miR-431-5p靶向Smad4影响人牙髓干细胞增殖、分化的实验研究

口腔生物医学 ›› 2022, Vol. 13 ›› Issue (4): 229-233.

miR-431-5p靶向Smad4影响人牙髓干细胞增殖、分化的实验研究

张建¹,吴建坤²,刘丽丽²,林秀雅²,刘学丽²

1. 沧州市中心医院
2. 沧州市中心医院口腔科

收稿日期:2022-07-01 修回日期:2022-08-29 出版日期:2022-12-25 发布日期:2022-12-24
通讯作者: 张建 E-mail:zhangjiancangzhou1@163.com
基金资助:
河北省沧州市重点研发计划自筹项目

Experimental study about the effects of miR-431-5p on the proliferation and differentiation of human dental pulp stem cells by targeting Smad4

Received:2022-07-01 Revised:2022-08-29 Online:2022-12-25 Published:2022-12-24

摘要/Abstract

摘要： 目的：探讨miR-431-5p对人牙髓干细胞（DPSCs）增殖、分化的调控作用及机制。方法：分别将miR-431-5p mimic（miR-431-5p mimic组）、Smad4过表达载体（pc-Smad4组）及二者共转染（pc-Smad4+miR-431-5p mimic组）DPSCs细胞。采用双荧光素酶报告基因实验验证miR-431-5p与Smad4靶向关系；qRT-PCR检测转染效率；CCK-8实验检测细胞增殖能力；茜素红染色观察钙化结节；碱性磷酸酶（ALP）染色试剂盒和ALP活性检测ALP表达及活性；Western blot检测成牙分化标志物骨钙蛋白（OCN）及Smad4蛋白表达。结果：共转染miR-431-5p mimic和野生型Smad4报告基因载体后，DPSCs细胞的荧光素酶活性显著降低（P<0.05）。与mimic-NC组比较，miR-431-5p mimic组miR-431-5p表达上调，Smad4表达下调，细胞增殖能力增加，钙化结节数目减少，ALP活性降低，OCN蛋白表达下调（均P<0.05）；pc-Smad4组细胞增殖能力降低（P<0.05），钙化结节数目增加，ALP活性增加，OCN蛋白表达上调（均P<0.05）。与pc-Smad4组比较，pc-Smad4+miR-431-5p mimic组细胞增殖能力显著增加，钙化结节数目减少，ALP活性降低，OCN蛋白表达下调（均P<0.05）。结论：miR-431-5p可通过靶向抑制Smad4蛋白表达抑制DPSCs成牙分化并促进其增殖。

关键词: 牙髓干细胞, miR-431-5p, Smad4, 增殖, 成骨分化

Abstract: Objective:To explore the regulation effect and mechanism of miR-431-5p on the proliferation and differentiation of human dental pulp stem cells (DPSC). Methods:The third molars of healthy people were collected to isolate and culture DPSC. In the experiments, mimic-NC group, miR-431-5p mimic group, pc-Smad4 group and pc-Smad4+miR-431-5p mimic group were set up. DPSC were transfected with lipid plastid transfection. The targeted relationship between miR-431-5p and Smad4 was verified by double luciferase reporter gene assay. The cells proliferation was detected by CCK-8. The calcified nodules were observed by alizarin red staining. The expression and activity of alkaline phosphatase (ALP) were detected by ALP staining kits and ALP activity test. The expressions of osteogenic differentiation marker osteocalcin (OCN) and Smad4 were detected by Western blot. Results:After co-transfection of miR-431-5p mimic and wild-type Smad4 reporter gene vector, luciferase activity of DPSC was significantly reduced (P<0.01), while which was not significantly changed after co-transfection of miR-431-5p mimic and mutant Smad4 reporter gene vector (P>0.05). Compared with mimic-NC group, expression of miR-431-5p was up-regulated, expression of Smad4 was down-regulated, ability of cells proliferation was increased, staining of calcified nodules was light, ALP activity was reduced, and expression of OCN protein was down-regulated in miR-431-5p mimic group (P<0.05). Compared with mimic-NC group, ability of cells proliferation was decreased, staining of calcified nodules was deepen, ALP activity was increased, and the expression of OCN was up-regulated in pc-Smad4 group (P<0.05). Compared with pc-Smad4 group, ability of cells proliferation was significantly increased, staining of calcified nodules was light, ALP activity was decreased, and the expression of OCN protein was down-regulated in pc-Smad4+miR-431-5p mimic group (P<0.05). Conclusions:The miR-431-5p can inhibit osteogenic differentiation of DPSC and promote their proliferation by inhibiting the expression of Smad4 protein.

Key words: dental pulp stem cell, miR-431-5p, Smad4, proliferation, osteogenic differentiation

张建吴建坤刘丽丽林秀雅刘学丽. miR-431-5p靶向Smad4影响人牙髓干细胞增殖、分化的实验研究[J]. 口腔生物医学, 2022, 13(4): 229-233.

参考文献 13

[1]	赵振宇, 李影, 王港, 等. p38丝裂原活化蛋白激酶对人乳牙牙髓干细胞成骨分化能力的影响[J]. 中华老年口腔医学杂志, 2019, 17(3):7-12.
[2]	孙帅, 李军. 骨形态发生蛋白调控牙髓干细胞成牙本质分化的研究进展[J]. 中华实用诊断与治疗杂志, 2020, 34(11):107-110.
[3]	Lu X , Chen X , Xing J , et al. miR-140-5p regulates the odontoblastic differentiation of dental pulp stem cells via the Wnt1/β-catenin signaling pathway[J]. Stem Cell Res Ther, 2019, 10(1):226.
[4]	Sun DG, Xin BC, Wu D, et al. miR‐140‐5p‐mediated regulation of the proliferation and differentiation of human dental pulp stem cells occurs through the lipopolysaccharide/toll‐like receptor 4 signaling pathway[J]. Eur J Oral Sci, 2017, 125(6):419-425.
[5]	Qiu Z , Lin S , Hu X , et al. Involvement of miR‐146a‐5p/neurogenic locus notch homolog protein 1 in the proliferation and differentiation of STRO‐1+ human dental pulp stem cells[J]. Eur J Oral Sci, 2019, 127(4):294-303.
[6]	吴韫慧, 郭磊, 蒋翠霞,等. 人牙髓干细胞增殖分化过程中的miR-431[J]. 中国组织工程研究, 2019, 23(21):49-54.
[7]	Xu CM , Chen LX , Gao F , et al. MiR-431 suppresses proliferation and metastasis of lung cancer via down-regulating DDX5[J]. Eur Rev Med Pharmacol Sci, 2019, 23(2):699-707.
[8]	Wang Y , Zhang K , Yuan X , et al. miR-431-5p regulates cell proliferation and apoptosis in fibroblast-like synoviocytes in rheumatoid arthritis by targeting XIAP[J]. Arthritis Res Ther, 2020, 22(1):231.
[9]	姜力铭, 宋戈, 夏商, 等. 转化生长因子β1对人牙髓干细胞成骨分化作用研究[J]. 中国实用口腔科杂志, 2018, 11(9):4-7.
[10]	刘亚楠, 李祥伟, 孙宏晨,等. bFGF调控DPSCs迁移,增殖和分化的研究进展[J]. 口腔医学, 2017, 37(8):746-750.
[11]	李鹏, 张晓莉, 艾力麦尔丹·艾尼瓦尔,等. 骨形成蛋白-2与转化生长因子-β3对牙髓干细胞成骨向分化的对比研究[J]. 中华实用诊断与治疗杂志, 2019, 33(1):7-10.
[12]	Hu X, Zhong Y, Kong Y, et al. Lineage-specific exosomes promote the odontogenic differentiation of human dental pulp stem cells (DPSCs) through TGFβ1/smads signaling pathway via transfer of microRNAs[J]. Stem Cell Res Ther, 2019, 10(1):170.
[13]	木合塔尔·霍加. 转化生长因子β3促进兔牙髓干细胞成骨向分化机制的初步研究[J]. 口腔医学, 2019, 39(2):97-102.

[1]	陈安琪吴清霖吴勇敏林媛媛赖颖真. PLA/PVP-GEL复合支架对MC3T3-E1细胞生物学行为的影响及成骨性能的初探[J]. 口腔生物医学, 2022, 13(4): 234-240.
[2]	博梦王林孙晓琳. 自产氧抗菌光动力纳米体系对炎症调节和成骨分化的影响[J]. 口腔生物医学, 2022, 13(4): 208-213.
[3]	吴桐洪高英周齐悦陈晨谢海峰. 氧化锆表面接枝绿原酸-壳聚糖复合物促成骨效果[J]. 口腔生物医学, 2022, 13(3): 142-147.
[4]	潘新华李烿烿叶露露朱国培. 全反式维甲酸联合阿帕替尼抑制口腔鳞癌细胞增殖体外研究[J]. 口腔生物医学, 2022, 13(1): 15-19.
[5]	徐瑞王锐高雪峰郭雨婷吕学超. 黄精多糖对乳牙牙髓干细胞增殖及成骨向分化的影响[J]. 口腔生物医学, 2022, 13(1): 40-44.
[6]	方媛春孙颖胡永. 具核梭杆菌感染的巨噬细胞对人舌鳞癌细胞系Cal-27细胞增殖与迁移的影响[J]. 口腔生物医学, 2021, 12(3): 166-169.
[7]	王桂芳蒋欣泉. 掺镁微弧氧化钛表面促进大鼠BMSCs成骨分化的研究[J]. 口腔生物医学, 2021, 12(2): 76-81.
[8]	史凡黄鉴栎刘梅王丹章非敏. 碳点示踪并促进大鼠骨髓间充质干细胞成骨分化的初步研究[J]. 口腔生物医学, 2021, 12(1): 5-11.
[9]	徐晓旭洪蕾蕾袁礼婵王晓亮马俊青. 二氧化钛纳米管负载京尼平交联的壳聚糖水凝胶体外生物活性的研究[J]. , 2020, 11(3): 164-167.
[10]	苏晓霞雷凤珍王芮林亭廷王菲廖立. 炎症微环境对于牙周膜干细胞与脐带间充质干细胞再生功能的影响[J]. , 2020, 11(2): 71-75.
[11]	范瑞瑞李欣聪刘玉孙卫斌郭婷. 聚己内酯/Ⅰ型胶原/氟磷灰石复合支架的制备及生物相容性研究[J]. , 2019, 10(1): 12-16.
[12]	石安源鲍东昱童昕秦海燕王斌. SB431542对人牙龈间充质干细胞体外成骨分化的影响[J]. , 2019, 10(1): 6-11.
[13]	杨昊清范志朋. SFRP2对脂肪干细胞成骨分化的作用研究[J]. , 2018, 9(3): 120-124.
[14]	王红魏福兰王春玲. miR-21研究进展[J]. , 2018, 9(2): 106-109.
[15]	周五超甘烟紫习伟宏. 多孔TiO2修饰钛植体对骨髓间充质干细胞粘附和成骨分化的影响[J]. , 2018, 9(2): 74-78.