摘要
目的:探讨CircPTTG1IP调控miR-223-3p/SMAD3轴对类风湿关节炎(RA)成纤维样滑膜(FLS)细胞增殖、迁移和炎症反应的影响。方法:实时荧光定量聚合酶链反应(qRT-PCR)、蛋白质印迹法(Western blot)检测正常FLS细胞和RA-FLS细胞中CircPTTG1IP、miR-223-3p表达以及SMAD3蛋白表达;双荧光素酶实验验证Circ-PTTG1IP与miR-223-3p、miR-223-3p与SMAD3的关系。将RA-FLS细胞分为RA-FLS组、si-NC组、si-PTTG1IP组、si-PTTG1IP+anti-NC组、si-PTTG1IP+anti-miR-223-3p组,qRT-PCR检测RA-FLS中Circ-PTTG1IP、miR-223-3p表达;CCK8检测RA-FLS增殖情况;流式细胞术检测RA-FLS凋亡率;Transwell实验检测RA-FLS细胞迁移及侵袭情况;Western blot检测SMAD3、EMT相关蛋白表达;ELISA法检测IL-2、IL-6水平。结果:与FLS组比较,RA-FLS组CircPTTG1IP、SMAD3上调,miR-223-3p下调;与RA-FLS组、si-NC组比较,si-PTTG1IP组OD值,RA-FLS细胞迁移、侵袭数量,Circ-PTTG1IP、SMAD3、N-cadherin、Vimentin蛋白表达,IL-6、MMP-2水平均显著降低(P<0.05),miR-223-3p表达、细胞凋亡率、E-cadherin蛋白表达以及IL-2水平均显著升高(P<0.05);miR-223-3p沉默减轻了上述指标的变化;Circ-PTTG1IP靶向调控miR-223-3p/SMAD3轴。结论:沉默Circ-PTTG1IP可能通过上调miR-223-3p来抑制SMAD3表达,进而抑制RA-FLS增殖、迁移和炎症反应。
Objective:To investigate the impacts of CircPTTG1IP on the proliferation,migration and inflammatory response of fibroblast like synovial(FLS)cells in rheumatoid arthritis(RA)by regulating miR-223-3p/SMAD3 axis.Methods:Real-time quantitative fluorescence polymerase chain reaction(qRT-PCR)and western blot were used to detect the expression of CircPTTG1IP,miR-223-3p and SMAD3 protein in normal FLS cells and RA-FLS cells.The relationships between Circ-PTTG1IP and miR-223-3p,miR-223-3p and SMAD3 were verified by dual luciferase assay.RA-FLS cells was divided into RA-FLS group,si-NC group,si-PTTG1IP group,si-PTTG1IP+anti-NC group,and si-PTTG1IP+anti-miR-223-3p group.The expression of CircPTTG1IP and miR-223-3p in RA-FLS was detected by qRT-PCR;CCK8 was used to detect the proliferation of RA-FLS cells;flow cytometry was used to detect the apoptosis rate of RA-FLS cells;Transwell assay was used to detect migration and invasion of RAFLS cells;Western blot was used to detect the expression of SMAD3 and EMT related proteins;ELISA was used to detect the levels of IL-2 and IL-6.Results:Compared with FLS group,CircPTTG1IP and SMAD3 were upregulated,and miR-223-3p was down-regulated in RA-FLS group;Compared with RA-FLS group and si-NC group,the OD value,the numbers of RA-FLS cells migration and invasion,CircPTTG1IP,SMAD3,N-cadherin,Vimentin protein expression,IL-6 and MMP-2 levels in si-PTTG1IP group were significantly decreased(P<0.05),the expression of miR-223-3p,apoptosis rate,the protein expression of E-cadherin and the level of IL-2 were significantly increased(P<0.05);miR-223-3p silencing alleviated the changes of the above indicators;CircPTTG1IP targeted and regulated the miR-223-3p/SMAD3 axis.Conclusion:Silencing CircPTTG1IP may inhibit the expression of SMAD3 by up-regulating miR-223-3p,thereby inhibiting the proliferation,migration and inflammatory response of RA-FLS.
作者
冷冬月
方兴刚
李旭峰
LENG Dongyue;FANG Xinggang;LI Xufeng(Department of Integrated Traditional Chinese and Western Medicine,Taihe Hospital(Affiliated Hospital of Hubei University of Medicine),Shiyan 442000,China;Department of Traditional Chinese Medicine,Shiyan People's Hospital,Shiyan 442000,China)
出处
《现代医学》
2023年第4期454-461,共8页
Modern Medical Journal
基金
湖北省卫生健康委2021—2022年度科研项目(WJ2021F032)。
