miR-214 inhibits the osteogenic differentiation of dental follicle cells in vitro

doi:10.12016/j.issn.2096-1456.2020.03.002

Abstract

Abstract:

Objective To investigate the effect of miR-214 on the osteogenic differentiation of dental follicle cells (DFCs). Methods Purified DFCs were cultured in vitro by bidirectional differential passage, with the untransfected DFCs as the control group (DFCs group). The expression of miR-214-3p in DFCs was upregulated and downregulated by transfection of miR-214-3p(miR-214 mimics group) or miR-214-3p inhibitors(miR-214 inhibitor group) into DFCs. The expression levels of miR-214, alkaline phosphatase (ALP), osteonectin (OSN) and runt-related transcription factor-2(RUNX-2) were detected by qRT-PCR after 7 days of osteogenesis induction, the protein expression levels of RUNX-2 and β-catenin were detected by western blot, and the formation of mineralized nodules was observed with alizarin red staining after 14 days of osteogenesis induction. Results Compared with the DFCs group, in the miR-214 mimics group, the expression of miR-214 was upregulated after 7 days of osteogenesis induction. The mRNA expression of ALP, OSN and RUNX-2 in the miR-214 mimics group was lower than that in the DFCs group, but only ALP in the two groups was statistically significant (P > 0.05); the mRNA expression of ALP, OSN and RUNX-2 in the miR-214 inhibitor group was higher than that in the DFCs group, and the difference was statistically significant (P < 0.05). The protein expression of RUNX-2 and β-catenin in the miR-214 mimics group was lower than that in the miR-214 inhibitor group. The number of calcified nodules in the miR-214 mimics group was significantly less than that in the DFCs group, while that in the miR-214 inhibitor group was significantly higher than that in the DFCs group. Conclusion The upregulation of miR-214 can downregulate the expression of β-catenin, can inhibit the expression of ALP, OSN and RUNX-2 related to osteogenesis, and can inhibit osteogenic differentiation. The downregulation of miR-214 demonstrated the opposite results; miR-214 may downregulate the expression of β-catenin and inhibit the osteogenic differentiation of DFCs.

Key words: dental follicle cells, bidirectional differential passage, miR-214, Wnt/β-catenin, osteogenic differentiation, alkaline phosphatase, osteonectin, mineralized nodule

摘要：

目的探讨miR-214对于牙囊细胞（dental follicle cells,DFCs）成骨分化的影响。方法双向差速传代法体外培养提纯牙囊细胞,以未转染的DFCs为对照（DFCs组）,通过向DFCs中转染miR-214-3p mimics（miR-214 mimics 组）或miR-214-3p inhibitor（miR-214 inhibitor组）分别上调和下调DFCs中的miR-214的表达。成骨诱导7 d,qRT-PCR检测miR-214表达及相关成骨基因碱性磷酸酶（alkaline phosphatase,ALP）、骨粘连蛋白（osteonectin,OSN）、成骨相关转录因子2（runt-related transcription factor-2,RUNX-2）的mRNA表达,免疫蛋白印迹检测各组RUNX-2、β-catenin蛋白表达;成骨诱导14 d茜素红染色观察矿化结节形成的情况。结果骨诱导7 d,相较DFCs组,miR-214 mimics组miR-214的表达上调,miR-214 mimics组成骨相关基因ALP、OSN及RUNX-2的mRNA表达均低于DFCs组,但仅ALP在两组的差异具有统计学意义（P＞0.05）,而miR-214 inhibitor组成骨相关基因ALP、OSN、RUNX-2的mRNA表达均高于DFCs组,且差异均具有统计学意义（P＜0.05）。miR-214 mimics组RUNX-2、β-catenin的蛋白表达均低于miR-214 inhibitor组。miR-214 mimics组钙化结节明显少于DFCs组,而miR-214 inhibitor组钙化结节却明显多于DFCs组。结论 miR-214 上调可使β-catenin表达下调,并抑制成骨相关基因 ALP、OSN及RUNX-2的表达,抑制成骨分化;下调miR-214,结果相反;miR-214可能是通过下调β-catenin的表达,抑制DFCs的成骨分化。

关键词: 牙囊细胞, 双向差速传代法, miR-214, Wnt/β-catenin, 成骨分化, 碱性磷酸酶, 骨粘连蛋白, 成骨相关转录因子2, 矿化结节

CLC Number:

WANG Zhiheng,ZUO Jie,WANG Mengqi,ZHU Shaojun,LIU Yishan. miR-214 inhibits the osteogenic differentiation of dental follicle cells in vitro[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2020, 28(3): 146-152.

王智亨,左婕,王梦琪,朱少军,刘奕杉. miR-214抑制牙囊细胞成骨分化的体外研究[J]. 口腔疾病防治, 2020, 28(3): 146-152.

Figures/Tables 7

Table 1 qRT-PCR target gene primer sequence

Primer	Sequence（5′→3′）
miR-214-3P-F miR-214-3P-R	5′-GCATCCACAGCAGGCACA-3′ 5′-CACTTCCTCAGCACTTGTTGGTAT-3′
U6-F U6-R	5′-CAGCACATATACTAAAATTGGAACG-3′ 5′-CCTACTGTGCGTTTAAGCA-3′
GAPDH-F GAPDH-R	5′-GACATGCCGCCTGGAGAAC-3′ 5′-TGATTTCCCGTAGGACCCGA-3′
RUNX-2-F RUNX-2-R	5′-CCATGCACGGTTGACAGACT-3′ 5′-GTAGTCGCAGTTGTGGTAGT-3′
ALP-F ALP-R	5′-GAGGAGACACGGCTGGAGAT-3′ 5′-CGGATGAACACACCGCACTT-3′
OSN-F OSN-R	5′-ATGACGACGACGATGACGAC-3′ 5′-CAGGAAGTGACGGTCGTGTG-3′

Figure 1 Primary DFCs and DFCs purified to the third generation by bidirectional differential passaging a: the primary DFCs; b: the third generation DFCs; DFCs: dental follicle cells

Figure 2 Expression of miR-214 1, 2 and 3 days after transfection of the miR-214 mimics and miR-214 inhibitor

Figure 3 Expression of miR-214 after 7 days of osteogenesis induction after the transfection of the miR-214 mimics and miR-214 inhibitor

Figure 4 Expression of ALP, OSN, RUNX-2 after 7 days of osteogenesis induction after the transfection of the miR-214 mimics and miR-214 inhibitor

Figure 5 Expression of RUNX-2 andβ-catenin after 7 days of osteogenesis induction in the miR-214 mimics, miR-214 inhibitor, DFCs groups mimics: miR-214 mimics group; inhibitor: miR-214 inhibitor group; DFCs: the DFCs group

Figure 6 Images of mineralized nodules by alizarin red staining after 14 days of osteogenesis induction in the 3 groups a: miR-214 mimics group; b: normal DFCs group; c: miR-214 inhibitor group

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