红景天苷通过miR-26a-5p靶向调控JAG1/Notch3信号通路改善瘢痕疙瘩

Salidroside improves keloid by targeting JAG1/Notch3 signaling pathway through miR-26a-5p

为探讨红景天苷通过miR-26a-5p靶向调控JAG1/Notch3信号通路对瘢痕疙瘩改善作用的确切机制,自2019年10月至2021年1月,收集武汉市中医医院10例经组织病理学检查确诊为瘢痕疙瘩的患者样本为研究对象,记为瘢痕疙瘩组,并采集相邻正常皮肤组织,记为正常组。分离上述原代瘢痕疙瘩成纤维细胞,将传至4~8代的细胞分为对照组(正常培养)、红景天苷低剂量组(10μmol/L红景天苷)、红景天苷中剂量组(20μmol/L)、红景天苷高剂量组(40μmol/L)、miR-NC组(40μmol/L红景天苷+miR-NC)、miR-26a-5p inhibitor组(40μmol/L红景天苷+miR-26a-5p inhibitor)、sh-RNA组(40μmol/L红景天苷+miR-26a-5p inhibitor+sh-RNA)和sh-JAG1组(40μmol/L红景天苷+miR-26a-5p inhibitor+sh-JAG1),各组细胞经转染后给予红景天苷或直接给予对应剂量的红景天苷处理24 h。qRT-PCR法检测成纤维细胞miR-26a-5p和JAG1的mRNA相对表达量;MTT法检测细胞的增殖活力;平板克隆实验检测细胞的克隆增殖能力;FACS检测细胞的凋亡情况;Transwell法检测细胞的迁移、侵袭能力;荧光素酶报告基因实验验证miR-26a-5p与JAG1的靶向互作关系;Western blotting检测瘢痕组织增生相关蛋白的表达。结果显示,与正常组相比,瘢痕疙瘩组miR-26a-5p表达显著下降(P<0.05),JAG1表达显著升高(P<0.05)。与0μmol/L组相比,10、20、40、80和160μmol/L红景天苷呈浓度依赖性地抑制瘢痕疙瘩成纤维细胞存活率(均P<0.05)。为保证瘢痕疙瘩成纤维细胞具有一定的存活率,并保证红景天苷的作用效果,后续使用10、20和40μmol/L浓度展开实验(经荧光素酶报告基因实验验证miR-26a-5p与JAG1存在结合序列)。与对照组相比,红景天苷低、中、高剂量组miR-26a-5p表达、细胞凋亡率均显著升高,JAG1表达、细胞克隆增殖数、迁移和侵袭数以及α-SMA、collagenⅠ、collagenⅢ、JAG1和Notch3蛋白表达量均显著降低(均P<0.05);与红景天苷高剂量组和miR-NC组相比,miR-26a-5p inhibitor组miR-26a-5p表达量和细胞凋亡率显著降低,而JAG1表达、细胞克隆数、迁移和侵袭数以及α-SMA、collagenⅠ、collagenⅢ、JAG1和Notch3蛋白表达量显著升高(均P<0.05);抑制JAG1表达可对细胞产生抑制增殖、迁移和侵袭作用,促进细胞凋亡。由此,瘢痕疙瘩组织中miR-26a-5p表达下调,JAG1表达上调,红景天苷可通过上调miR-26a-5p表达靶向抑制JAG1/Notch3信号通路,抑制瘢痕疙瘩成纤维细胞增殖、迁移和侵袭,促进细胞凋亡,达到改善瘢痕疙瘩的效果。

To investigate the mechanism by which salidroside improves keloid on regulating JAG1/Notch3 signaling pathway through miR-26a-5p,10 keloid patients diagnosed by histopathology in Wuhan Hospital of Traditional Chinese Medicine were enrolled as the research objects from October 2019 to January 2021.The keloid tissues removed by surgery were used in the keloid group,and the adjacent healthy skin tissues were used in the normal group.Primary keloid fibroblasts were isolated and cultured,and the cells of generation 4~8 were divided into 8 groups:control group(normal culture),salidroside low-dose group(10μmol/L salidroside),salidroside medium-dose group(20μmol/L salidroside),salidroside high-dose group(40μmol/L salidroside),miR-NC group(40μmol/L salidroside+miR-NC),miR-26a-5p inhibitor group(40μmol/L salidroside+miR-26a-5p inhibitor),sh-RNA group(40μmol/L salidroside+miR-26a-5p inhibitor+sh-RNA)and sh-JAG1 group(40μmol/L salidroside+miR-26a-5p inhibitor+sh-JAG1).Cells in each group were treated with salidroside for 24 h after transfection or directly treated with the corresponding dosage of salidroside for 24 h.The relative mRNA expressions of miR-26a-5p and JAG1 in tissues and cells were quantified using qRT-PCR.Cell proliferating viability was measured by the MTT assay and cell cloning capacity was detected using the plate cloning method.Cell apoptosis was detected by FACS and cell migration and invasion were assessed by Transwell method.Luciferase reporter system was utilized to examine the targeting interaction between miR-26a-5p and JAG1.Western blotting was used to detect the expressions of tissue hyperplasia-related proteins.The results showed that,compared to that of the normal group,the expression of miR-26a-5p in keloid group was significantly decreased(P<0.05),while the JAG1 expression significantly increased(P<0.05).Compared to the 0μmol/L salidroside group,10,20,40,80,and 160μmol/L salidroside treatment inhibited the survival rate of keloid fibroblasts in a concentration-dependent manner(all with P<0.05).To ensure both the survival of keloid fibroblasts and the effect of salidroside,subsequent experiments were conducted with salidroside concentrations of 10,20,and 40μmol/L(It was proved that miR-26a-5p combines to JAG1).In the three salidroside groups,compared to those of the control group,the expression of miR-26a-5p and the apoptosis rate increased dependently,while the expression of JAG1,the numbers of cell clones,cell migrations,and invasions,and the expression ofα-SMA,collagenⅠ,collagenⅢ,JAG1,and Notch3 proteins decreased significantly(all with P<0.05).Compared to those of the salidroside high-dose group and the miR-NC group,the expression of miR-26a-5p and the cell apoptosis rate in the miR-26a-5p inhibitor group reduced significantly,while the expression of JAG1,the numbers of cell clones,migration and invasion,and the expression ofα-SMA,collagenⅠ,collagenⅢ,JAG1 and Notch3 proteins increased significantly(all with P<0.05).Inhibition of JAG1 expression suppressed the effect of miR-26a-5p inhibitor on promoting cell proliferation,migration and invasion,and on inhibiting cell apoptosis.In conclusion,the expression of miR-26a-5p is down-regulated and JAG1 is up-regulated in keloid tissue,salidroside can inhibit the JAG1/Notch3 signaling pathway and the proliferation,migration and invasion of keloid fibroblasts,while promoting cell apoptosis,which in turn improves keloid by up-regulating the expression of miR-26a-5p.