Mechanism of miR-149-3p Targeted Regulation of Akt1-mediated Osteogenic Differentiation of Periodontal Ligament Stem Cells
Objective To explore the role and mechanism of miR-149-3p targeting regulating protein kinase B(Akt1)in the osteogenic differentiation of periodontal ligament stem cells(PDLSCs).Methods The premolars or third molars of 18-25-year-old patients who were extracted for orthodontic treatment in the department of stomatology of au-thor's hospital in January 2022 were selected,the periodontal ligament tissues were isolated and human PDLSCs were ex-tracted.Human PDLSCs were identified by immunohistochemical staining of pan-cytokeratin and vimentin.Human PDLSCs were divided into control group,empty plasmid group,miR-149-3p inhibitor group and miR-149-3p mimic group.The human PDLSCs in control group were not treated,the empty plasmid,miR-149-3p inhibitor and miR-149-3p mimic groups were transfected into human PDLSCs according to the instructions of Lipofectamine 3000 in the empty plas-mid group,miR-149-3p inhibitor group and miR-149-3p mimic group.The mRNA expressions of miR-149-3p,alkaline phosphatase(ALP),runt-related transcription factor 2(Runx2)and Akt1 were detected by real-time reverse transcrip-tion polymerase chain reaction(RT-PCR).Protein expressions of ALP,Runx2 and Akt1 were detected by Western blot.Intracellular calcium ion was detected by Fluo-3 AM probe.Results The results of immunohistochemistry showed that the diaminobenzidine(DAB)staining of hu-man PDLSCs was positive for vimentin and negative for pan-cytokinetin which indicated the successful exrtaction of PDLSCs.Compared with the control group and the empty plasmid group,the expression of miR-149-3p in the miR-149-3p inhibitor group significantly decreased,the mRNA and protein expressions of ALP,Runx2,Akt1,and the intra-cellular calcium ion fluorescence intensity significantly increased(all P＜0.05);the expression of miR-149-3p in the miR-149-3p mimic group significantly increased,and the mRNA,protein expressions and the intracellular calcium ion fluores-cence intensity of ALP,Runx2 and Akt1 significantly decreased(all P＜0.05).Compared with miR-149-3p inhibitor group,miR-149-3p expression significantly increased in the miR-149-3p mimic group,mRNA,protein expressions and in-tracellular calcium ion fluorescence intensity of ALP,Runx2 and Akt1 significantly decreased(all P＜0.05).There was no significant difference between the control group and the empty plasmid group(P＞0.05).Conclusion Over-expressed miR-149-3p can target the expression of Akt1 protein,thus inhibiting the expression levels of osteogenic markers ALP and Runx2,decreasing calcium ion concentration in human PDLSCs,which is not conducive to the osteogenic differentia-tion of human PDLSCs.
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