microRNA21在人牙周韧带细胞成骨分化中的表达变化

doi:10.3877/cma.j.issn.1674-1366.2015.01.002

目的

研究微小RNA21（miR21）在人牙周韧带细胞（PDLC）成骨分化早期的表达变化，探讨其对PDLC成骨分化的作用及可能的调控机制。

方法

培养PDLC，免疫细胞化学染色鉴定其来源。取第3~4代细胞矿化培养7、14、21 d进行碱性磷酸酶（ALP）检测，21 d进行茜素红染色；分别培养4 h和1、3、7 d，实时荧光定量聚合酶链反应（PCR）检测miR21、靶基因萌牙2（SPRY2）和Runt相关转录因子2（RUNX2）；Western blot检测磷酸化细胞外调节蛋白激酶（ERK）1/2、磷酸化p38、SPRY2和RUNX2。对照组为正常培养细胞。

结果

培养的PDLC来源于间充质，7、14、21 d的ALP活性明显增高（t_{7 d}= 2.707，P_{7 d}= 0.011；t_{14 d}= 8.189，P_{14 d}= 0.001；t_{21 d}= 3.546，P_{21 d}= 0.024），矿化诱导21 d茜素红染色可见矿化结节，具有成骨分化能力；实时荧光定量PCR结果显示，miR21在矿化诱导3、7 d表达上升，SPRY2的表达则呈下降趋势（F_miR21= 14.567，P_miR21 < 0.05，F_SPRY2= 7.765，P_SPRY2= 0.004）；Western blot结果显示，ERK-丝裂原活化蛋白激酶（MAPK）信号通路在矿化诱导第1天始激活，而后稳步上升（t_{1 d}= 14.378，P_{3 d} < 0.05，t_{3 d}= 6.558，P_{3 d}= 0.003，t_{7 d}= 10.685，P_{7 d} < 0.05）；p38 MAPK信号通路活性在诱导4 h时被激活，而后与对照组活性无差异，在第7天时有所升高（t_{4 h}= 18.803，P_{4 h} < 0.05，t_{7 d}= 9.643，P_{7 d}= 0.001）。

结论

miR21与PDLC成骨分化相关，其调控机制可能与ERK-MAPK、p38 MAPK信号通路相关；miR21可能靶向SPRY2调控ERK-MAPK信号通路活性，实验结果为进一步研究miR21的功能作用与调控机制提供理论依据。

关键词: 微小RNA21, 牙周韧带细胞, 成骨分化, 丝裂原活化蛋白激酶信号通路

Objective

To investigate the expression of microRNA21 (miR21) and explore its function and possible mechanism during osteogenic differentiation in human periodontal ligament cells (PDLCs) .

Methods

PDLCs were isolated from human periodontal ligament tissue and immuncytochemical staining was used to identify their origin. Cells from the 3rd to 4th passage were cultivated in mineralizing medium for 7 days, 14 days and 21 days respectively. Alkaline phosphatase (ALP) experiment and alizarin red (AR) staining were used to identify osteogenic differentiation capacity of PDLCs. The cells were cultured for 4 hours, 1 day, 3 days and 7 days respectively. Expression of miR21, Sprouty 2 (SPRY2) and Runx2 were tested by quantitative real-time PCR and the phosphorylated ERK1/2, phosphorylated p38, SPRY2 and Runx2 were measured by western blot. Cells without osteogenic induction served as controls.

Results

The cultured PDLCs were derived from mesenchyma. Compared to the control groups, AR staining showed obvious mineralization nodules, and ALP activities were significantly higher in 7 d-, 14 d- and 21 d-osteogenic induction groups (P < 0.05) . MiR21 started up-regulate since 3rd day, and remained high expressed level until 7 days in osteogenic induction groups. Expression of SPRY2 reversed to which of miR21 in the induction groups. The results of western blot showed that both ERK-MAPK and p38 MAPK pathway were activated. ERK-MAPK pathway was activated since the 1st day and sustained during the early osteogenesis period of PDLCs. At the 4th hour of induction, the activity of p38 MAPK was obviously enhanced, then returned to which of the control groups, and up regulated again at 7 days of mineralization cultivation.

Conclusions

MiR21 was related to osteogenic differentiation of PDLCs and probably modulated ERK-MAPK and p38 MAPK pathways. MiR21 might target SPRY2 as a regulator of ERK-MAPK pathway during osteogenic differentiation of PDLCs. This result can provide basis for further research on its fine mechanism.

Key words: MicroRNA21, Periodontal ligament cells, Osteogenic differentiation, Mitogen-activated protein kinases signaling pathway

表1 待测基因的引物序列及大小

基因	引物序列	产物大小（bp）
miR21	F:5 ′ -ACACTCCAGCTGGGTAGCTTATCAGACTGATG-3 ′	72
?	R:5 ′ -CTCAACTGGTGTCGTGGA-3 ′	?
SPRY2	F:5 ′ -CGATTGCTCGGAAGTTGGTC-3 ′	163
?	R:5 ′ -TCCAGCAGGCTTAGAACACA-3 ′	?
RUNX2	F:5 ′ -GATGGTACGGTGGTGACTGT-3 ′	160
?	R:5 ′ -CAGTGATGGTCAGGGTGAAA-3 ′	?

表1 待测基因的引物序列及大小

基因	引物序列	产物大小（bp）
miR21	F:5 ′ -ACACTCCAGCTGGGTAGCTTATCAGACTGATG-3 ′	72
?	R:5 ′ -CTCAACTGGTGTCGTGGA-3 ′	?
SPRY2	F:5 ′ -CGATTGCTCGGAAGTTGGTC-3 ′	163
?	R:5 ′ -TCCAGCAGGCTTAGAACACA-3 ′	?
RUNX2	F:5 ′ -GATGGTACGGTGGTGACTGT-3 ′	160
?	R:5 ′ -CAGTGATGGTCAGGGTGAAA-3 ′	?

图1 人牙周韧带细胞培养、来源与成骨分化能力检测

图2 miR21、SPRY2、RUNX2实时荧光定量PCR结果

图3 Western blot检测矿化组与对照组ERK-MAPK、p38 MAPK通路活性变化

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Expression of microRNA21 in human periodontal ligament cells osteogenic differentiation

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Expression of microRNA21 in human periodontal ligament cells osteogenic differentiation