摘要
目的探讨hsa_circ_0001859靶向miR-16抑制类风湿关节炎(RA)滑膜成纤维细胞(RASFs)增殖和转移的机制研究。方法选取2019年6月-2020年7月于成都八一骨科医院进行治疗的RA患者40例为RA组,同时选取本院体检中心健康志愿者40例为对照组。培养RASFs细胞,将si-NC、si-hsa_circ_0001859、pcDNA-NC、pcDNA-hsa_circ_0001859、miR-NC、miR-16 mimics、si-hsa_circ_0001859+anti-miR-NC、si-hsa_circ_0001859+anti-miR-16分别转染至RASFs,记为si-NC组、si-hsa_circ_0001859组、pcDNA-NC组、pcDNA-hsa_circ_0001859组、miR-NC组、miR-16组、si-hsa_circ_0001859+anti-miR-NC组、si-hsa_circ_0001859+anti-miR-16组。RT-qPCR检测hsa_circ_0001859、miR-16表达水平;四甲基偶氮唑蓝(MTT)检测细胞活性;Transwell检测细胞增殖、迁移和侵袭;Western blot法检测细胞周期蛋白D1(Cyclin D1)、基质金属蛋白酶-2(MMP-2)、MMP-9蛋白表达;双荧光素酶报告实验检测hsa_circ_0001859和miR-16的靶向关系。结果RA组织中hsa_circ_0001859表达水平(2.66±0.36)较对照组(1.00±0.13)明显升高,miR-16表达水平(0.40±0.04)较对照组(1.00±0.15)明显降低,差异均有统计学意义(P均<0.05)。si-hsa_circ_0001859组hsa_circ_0001859表达水平、细胞存活率、细胞迁移和侵袭数量、Cyclin D1、MMP-2、MMP-9蛋白表达[(0.38±0.03)、(46.93±6.39)%、(109.38±17.39)个、(138.95±35.73)个、(0.37±0.03)、(0.40±0.04)、(0.38±0.03)]较si-NC组[(1.00±0.10)、(98.54±9.35)%、(247.98±35.73)个、(223.94±22.48)个、(0.90±0.09)、(0.90±0.08)、(0.89±0.08)]降低,差异均有统计学意义(P均<0.05)。miR-16组miR-16表达水平[(2.57±0.25)]较miR-NC组[(1.02±0.11)]升高,细胞存活率、细胞迁移及侵袭数量、Cyclin D1、MMP-2、MMP-9蛋白表达[(48.48±5.11)%、(126.94±11.14)个、(118.40±12.23)个、(0.35±0.05)、(0.41±0.04)、(0.38±0.03)]较miR-NC组[(99.98±10.56)%、(284.59±33.68)个、(249.47±25.78)个、(0.89±0.08)、(0.92±0.09)、(0.89±0.08)]降低,差异均有统计学意义(P均<0.05)。miR-16 mimics组WT-hsa_circ_0001859的相对荧光素酶活性[(0.40±0.04)]较miR-NC组[(1.00±0.10)]降低,差异有统计学意义(P<0.05)。si-hsa_circ_0001859组miR-16表达水平[(2.75±0.31)]较si-NC组[(1.00±0.10)]升高,pcDNA-hsa_circ_0001859组miR-16表达水平[(0.39±0.03)]较pcDNA-NC组[(1.00±0.09)]降低,差异均有统计学意义(P均<0.05)。si-hsa_circ_0001859+anti-miR-16组miR-16表达水平[(0.36±0.04)]较si-hsa_circ_0001859+anti-miR-NC组[(1.01±0.11)]降低,细胞存活率、细胞迁移和侵袭数量、Cyclin D1、MMP-2、MMP-9蛋白表达[(98.32±9.11)%、(255.76±30.03)、(279.74±33.92)、(0.90±0.09)、(0.91±0.08)、(0.88±0.08)]较si-hsa_circ_0001859+anti-miR-NC组[(44.87±5.39)%、(98.58±8.21)、(102.46±14.79)、(0.37±0.03)、(0.39±0.03)、(0.35±0.03)]升高,差异均有统计学意义(P均<0.05)。结论抑制hsa_circ_0001859靶向提高miR-16表达,抑制RASFs增殖和转移。
Objective To study the mechanism of hsa_circ_0001859 inhibiting the proliferation and metastasis of synovial fibroblasts(RASFs)of rheumatoid arthritis(RA)by targeting miR-16.Methods Forty patients with RA who received treatment in Chengdu Bayi Orthopedic Hospital from June 2019 to July 2020 were selected as the RA group,and 40 healthy volunteers from the physical examination center of our hospital were selected as the control group.RASFs cells were cultured and si-NC,si-hsa_circ_0001859,pcDNA-NC,pcDNA-hsa_circ_0001859,miR-NC,miR-16 mimics,si-hsa_circ_0001859+anti-miR-NC,si-hsa_circ_0001859+anti-miR-16 were transfected into RASFs.They were recorded as si-NC group,si-hsa_circ_0001859 group,pcDNA-NC group,pcDNA-hsa_circ_0001859 group,miR-NC group,miR-16 group,si-hsa_circ_0001859+anti-miR-NC group,si-hsa_circ_0001859+anti-miR-16 group.RT-qPCR was used to detect the expression levels of hsa_circ_0001859 and miR-16.Cell viability was detected by methyl thiazolyl tetrazolium(MTT).Cell migration and invasion were detected by Transwell.The expression of Cyclin D1,matrix metalloproteinase-2(MMP-2)and MMP-9 proteins were detected by Western blot assay.Dual luciferase reporter assay was used to detect the targeting relationship between hsa_circ_0001859 and miR-16.Results The expression level of hsa_circ_0001859 in RA tissue(2.66±0.36)was significantly up-regulated compared with that in control group(1.00±0.13),and the expression level of miR-16(0.40±0.04)was significantly down-regulated compared with that in control group(1.00±0.15),the differences were statistically significant(all P<0.05).The expression level of hsa_circ_0001859,cell survival rate,number of cell migration and invasion,and the expressions of Cyclin D1,MMP-2 and MMP-9 proteins in si-hsa_circ_0001859 group[(0.38±0.03),(46.93±6.39)%,(109.38±17.39),(138.95±35.73),(0.37±0.03),(0.40±0.04),(0.38±0.03)]were lower than those in si-NC group[(1.00±0.10),(98.54±9.35)%,(247.98±35.73),(223.94±22.48),(0.90±0.09),(0.90±0.08),(0.89±0.08)],and the differences were statistically significant(all P<0.05).The expression level of miR-16 in miR-16 group(2.57±0.25)was higher than that in miR-NC group(1.02±0.11);cell survival rate,cell migration and invasion number,the expressions of Cyclin D1,MMP-2 and MMP-9 proteins in the miR-16 group[(48.48±5.11)%,(126.94±11.14),(118.40±12.23),(0.35±0.05),(0.41±0.04),(0.38±0.03)]were higher than those of miR-NC group[(99.98±10.56)%,(284.59±33.68),(249.47±25.78),(0.89±0.08),(0.92±0.09),(0.89±0.08)],and the differences were statistically significant(all P<0.05).The relative luciferase activity of WT-hsa_circ_0001859 in miR-16 mimics group(0.40±0.04)was lower than that in miR-NC group(1.00±0.10),and the difference was statistically significant(P<0.05).The expression level of miR-16 in si-hsa_circ_0001859 group(2.75±0.31)was higher than that in si-NC group(1.00±0.10);the expression level of miR-16 in pcDNA-hsa_circ_0001859 group(0.39±0.03)was lower than that in pcDNA-NC group(1.00±0.09),and the differences were statistically significant(all P<0.05).The expression level of miR-16 in si-hsa_circ_0001859+anti-miR-16 group(0.36±0.04)was lower than that in si-hsa_circ_0001859+anti-miR-NC group(1.01±0.11);cell survival rate,number of cell migration and invasion,the expression of Cyclin D1,MMP-2 and MMP-9 proteins in si-hsa_circ_0001859+anti-miR-16 group[(98.32±9.11)%,(255.76±30.03),(279.74±33.92),(0.90±0.09),(0.91±0.08),(0.88±0.08)]were higher than those of si-hsa_circ_0001859+anti-miR-NC group[(44.87±5.39)%,(98.58±8.21),(102.46±14.79),(0.37±0.03),(0.39±0.03),(0.35±0.03)],and the differences were statistically significant(all P<0.05).Conclusion Inhibition of hsa_circ_0001859 targeted to increase miR-16 expression and inhibit proliferation and metastasis of RASFs.
作者
邓明涛
郑雷锋
黄拯
DENG Mingtao;ZHENG Leifeng;HUANG Zheng(Department of Spine and Joint,Chengdu Bayi Orthopedic Hospital,Chengdu,Sichuan 610031,China)
出处
《热带医学杂志》
CAS
2023年第9期1223-1227,1344,共6页
Journal of Tropical Medicine
基金
四川省中医药管理局科研项目(2018BY013)
