Objective: To study the molecular mechanisms underlying the post-transcriptional regulation of secreted frizzle-related protein 1(SFRP1)gene in MC3T3-E1 murine pre-osteoblast cells using a newly constructed plasmid pMIR-REPORT-SFRP1UTR in which the expression of luciferase was regulated by the 3'UTR of mouse SFRP1 gene.
Methods: The mRNA and protein levels of SFRP1 gene in MC3T3-E1 cells were measured using real-time quantitative polymerase chain reaction and Western blotting at 0, 7 and 10 days respectively after osteogenic differentiation was induced in these cells using 50 mg/L of ascorbic acid. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)and β-actin served as controls, respectively. The 3'UTR of mouse SFRP1 gene was then amplified from total RNA isolated from MC3T3-E1 cells using PCR. The SFRP1 3'UTR fragment was then subcloned into the vector, pMIR-REPORT, resulting in a new plasmid named as pMIR-REPORT-SFRP1UTR. pMIR-REPORT-SFRP1UTR was transfected into MC3T3-E1 and NIH3T3 cells, and the luciferase levels were determined.Cells transfected with pMIR-REPORT-SFRP1UTR were assigned to pMIR-REPORT-SFRP1UTR group, and cells transfected with pMIRREPORT were assigned to pMIR-REPORT group.
Results: SFRP1 mRNA levels in MC3T3-E1 cells treated with ascorbic acid for 0, 7 and 10 days were 1.00±0.12, 1.97±0.34(P>0.05, vs. 0 d group)and 4.98±0.99(P<0.05, vs. 0 d group), respectively. After treated with ascorbic acid for 0, 7 and 10 days, the protein levels of SFRP1 in MC3T3-E1 cells were 0.94 ± 0.13,1.26 ± 0.16(P>0.05, vs. 0 d group)and 1.30 ± 0.15(P>0.05, vs. 0 d group), respectively. Luciferase levels in MC3T3-E1 cells transfected with pMIR-REPORT-SFRP1UTR and pMIRREPORT were 74 ± 10 and 487 ± 34, respectively(P<0.05). In contrast, luciferase levels in NIH3T3 cells transfected with pMIR-REPORT-SFRP1UTR and pMIR-REPORT were 2 240±175 and 2 707±117, respectively(P<0.05).
Conclusions: The 3'UTR of mouse SFRP1 gene was successfully amplified and subcloned into pMIRREPORT. The3'UTR of SFRP1 geneis involved in the cell-specific post-transcriptional regulation of SFRP1 gene.