Effects of ADAM28 shRNA interference vector on biologic functions of human periodontal ligament stem cells

Background/Objective: This study was to investigate the effects of a disintegrin and metalloproteinase 28 (ADAM28) shRNA interference vector on proliferation, differentiation, and apoptosis of human periodontal ligament stem cells (HPDLSCs) and action mechanism. Methods: After PGPU6/GFP/Neo ADAM28 shRNA1-4 were constructed and identified, and transfected into HPDLSCs for 48h, transfection efficiency and inhibition efficiency of ADAM28 shRNA1-4 were detected by QRT-PCR and Western blot. A group of ADAM28 interference vector with the highest inhibitory efficiency was selected to detect proliferation, differentiation and apoptosis of HPDLSCs. Methabenzthiazuron (MTT) and cell-cycle assay by flow cytometry (FCM) were used to test the HPDLSCs proliferation activity. Alkaline phosphatase (ALP) analysis by enzyme kinetics were performed to measure cytodifferentiation standard. Western blot was carried out to determine the influence of ADAM28-shRNA1 on HPDLSCs expressing CAP and Pax9. Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) analysis by FCM was performed to measure apoptosis rate and Western blot was used to detect the expression of Bax and Bcl-2 proteins. Results: PGPU6/GFP/Neo-ADAM28-shRNA1-4 interference vector were successfully constructed and efficiently transfected into HPDLSCs. PGPU6/GFP/Neo-ADAM28-shRNA1-4 all had significant inhibition efficiency, and ADAM28-shRNA1 displayed the highest level. Furthermore, ADAM28-shRNA1 could significantly suppress proliferation and differentiation, and induce apoptosis of HPDLSCs. ADAM28-shRNA1 was able to inhibit the expression levels of CAP and Pax9. Whereas, apoptosis rate of ADAM28-shRNA1 group presented the highest level and ADAM28-shRNA1 could significantly enhance expression levels of Bcl-2 and Bax proteins. Conclusion: ADAM28 may participate in root development as a major regulator of proliferation, differentiation and apoptosis of HPDLSCs.