中国生物工程杂志  2016, Vol. 36 Issue (2): 22-29

文章信息

宋琪玲, 施琼, 陈楚, 唐祖川, 刘红霞, 周一青, 张汝益, 严树涓, 翁亚光
SONG Qi-ling, SHI Qiong, CHEN Chu, TANG Zu-chuan, LIU Hong-xia, ZHOU Yi-qing, ZHANG Ru-yi, YAN Shu-juan, WENG Ya-guang
MiR-21协同BMP9促进间充质干细胞C3H10T1/2成骨分化
MiR-21 Synergizes with BMP9 in Osteogenic Differentiation During Mesenchymal Stem Cells Line C3H10T1/2
中国生物工程杂志, 2016, 36(2): 22-29
China Biotechnology, 2016, 36(2): 22-29
http://dx.doi.org/10.13523/j.cb.20160204

文章历史

收稿日期: 2015-07-27
修回日期: 2015-08-31
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引用本文 0
宋琪玲, 施琼, 陈楚, 唐祖川, 刘红霞, 周一青, 张汝益, 严树涓, 翁亚光. MiR-21协同BMP9促进间充质干细胞C3H10T1/2成骨分化[J]. 中国生物工程杂志, 2016, 36(2): 22-29.
SONG Qi-ling, SHI Qiong, CHEN Chu, TANG Zu-chuan, LIU Hong-xia, ZHOU Yi-qing, ZHANG Ru-yi, YAN Shu-juan, WENG Ya-guang. MiR-21 Synergizes with BMP9 in Osteogenic Differentiation During Mesenchymal Stem Cells Line C3H10T1/2[J]. China Biotechnology, 2016, 36(2): 22-29.
MiR-21协同BMP9促进间充质干细胞C3H10T1/2成骨分化
宋琪玲, 施琼, 陈楚, 唐祖川, 刘红霞, 周一青, 张汝益, 严树涓, 翁亚光     
重庆医科大学检验医学院 临床检验诊断学教育部重点实验室 重庆 400016
收稿日期: 2015-07-27; 修订日期: 2015-08-31;
基金项目: 国家自然科学基金(NSFC 31200971)、教育部博士点基金(20115503110009)、重庆市教育委员会科学技术研究项目(KJ120327)资助项目.
通讯作者, 电子信箱: yaguangweng@126.com
摘要: 目的:探讨miR-21与BMP9之间的关系,明确miR-21在BMP9诱导间充质干细胞成骨分化中的作用。方法:(1)Ad-BMP9感染C3H10T1/2细胞,Real-time-PCR检测miR-21表达。RT-PCR检测ALP的表达。(2)MiR-21转染C3H10T1/2细胞,Real-time-PCR检测miR-21和BMP9表达。(3)MiR-21和BMP9-CM处理C3H10 T1/2细胞,ALP活性和染色实验检测C3H10 T1/2细胞早期成骨能力。茜素红S染色实验检测钙盐沉积情况。(4)MiR-21和BMP9-CM处理C3H10 T1/2细胞,Real-time-PCR检测成骨分化相关因子ALP,OCN的表达。(5)MiR-21和BMP9-CM处理C3H10T1/2细胞,Western blot检测p-Smad1/5蛋白水平的表达。结果:(1)BMP9暂时降低miR-21的表达。MiR-21也可以暂时降低BMP9的表达。(2)MiR-21可以协同BMP9增强ALP和钙盐沉积。(3)MiR-21协同BMP9增加了p-Smad1/5蛋白水平的表达。结论:MiR-21与BMP9存在相互关系,两者可以互相调节表达。MiR-21可以协同BMP9促进间充质干细胞C3H10T1/2细胞成骨分化,这一过程与增强BMP9/Smad信号的激活程度有关。
关键词: MiR-21     骨形成蛋白9     C3H10T1/2细胞     成骨分化    
MiR-21 Synergizes with BMP9 in Osteogenic Differentiation During Mesenchymal Stem Cells Line C3H10T1/2
SONG Qi-ling, SHI Qiong, CHEN Chu, TANG Zu-chuan, LIU Hong-xia, ZHOU Yi-qing, ZHANG Ru-yi, YAN Shu-juan, WENG Ya-guang     
Key Laboratory of Clinical Laboratory Diagnostics of Ministry Education, Faculty of Laboratory Medicine, Chongqing Medicine University, Chongqing 400016, China
Abstract: Objective:(1) To study if there is a crosstalk between bone morphogenetic protein 9(BMP9) and microRNA-21(miR-21);(2) to investigate the effect of miR-21 on BMP9-induced osteogenic differentiation of mesenchymal stem cells line C3H10T1/2.Methods:(1) Using Ad-BMP9 infecting C3H10 T1/2 cells, the expression of miR-21 were detected by Real-time-PCR. After transfecting miR-21 mimic 1 day and 5 days, the expression of BMP were tested by Real-time-PCR.(2) In vitro, after transfecting miR-21, BMP9-CM treating C3H10T1/2, ALP staining and ALP activity were detected and the calcium deposition were detected by Alizarin Red S staining.(3)The expression of BMP9 downstream factors ALP and OCN were detected by Real-time-PCR.(4) The protein expression of p-Smad1/5 were detected by Western blot. Results:BMP9 can decrease the expression of miR-21 temporarily and miR-21 also decreases the expression of BMP9 temporarily. After transfecting miR-21 and treating C3H10T1/2 cell with BMP9-CM, the early osteogenic differentiation marker ALP activity and ALP staining and later osteogenic differentiation marker calcium deposition were enhanced. What's more, the expression of ostengenic factors ALP and OCN also increase during miR-21 combined with BMP9-CM. The protein expression of p-Smad1/5 were upregulation during the same process. Conclusion:There is a crosswalk between BMP9 and miR-21. MiR-21 synergizes with BMP9 promoting mesenchymal stem cells C3H10T1/2 osteogenic differentiation by increasing BMP9/Smad signaling pathway activated.
Key words: MiR-21     Bone morphogenetic protein9     C3H10 T1/2 cells     Osteogenic differentiation    

近年来较多研究表明，microRNA(miRNA)通过转录后调节其靶基因表达来调节干细胞分化能力[1]。MiR-21在干细胞分化中的作用也得到初步阐述。通过调节TGF-β信号通路，miR-21可以促进间充质干细胞(mesenchymal stem cells，MSCs)成脂分化[2]。在成骨分化过程中，miR-21可以靶向调节Spry1,Spry2，通过Erk-MAPK信号通路来促进间充质干细胞成骨分化 [3, 4, 5]。因此，miR-21在间充质干细胞成骨分化过程中发挥着重要作用。

BMP9(bone morphogenetic proteins，BMP9)属于TGF-β超家族成员，是目前已知具最强成骨能力的骨形态发生蛋白 [6, 7, 8, 9]。BMP9主要通过经典的Smad信号通路和非经典的MAPK信号通路促进间充质干细胞成骨分化[10]。但是microRNA参与BMP9调控间充质干细胞成骨分化鲜有报道[11]，miR-21参与BMP9调控间充质干细胞成骨分化目前还未见报道。更有意义的是，BMP/Smad信号通路中重要的Smad蛋白可以通过影响Drosha蛋白中P68的组装来影响成熟miR-21的表达[12]。因此，BMP9与miR-21的关系，miR-21是否参与BMP9诱导间充质干细胞成骨分化，miR-21是否影响BMP9/Smad经典信号通路是本文探讨的重点。这为临床治疗骨缺损疾病提供了一定的理论依据。

1 材料与方法 1.1 细胞及重组腺病毒

小鼠C3H10T1/2 细胞和人结肠癌细胞株HCT116购于ATCC公司；重组腺病毒Ad-GFP、Ad-BMP9(同时表达GFP)由本实验室前期构建保存。本实验所用的腺病毒是由穿梭质粒和骨架质粒一起在HEK-293细胞中包装而成，Ad-BMP9这一腺病毒的穿梭质粒pAdTRACK上插入了BMP9的CDS区，而pAdTRACK这个载体前期掺入了一个表达GFP标记的独立表达框以便于评估腺病毒的转导效率。因此，当细胞感染Ad-BMP9时，我们可以用荧光显微镜观察绿色荧光来了解病毒的感染情况[13]。

1.2 miR-21模拟物及其阴性对照物

MiR-21模拟物(miR-21)及其阴性对照物(NC)购于上海吉玛制药技术有限公司，序列信息见表 1。

表 1 模拟物信息(小鼠) Table 1 Mimic sequence(Mus musculus)
Mimic nameMimic sequence(5′~3′)
mmu-miR-21 mimic(miR-21)UAGCUUAUCAGACUGAUGUUGA
mimics NC(NC)UUGUACUACACAAAAGUACUG
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1.3 材料及试剂

DMEM培养基和胎牛血清购于Hyclone公司；链霉素、青霉素、茜素红S染料、维生素C和β-磷酸甘油购于Sigma公司；转染试剂EntransterTM-R4000购于北京英格恩生物科技有限公司；碱性磷酸酶(ALP)定量及活性试剂盒购于BD公司；RNA提取试剂Trizol购于Invitrogen公司；M-MLV逆转录酶，RT-PCR、Real-time PCR试剂盒购于TaKaRa公司；PCR引物由上海百力格公司合成。P-smad1/5抗体购于Cell Signaling公司。Smad1/5/8抗体购于Santa Cruz公司。β-actin抗体和二抗购于北京中杉金桥生物技术有限公司。其他试剂均为进口分装或国产分析纯。

1.4 方 法 1.4.1 细胞培养

C3H10T1/2细胞和HCT116细胞培养于含有10%胎牛血清的DMEM高糖培养基中，其中含有100U/ml青霉素和100μg/ml链霉素,在37℃、5%CO2的饱和湿度培养箱中培养。

1.4.2 条件培养基的制备

接种HCT116细胞于60mm细胞培养皿中，细胞融合度达到60%~70%时加入适当滴度Ad-BMP9，6h后更换为无血清无双抗的DMEM高糖培养基培养，分别在24h、48h收集培养基并离心，4℃保存，以BMP9-CM作为标记，一周内用完。

1.4.3 ALP染色及活性测定

将C3H10T1/2细胞种入24孔板后，待细胞密度达到70%时加入不同的处理因素，培养C3H10T1/2细胞7d后，根据试剂盒说明书进行ALP活性测定。弃去培养基后加入0.1g/L色酚AS-MX(NaphtholAS-MX)碱性磷酸酶溶液和0.6 g/L 固牢蓝(Fast Blue RR Salt)混合液200μl进行ALP染色，避光30min后观察染色结果 。

1.4.4 茜素红S染色测定细胞钙盐沉积

C3H10T1/2细胞接种于24孔板，细胞融合度达到70%时加入不同的处理因素，6h后换液，同时加入工作浓度50μg/ml维生素C、10mmol/L的β-磷酸甘油，继续培养14d进行茜素红S染色：弃去孔板中的培养基，PBS洗3次后用0.05%戊二醛固定10 min，去离子水洗3次后加入0.4%茜素红S，染色5min，弃染液，去离子水终止反应并洗涤，显微镜下观察、成像。

1.4.5 PCR

六孔板培养C3H10T1/2细胞，细胞覆盖达到70%时加不同的处理因素，分别在适当时间点提取RNA,逆转录成cDNA进行RT-PCR(β-actin为内参)或Real-time PCR(microRNA检测U6为内参，一般基因检测β-actin为内参)。Real-time PCR数据分析采用比较CT法(ΔΔCT)，目的基因相对表达量=2-[(CT处理-CT内参)-(CT对照-CT内参)],所有数值取三次重复的平均值。所用引物序列见表 2。

表 2 引物信息(小鼠) Table 2 The sequence of Primer for PCR(Mus musculus)
GeneForward primer(5′to 3′)Reverse primer(5′to 3′)
miR-21TGGCGTAGCTTATCAGACTGAGTGCAGGGTCCGAGGT
U6CTCGCTTCGGCAGCACATATACTACGCTTCACGAATTTGCGTGTC
β-actinTGTTACCAACTGGGACGACAGGGGTGTTGAAGGTCTCAAA
BMP9GTACGCCTCCAACAGTCCTCCAGAACATCATAAACG
OCNTCTGACAAAGCCTTCATGTCCAAATAGTGATACCGTAGATGCG
ALPCCCCATGTGATGGCGTATCGGTAGGGAGAGCACAGC
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1.4.6 Western blot

细胞经不同处理因素处理后，用RIPA裂解液于冰上，离心取上清，BCA法检测上清总蛋白浓度，上清液加入适量load buffer煮沸10min，经过SDS-PAGE、转膜、封闭、孵一抗、洗膜、孵二抗、洗膜显影后，最后成像保存。

1.5 统计学分析

数据采用均数±标准差(x±s)表示，组间比较采用单因素方差分析，两组间比较采用t检验，统计所用软件为SPSS17.0，P＜0.5认为差异有统计学意义。

2 结 果 2.1 miR-21在BMP9诱导间充质干细胞C3H10T1/2的成骨分化中内源性表达暂时性降低

运用Ad-BMP9和Ad-GFP感染C3H10T1/2细胞5d(图 1a)。在Ad-BMP9组，ALP表达明显高于对照组(图 1b、1d)，表明Ad-BMP9诱导成骨分化成功。同时，在Ad-BMP9组，miR-21表达明显低于对照组(P＜0.01)(图 1c)。以上结果表明，在BMP9诱导C3H10T1/2的成骨分化的第5d，miR-21内源性表达暂时性降低。

图 1 miR-21在BMP9诱导C3H10T1/2细胞成骨分化中的表达 Fig. 1 The temporary downregulation of miR-21 during BMP9-induced osteogenic differentiation of C3H10 T1/2 cell (a)Ad-BMP9 and Ad-GFP can infect C3H10 T1/2 cell(×100) (b)Expression of ALP detected by RT-PCR (c)Expression of miR-21 detected by Real-time PCR. *** P<0.001 compared with Ad-GFP group,Blank group (d)The ALP mRNA expression were quantitated ** P<0.01 compared with Ad-GFP group; ** P<0.01 compared with Blank group
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2.2 在间充质干细胞C3H10T1/2中，过表达miR-21使得BMP9内源性表达降低

用miR-21寡核苷酸模拟物转染C3H10T1/2细胞24h，miR-21表达明显升高(图 2a)。在第1d和第5d检测BMP9的表达，过表达miR-21时，BMP9表达较对照组明显下降(图 2b,图 2c)。miR-21可以暂时降低BMP9的内源性表达。

图 2 过表达miR-21，BMP9在C3H10T1/2细胞中的表达变化 Fig. 2 The temporary downregulation of BMP9 mRNA by transferring miR-21 into C3H10 T1/2 cell (a)Expression of miR-21 detected by Real-time PCR *** P<0.001 compared with NC group (b)Expression of BMP9 at 1day detected by Real-time PCR ** P<0.01 compared with NC group (c)Expression of BMP9 at 5day detected by Real-time PCR * P<0.5 compared with NC group
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2.3 miR-21促进了BMP9诱导的C3H10T1/2细胞早期成骨分化

过表达miR-21后，再联用BMP9-CM处理C3H10T1/2细胞7天后，与对照组相比，ALP染色明显增强(图 3a，图 3b)，ALP活性升高(图 3c)。

图 3 miR-21促进了BMP9诱导的C3H10T1/2细胞早期成骨分化 Fig. 3 Overexpression miR-21 enhances BMP9-induced early osteogenic differentiantion of C3H10T1/2 cell (a)ALP staining results were recorded in higher magnifications(×100) (b) ALP staining results were recorded in lower magnifications (c)ALP activity determined by ALP quantitative assay * P<0.5 compared with NC+BMP9 group; ** P<0.01 compared with miR-21 group
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2.4 miR-21促进了BMP9诱导的C3H10T1/2细胞晚期成骨分化

过表达miR-21后，BMP9-CM处理C3H10T1/2细胞14d，钙盐沉积较对照组明显增加(图 4a，图 4b)。

图 4 miR-21促进了BMP9诱导的C3H10T1/2细胞晚期成骨分化 Fig. 4 Overexpression miR-21 enhances BMP9-induced later osteogenic differentiantion of C3H10T1/2 cell (a) Alizarin Red S staining results were recorded in higher magnifications(×100) Calcium precipitation was detected by Alizarin Red S staining (b) Alizarin Red S results staining were recorded in lower magnifications
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2.5 miR-21促进了BMP9诱导的成骨相关因子的表达

过表达miR-21后(图 5a)，联合BMP9-CM处理C3H10T1/2细胞5d，成骨相关因子ALP和OCN mRNA表达水平增高，单独高表达miR-21不能使ALP，OCN mRNA表达升高(图 5b)。

图 5 miR-21促进了BMP9诱导的成骨相关因子的表达 Fig. 5 BMP9-induced expression of osteogenesis related genes (a)Expression of miR-21 detected by Real-time PCR *** P<0.001 compared with NC+BMP9 group. Ns present no significant difference compared with miR-21 group (b) Expression of ALP and OCN detected by Real-time PCR * P<0.5,** P<0.01 compared with NC+BMP9 group ** P<0.01,* P<0.5 compared with miR-21 group
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2.6 miR-21对BMP9/Smad信号通路的影响

过表达miR-21，联用BMP9-CM处理C3H10T1/2细胞，p-Smad1/5表达升高(图 6a,图 6c)。单独高表达miR-21不能使p-Smad1/5表达升高(图 6b，图 6d)。

图 6 miR-21对BMP9/Smad信号通路的影响 Fig. 6 miR-21 heighten BMP9-Smad Signaling in C3H10T1/2 cell (a)and(b)The p-Smad1/5 protein expression was analyzed by Western blot;(c)and(d)The p-Smad1/5 protein expression were quantitated. ** P<0.01 compared with NC+BMP9 group. Ns present no significant difference compared with miR-21 group
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3 讨 论

骨缺损疾病，是以骨量下降、骨组织微结构退变为特征的一大类骨疾病，包括骨创伤、代谢性骨疾病、退化性骨疾病[9]。增加骨量，恢复骨组织微结构是治疗骨缺损疾病的关键。大量研究表明，BMP9是目前已知骨形态发生蛋白家族BMP2-BMP15中成骨能力最强者[6, 7, 8, 9]。BMP9是通过激活下游一系列信号分子发挥生物学效应，这些信号分子包括microRNAs。尽管已知BMP9具有极强成骨能力，但microRNA参与BMP9调控间充质干细胞成骨分化过程目前鲜有报道。最近研究表明，miR-21可以靶向DOCK家族调节成脂干细胞成脂分化[2]。MiR-21也可以通过靶向于Spry1，Spry2来激活MAPK-ERK信号通路，从而促进间充质干细胞成骨分化[3, 4]。这提示我们miR-21具有调节间充质干细胞分化的能力。结合以上两点，本课题重点阐述了miR-21与BMP9的相互关系，miR-21在BMP9诱导间充质干细胞C3H10T1/2细胞成骨分化中的作用，以及这一作用是否通过BMP9/Smad信号通路来实现。

BMP9可以暂时性降低miR-21的表达，miR-21也可以暂时性降低BMP9mRNA水平。这一结果说明BMP9与miR-21存在相互调控关系。而两者的相互调控可能对于维持骨形成平衡，防止过度成骨有着重要作用。为了更加明确两者之间的上下游关系，需要从浓度梯度和时间梯度两方面进行探讨miR-21与BMP9的调控关系[14]。 MiR-21协同BMP9增强了间充质干细胞C3H10T1/2细胞ALP以及钙盐沉积。同时，miR-21可以协同BMP9促进成骨分化相关因子ALP、OCN的表达。提示miR-21促进了BMP9诱导的间充质干细胞C3H10T1/2细胞成骨分化。这一过程可能与miR-21调节靶基因有关。结合生物信息学分析Target Scan(http://www.targetscan.org)，PicTar(http://pictar.bio.nyu.edu)和miRanda(http://www.microrna.org)，发现miR-21可以靶向BMP9/Smad信号通路中的抑制性Smad-Smad7。Smad7可以通过抑制p-Smad1/5/8从而减弱BMP9/Smad信号通路的激活[7, 15]。因此，本课题将后续探讨miR-21对Smad7是否存在转录后调控作用，并且通过双荧光素酶报告实验证明miR-21与Smad7是否存在直接靶向作用[14, 16]。

MiR-21的协同BMP9成骨效应与增强了BMP9/Smad信号通路的激活程度有关。BMP9/Smad信号通路的主要效应是由于BMP9与细胞膜上BMPⅡR、BMPΙR结合以后，激活胞浆中的Smad1/5/8使其磷酸化，p-Smad1/5/8入核与Smad4结合进而促进成骨相关转录因子的表达[17]。结合靶基因的预测，我们猜测miR-21极有可能靶向Smad7进而协同BMP9促进了BMP9/Smad信号通路的激活。因此，我们将对miR-21靶基因，靶基因在BMP9诱导间充质干细胞成骨分化中的效应以及动物实验深入分析miR-21在BMP9诱导间充质干细胞成骨分化中的作用。

参考文献
[1] Lian J B,Stein G S,van Wijnen A J,et al. MicroRNA control of bone formation and homeostasis. Nat Rev Endocrinol,2012,8(4):212-227.
[2] Kim Y J,Hwang S J,Bae Y C,et al. MiR-21 regulates adipogenic differentiation through the modulation of TGF-beta signaling in mesenchymal stem cells derived from human adipose tissue. Stem Cells,2009,27(12):3093-3102.
[3] Yang N,Wang G,Hu C,et al. Tumor necrosis factor α suppresses the mesenchymal stem cell osteogenesis promoter miR-21 in estrogen deficiency-induced osteoporosis. J Bone Miner Res,2013,28(3):559-573.
[4] 杨楠,周威,王光,等.Spry1在miR-21调控人骨髓间充质干细胞成骨分化中的作用.中国组织工程研究,2014,18(32):5085-5090. Yang N,Zhou W,Wang G,et al. Role of Spry1 in osteogenic differentiation of human bone marrow mesenchymal stem cells under miR-21 regulation. Chinese Journal of Tissue Engineenng Research,2014,18(32):5085-5090.
[5] Mei Y,Bian C,Li J,et al. miR-21 modulates the ERK-MAPK signaling pathway by regulating SPRY2 expression during human mesenchymal stem cell differentiation. J Cell Biochem,2013,114(6):1374-1384.
[6] Kang Q,Sun M H,Cheng H,et al. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther,2004,11(17):1312-1320.
[7] Lamplot J D,Qin J,Nan G,et al. BMP9 signaling in stem cell differentiation and osteogenesis. Am J Stem Cells,2013,2(1):1-21.
[8] Luther G,Wagner E R,Zhu G,et al. BMP-9 induced osteogenic differentiation of mesenchymal stem cells:molecular mechanism and therapeutic potential. Curr Gene Ther,2011,11(3):229-240.
[9] Cheng H,Jiang W,Phillips F M,et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins(BMPs). J Bone Joint Surg Am,2003,85-A(8):1544-1552.
[10] Davis B N,Hilyard A C,Lagna G,et al. SMAD proteins control DROSHA-mediated microRNA maturation. Nature,2008,454(7200):56-61.
[11] 李宝林,白慧丽,张汝益,等. miR-30a抑制BMP9诱导间充质干细胞C3H10T1/2的成骨分化. 中国生物工程杂志,2013,33(11):14-20. Li B L,Bai H L,Zhang R Y,et al.BMP9-induced osteogenic differentiationis partially inhibited by miR-30a in mesenchymal stem cells Line C3H10T1/2. China Biotechnology,2013,33(11):14-20.
[12] Wu J Y,Aarnisalo P,Bastepe M,et al. Gsa enhances commitment of mesenchymal progenitors to the osteoblast lineage but restrains osteoblast differentiation in mice. J Clin Invest,2011,121(9):3492-3504.
[13] Luo J,Deng Z L,Luo X,et al. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc,2007,2(5):1236-1247.
[14] Wang X,Guo B,Li Q,et al. miR-214 targets ATF4 to inhibit bone formation. Nat Med,2013,19(1):93-100.
[15] Liu Y,Liu Y,Zhang R,et al. All-trans retinoic acid modulates bone morphogenic protein 9-induced osteogenesis and adipogenesis of preadipocytes through BMP/Smad and Wnt/β-catenin signaling pathways. Int J Biochem Cell Biol,2014,47:47-56.
[16] Krichevsky A M,Gabriely G. miR-21:a small multi-faceted RNA. J Cell Mol Med,2009,13(1):39-53.
[17] Naznin Akhtar,Hossen Mohammad Jamil,Rajat Suvra Banik,et al.TGF-β/BMP signaling and other molecular events:regulation of osteoblastogenesis and bone formation. Bone Research,2015,3:15005.