红景天苷调节miR-26a-5p/JAG1/Notch-1信号轴对瘢痕疙瘩成纤维细胞生物学功能的影响

Influence of salidroside on the biological functions of keloid fibroblasts by regulating the miR-26a-5p/JAG1/Notch-1 axis

目的探讨红景天苷(SAL)调节微小RNA-26a-5p(miR-26a-5p)/JAG1/神经源性基因Notch同源蛋白1(Notch-1)信号轴对瘢痕疙瘩(KD)成纤维细胞(HKF)生物学功能的影响。方法将HKF细胞随机分为对照组(Control组)、SAL低浓度组(SAL-L组,50μmol/L SAL)、SAL高浓度组(SAL-H组,100μmol/L SAL)、SAL高浓度+miR-NC组(SAL-H+miR-NC组)、SAL高浓度+miR-26a-5p mimics组(SAL-H+miR-26a-5p mimics组)、SAL高浓度+NC-inhibitor组(SAL-H+NC-inhibitor组)、SAL高浓度+miR-26a-5p inhibitor组(SAL-H+miR-26a-5p inhibitor组)。CCK-8法检测细胞增殖;划痕实验检测细胞迁移;Transwell实验检测细胞侵袭;流式细胞术检测细胞凋亡与细胞周期;RT-qPCR检测各组细胞中的miR-26a-5p、JAG1和Notch-1 mRNA的表达;Western blot检测细胞中JAG1和Notch-1蛋白表达;双荧光素酶报告基因实验验证miR-26a-5p与JAG1的关系。结果与Control组相比,SAL-H组HKF细胞增殖能力(24 h)、迁移距离、侵袭细胞个数、S期细胞比例、JAG1和Notch-1 mRNA和蛋白表达显著减少(P<0.05),细胞凋亡率、G0/G1期细胞比例、miR-26a-5p表达显著增加(P<0.05);与SAL-H组和SAL-H+miR-NC组相比,SAL-H+miR-26a-5p mimics组相应指标变化与上述变化相同;miR-26a-5p inhibitor减弱了SAL-H对HKF细胞增殖、迁移、侵袭的抑制作用,减弱了HKF细胞凋亡。miR-26a-5p靶向负调控JAG1表达。结论SAL可能通过上调miR-26a-5p,靶向抑制JAG1/Notch-1信号轴抑制HKF细胞增殖、迁移、侵袭,促进细胞凋亡,改善KD。

Objective To explore the influence of salidroside(SAL)on the biological functions of fibroblasts(HKF)in keloid disease(KD)by the regulation of the microRNA-26a-5p(miR-26a-5p)/JAG1/neurogenic gene Notch homologous protein 1(Notch-1)axis.Methods HKF cells were treated with blank control(control group),50μmol/L SAL(SAL-L group),100μmol/L SAL(SAL-H group),100μmol/L SAL and transfection of miR-NC(SAL-H+miR-NC group),100μmol/L SAL and transfection of miR-26a-5p mimics(SAL-H+miR-26a-5p mimics group),100μmol/L SAL and transfection of NC inhibitor(SAL-H+NC inhibitor group)and 100μmol/L SAL and transfection of miR-26a-5p inhibitor(SAL-H+miR-26a-5p inhibitor group).Cell proliferation,migration and invasion were detected by Cell Counting Kit-8(CCK-8),wound healing and Transwell assay,respectively.Cell apoptosis and cell cycle progression were detected by flow cytometry.The mRNA levels of miR-26a-5p,JAG1 and Notch1 were measured by real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR).Protein levels of JAG1 and Notch1 were measured by Western blot.The target binding between miR-26a-5p and JAG1 was identified by dual-luciferase reporter assay.Results Compared with those of control group,significantly decreased proliferative rate at 24h,migratory distance,number of invasive cells and the proportion of HFK cells in S phase,downregulated JAG1 and Notch1,increased apoptotic rate and the proportion of HFK cells in G0/G1 phase,and upregulated miR-26a-5p were detected in SAL-H group(all P<0.05).The above indexes were similar between SAL-H group,SAL-H+miR-NC group and SAL-H+miR-26a-5p mimics group.Knockdown of miR-26a-5p attenuated the inhibitory effects of SAL-H on the proliferation,migration and invasion of HFK cells,which reduced cell apoptosis.MiR-26a-5p negatively regulated JAG1.Conclusion SAL protects the biological function of KD by inhibiting the proliferation,migration and invasion,and promoting cell apoptosis of HKF cells by targeting the JAG1/Notch-1 axis via upregulating miR-26a-5p.