To investigate the role of methyltransferase-like 3 (METTL3) in osteogenic and adipogenic differentiation of periodontal mesenchymal stem cells (PDLSCs) .

Flow cytometry and colony formation assay were used to identify the surface markers and proliferation ability of PDLSCs. The osteogenic and adipogenic differentiation potential of PDLSCs was detected by alizarin red staining and oil red O staining, respectively. Human periodontal mesenchymal stem cells (hPDLSCs) and periodontal mesenchymal stem cells in patients with periodontitis (pPDLSCs) were used to construct METTL3 overexpression and knockdown models, respectively. The changes of osteogenesis and adipogenesis were detected by RT-PCR, Western blot, alizarin red staining and oil red O staining at mRNA level, protein level and macroscopic level, respectively. Two samples were compared using independent sample t test, and multiple groups were compared using One-Way ANOVA.

Flow cytometry showed that hPDLSCs and pPDLSCs were positive for CD29 (100.0%, 98.0%), CD105 (100.0%, 99.5%) and CD146 (31.5%, 17.8%), and negative for CD34 (1.3%, 0.4%) and CD45 (1.4%, 0.4%). The results of colony formation experiment showed that the number of colonies formed of hPDLSCs and pPDLSCs was 55 ± 5 and 72 ± 8, respectively, and the cell proliferation ability of hPDLSCs was lower than that of pPDLSCs (t = 3.16, P = 0.034). Alizarin red staining and oil red O staining showed that both cells had osteogenic and adipogenic differentiation ability. hPDLSCs had stronger osteogenic differentiation ability (t = 27.77, P<0.001), while pPDLSCs had stronger adipogenic differentiation ability (t = 5.02, P = 0.007). In the lentivirus transfection model, after 7 days of osteogenic induction culture, the mRNA expression level of Runx2 in the METTL3 overexpression group was higher than that in the overexpression control group. The expression level of Runx2 in the METTL3 overexpression group was 3.63 ± 1.15 and 1.61 ± 0.38 for hPDLSCs and pPDLSCs, respectively, which was 3.39 times (t = 3.777, P = 0.020) and 1.71 times (t = 2.948, P = 0.042) of the control group. The expression level of Runx2 in the METTL3 knockdown group was 0.16 ± 0.03 and 0.26 ± 0.07 for hPDLSCs and pPDLSCs, respectively, which was 0.15 times (t = 9.669, P<0.001) and 0.26 times (t = 8.767, P<0.001) of the control group. Runx2 protein expression level changed in the same way. After 21 days of osteogenic induction culture, the transfected cells were stained with alizarin red. The results showed that the METTL3 overexpression group had deeper staining and larger calcified nodules than the overexpression control group. The quantitative analysis results showed that the values of METTL3 overexpression group in hPDLSCs and pPDLSCs were 28.47% ± 3.82% and 8.55% ± 0.43%, which were 1.78 times (t = 5.012, P = 0.007) and 1.76 times (t = 7.293, P = 0.002) of the METTL3 overexpression control group. The staining was lighter and the calcified nodules were smaller in the knockdown group. The results of quantitative analysis showed that Runx2 protein expression level in the METTL3 knockdown group was 6.36% ± 2.00% and 3.78% ± 0.56% for hPDLSCs and pPDLSCs, respectively, which was 0.35 times (t = 4.444, P = 0.011) and 0.43 times (t = 5.337, P = 0.006) of the knockdown control group. The transfected cells were cultured for lipid induction for 21 days, and then stained with oil red O. The results showed that the size and number of lipid droplets in the METTL3 overexpression group were less than that in the overexpression control group. The quantitative analysis results showed that the values of lipid droplets in the METTL3 overexpression group in hPDLSCs and pPDLSCs were 0.89% ± 0.11% and 1.10% ± 1.15%, which were 0.24 times (t = 5.454, P = 0.006) and 0.49 times (t = 2.935, P = 0.043) of the control. The lipid droplets in the METTL3 knockdown group were larger and more than those in the knockdown control group. The quantitative analysis results showed that the values of lipid droplets in the METTL3 knockdown group in hPDLSCs and pPDLSCs were 3.60% ± 1.08% and 5.34% ± 1.31%, which were 1.94 times (t = 2.794, P = 0.049) and 2.93 times (t = 4.131, P = 0.015) of the control group, respectively.

The METTL3 expression level in pPDLSCs was lower than that in hPDLSCs. Overexpression of METTL3 can promote the osteogenic differentiation of hPDLSCs and pPDLSCs, and inhibit the adipogenic differentiation.