH)-transfected hADSCs. **P<0.01 versus miR-control IH. (d and e) Downregulation or upregulation of p21 (a protein that in humans is encoded by the CDKN1A gene) expression. Cells were transfected with indicated miRNA after transfection with siR-control or siR-CDKN1A. Forty-eight hours after cotransfection of miRNA and siRNA, the protein levels of p21 were analyzed by western blotting. (f and g) Effect of CDKN1A siRNA on proliferation. hADSCs with or without silencing of CDKN1A expression were transfected with miR-control or miR-302d (f), and with miR-control IH or miR-302d IH (g). Cells were counted at different time points. *P<0.05, **P<0.01 versus miR-control (or miR-control IH). ^#P<0.05, ^##P<0.01 versus siR-control. ^&P<0.05 versus miR-302d (or miR-302dIH)-transfected siR-control. (h) Effect of CDKN1A siRNA on oxidant-induced hADSCs death. Cells were transfected with siR-CDKN1A and/or miR-302d and were then treated with CoCl₂ or SIN-1. Cell viability was determined by trypan blue exclusion. (i) The CDKN1A mRNA level in oxidant-treated hADSCs after transfection of miR-302d. The CDKN1A mRNA level was confirmed by real-time PCR at 48 h after transfection. *P<0.05, **P<0.01 versus CoCl₂ or SIN-1 treated miR-control. ^$P<0.05 versus untreated control. (j) Schematic diagram of luciferase reporter vector. The CDKN1A 3'UTR sequences containing the predicted miR-302d binding site or mutated binding site (mut) were inserted into the pMIR-REPORT vector. The box shows the sequence alignment of miR-302d and its predicted binding site in 3'UTR of CDKN1A. miR-control or miR-302d was cotransfected with pMIR-CDKN1A or pMIR-CDKN1A mut luciferase constructs into hADSCs, respectively. Relative luciferase activities were analyzed at 72 h post transfection. **P<0.01 versus miR-control. Data are represented the mean±S.D. of four independent experiments