miR⁃431⁃5p靶向Smad4影响人牙髓干细胞增殖、分化的实验研究

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

目的:探讨miR⁃431⁃5p对人牙髓干细胞(DPSC)增殖、分化的调控作用及机制。方法:收集人健康第三磨牙,分离、培养DPSC,采用双荧光素酶报告基因实验验证miR⁃431⁃5p与野生型Smad4和突变型Smad4靶向关系。实验分成mimic⁃NC、miR⁃431⁃5p mimic、pc⁃Smad4和pc⁃Smad4+miR⁃431⁃5p mimic组,各组DPSC均采用脂质体转染法进行细胞转染。CCK⁃8实验检测DPSC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检测成骨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,DPSC细胞的荧光素酶活性显著降低(P<0.01);而共转染miR⁃431⁃5p mimic和突变型Smad4报告基因载体后,DPSC的荧光素酶活性无显著改变(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,钙化结节染色较浅,ALP活性降低,OCN蛋白表达下调(P<0.05);pc⁃Smad4组细胞增殖能力降低,钙化结节染色加深,ALP活性增加,OCN蛋白表达上调(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,钙化结节染色较浅,ALP活性降低,OCN蛋白表达下调(P<0.05)。结论:miR⁃431⁃5p可通过靶向抑制Smad4蛋白表达抑制DPSC成骨分化并促进其增殖。

Objective:To explore the regulation effect and mechanism of miR⁃431⁃5p on the proliferation and differentiation of hu⁃man dental pulp stem cell(DPSC).Methods:The third molars of healthy people were collected to isolate and culture DPSC.The tar⁃geted relationship between miR⁃431⁃5p and Smad4 was verified by double luciferase reporter gene assay.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 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,the expression of miR⁃431⁃5p was up⁃regulated,the expression of Smad4 was down⁃regulated,the 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,the ability of cells proliferation was de⁃creased,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,the ability of cells proliferation was significantly increased,staining of calci⁃fied 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.