[Effect of WW-domain transcription regulator 1 on aging regulation of human dental pulp stem cells].

Abstract

Objective: Investigating the changes of phenotype and moleculars associated with aging with the increase of passage times of human dental pulp stem cells (hDPSC), to explore the role of WW-containing transcriptional regulator 1 (WWTR1) in the aging mechanism. Methods: hDPSCs were cultured by tissue block method, and were divided into 4 groups according to the age, algebra, cell knockdown and overexpression of WWTR1 in hDPSCs. Group Ⅰ: hDPSCs from human teeth were further divided into youth group (15-25 years old) and group middle-aged group (40-50 years old) according to different ages. Group Ⅱ: according to different passage, hDPSCs were divided into young cells group (hDPSCs were transmitted to P3 generation), and old cells group (hDPSCs were transmitted to P10 generation). Group Ⅲ: hDPSCs were knocked down of WWTR1, which were further divided into knockdown group and knockdown carrier group. Group Ⅳ: hDPSCs were overexpressed of WWTR1, which were further divided into overexpression group and overexpression carrier group. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the changes of WWTR1 expression in groups Ⅰ and Ⅱ, and cell counting kit-8 (CCK-8) was used for groups Ⅱ, Ⅲ, and Ⅳ. Cell proliferation capacity was detected by CCK-8 assay. The ability of osteogenic differentiation was detected by alizarin red staining. Cell senescence positive rate was detected by age-related β-galactosidase staining. The expression levels of age-related genes p53 and p21 were detected by RT-qPCR. Results: The proportion of senescent cells increased gradually with continuous culture. The proliferation and osteogenic differentiation of hDPSCs in the old group were significantly lower than those in the young group (P<0.001). The expression levels of senescence related genes p53 (2.09±0.24) and p21 (4.91±0.54) in old cell group were higher than those in young cell group respectively [p53: (1.08±0.09) and p21: (1.09±0.08)] (P<0.01, P<0.001). The WWTR1 expression levels of hDPSCs in middle-aged group and old cells group were both decreased compared with those in young group and young cells group (P<0.01). The proportion of senescent cells in knockdown group (44.50±2.42) was higher than that in knockdown carrier group (22.27±0.56) (P<0.001). After knocking down WWTR1 in hDPSCs, the expression levels of age-related genes p53 and p21 were up-regulated (P<0.001), and the abilities of proliferation and osteogenic differentiation in the knockdown group were lower than those in the knockdown carrier group (P<0.001). The proportion of senescent cells in overexpression empty carrier group (20.40±0.79) was higher than that in overexpression group (10.07±0.61) (P<0.001). After WWTR1 overexpression ins hDPSCs, the expression levels of age-related genes p53 and p21 were down-regulated, and the proliferation and osteogenic differentiation ability in overexpression group were higher than those in overexpression carrier group (P<0.001). Conclusions: WWTR1 can inhibit the expression levels of age-related genes p53 and p21, thus delaying the aging process as well as promoting the proliferation and osteogenic differentiation of hDPSCs.