ASH1L Effects on Mesenchymal Stem Cells Proliferation and Apoptosis

Objectives: To investigate the role of absent, small, or homeotic-like (ASH1L) in regulating bone marrow stromal cells (MSCs) proliferation..
Methods: MSCs were transfected with stealth siRNA to knock down ASH1L expression. ASH1L-fragment 1, 2, 3 vectors encoding 1-880 amino acids (aa), 881-1,885aa, and 1,886-2,958aa of Ash1lrespectively, and ASH1LΔN (N2212I) mutant abolishing H3K4 methytransferase activity, were constructed. We detected the living and proliferating cells with CCK8 and EdU assay respectively. Flow cytometry was performed to reflect cell cycle and apoptosis of MSCs. Western blot analysis was used to detect the cell cycle and apoptosis related proteins.
Results: In ASH1L knockdown MSCs, the cell cycles of MSC have no change, but there are fewer viable and proliferating cells and more apoptotic cells, compared with negative control MSCs. Consistent with above, apoptosis related protein P53 was elevated in ASH1L knock down MSCs, and cell cycle related protein such as CYCLINA2, CYCLINB1, CYCLIND1 remained unaffected by ASH1L.
In ASH1L overexpressed MSCs, there were more proliferating and fewer apoptotic cells in Ash1l-fragment 1 group, and overexpression of ASH1L-fragment 1 could attenuated P53 expression.
Conclusions: ASH1L had no significant influence on proliferation of MSCs while inhibited apoptosis. The regulating functional domain lies in ASH1L-fragment 1 and the detail mechanism is being studied in our programme.