[1]王最,徐佳妮,吴良邦,等.骨关节炎软骨损伤中miRNA-214的作用机制及靶向基因研究[J].中医正骨,2023,35(03):6-14,30.
　WANG Zui,XU Jiani,WU Liangbang,et al.Mechanism and targeted genes of miRNA-214 in osteoarthritis-induced cartilage injury[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2023,35(03):6-14,30.
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骨关节炎软骨损伤中miRNA-214的作用机制及靶向基因研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第35卷
期数:
2023年03期
页码:
6-14,30
栏目:
基础研究
出版日期:
2023-03-20

文章信息/Info

Title:
Mechanism and targeted genes of miRNA-214 in osteoarthritis-induced cartilage injury
作者:
王最1; 徐佳妮1; 吴良邦1; 钱钧2
(1.解放军联勤保障部队第九〇三医院,浙江 杭州 310004; 2.衢州市人民医院,浙江 衢州 324000)
Author(s):
WANG Zui1; XU Jiani1; WU Liangbang1; QIAN Jun2
1.The 903rd Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army,Hangzhou 310004,Zhejiang,China 2.Quzhou People's Hospital,Quzhou 324000,Zhejiang,China
关键词:
骨关节炎;  软骨疾病;  微RNAs;  基因表达;  大鼠;  动物实验
Keywords:
osteoarthritis;  cartilage diseases;  microRNAs;  gene expression;  rats;  animal experimentation
摘要:
目的:探讨骨关节炎(osteoarthritis,OA)软骨损伤中微小RNA(micro RNA,miRNA)-214的作用机制及靶向基因。方法:①OA患者膝关节损伤软骨组织中miRNA-214表达量检测。根据软骨损伤程度Outerbridge分级标准将收集的OA患者膝关节软骨组织进行分级,分别提取各软骨组织的总RNA,逆转录cDNA后采用实时定量PCR检测损伤软骨组织中miRNA-214的表达量。②miRNA-214在OA大鼠软骨损伤中的作用机制分析。采用手术剪断大鼠前交叉韧带的方法建立大鼠OA模型。将40只造模成功的大鼠随机分为miRNA-214高表达组、miRNA-214低表达组、阴性对照组及模型组,将10只接受手术但不剪断前交叉韧带的大鼠纳入假手术组。设计、合成miRNA-214模拟物、miRNA-214抑制剂及miRNA-214阴性对照序列,构建慢病毒表达载体,完成病毒包装。在miRNA-214高表达组、miRNA-214低表达组、阴性对照组、模型组及假手术组大鼠右侧膝关节腔内分别注射100 μL miRNA-214模拟物慢病毒混悬液、100 μL miRNA-214抑制剂慢病毒混悬液、100 μL miRNA-214阴性对照慢病毒混悬液、100 μL生理盐水、100 μL生理盐水。干预后4周,采集各组大鼠腹主动脉血,检测血清白细胞介素(interleukin,IL)-17、IL-23水平; 制备各组大鼠膝关节软骨组织石蜡切片,HE染色后观察软骨组织病理学改变; 采用免疫印迹法检测各组大鼠膝关节软骨组织中聚集蛋白聚糖、Ⅱ型胶原蛋白(collagenⅡ,ColⅡ)、基质金属蛋白酶(matrix metalloproteinase,MMP)-13的蛋白相对表达量。③OA软骨损伤相关的miRNA-214靶向基因分析。检索miRNA靶基因数据库中miRNA-214的靶向基因,根据文献资料筛选与骨代谢相关的人类miRNA-214靶向基因。采用双荧光素酶实验验证miRNA-214对活化转录因子4(activating transcription factor 4,ATF4)的靶向性。采用免疫印迹法检测各组大鼠右侧膝关节软骨组织中ATF4的蛋白相对表达量。结果:①OA患者膝关节损伤软骨组织中miRNA-214表达量检测结果。共收集38例OA患者的膝关节软骨组织,其中Ⅲ级28例、Ⅳ级10例。Ⅳ级损伤软骨组织的miRNA-214相对表达量高于Ⅲ级(1.67±0.40,0.51±0.16,t=12.941,P=0.000)。②大鼠血清炎症因子水平检测结果。5组大鼠血清IL-17、IL-23水平比较,组间差异有统计学意义[(62.94±8.12)pg·mL-1,(45.19±5.34)pg·mL-1,(44.63±6.67)pg·mL-1,(24.74±3.90)pg·mL-1,(18.15±2.33)pg·mL-1,F=94.153,P=0.000;(72.39±10.26)pg·mL-1,(48.70±5.87)pg·mL-1,(47.59±5.76)pg·mL-1,(27.54±4.05)pg·mL-1,(18.13±3.05)pg·mL-1,F=111.701,P=0.000]。miRNA-214 高表达组、阴性对照组、模型组及miRNA-214低表达组大鼠血清IL-17、IL-23水平均高于假手术组(P=0.000,P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000,P=0.000); miRNA-214高表达组大鼠血清IL-17、IL-23水平均高于阴性对照组、模型组及miRNA-214低表达组(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000); miRNA-214低表达组大鼠血清IL-17、IL-23水平均低于阴性对照组和模型组(P=0.000,P=0.000; P=0.000,P=0.000); 阴性对照组大鼠血清IL-17、IL-23水平与模型组比较,组间差异均无统计学意义(P=0.838,P=0.675)。③大鼠软骨组织病理学检查结果。HE染色结果显示,假手术组软骨组织潮线清晰,结构完整,软骨细胞分布均匀; 阴性对照组、模型组软骨组织潮线模糊不清,结构严重破坏,软骨细胞聚集; miRNA-214高表达组软骨组织损伤情况较阴性对照组、模型组更为严重; miRNA-214低表达组软骨组织潮线可辨识,结构较为完整,软骨细胞聚集现象较阴性对照组、模型组及miRNA-214高表达组有所改善。④大鼠软骨组织聚集蛋白聚糖、ColⅡ、MMP-13蛋白表达量检测结果。5组大鼠软骨组织聚集蛋白聚糖、ColⅡ、MMP-13蛋白表达量比较,组间差异均有统计学意义(0.08±0.02,0.14±0.03,0.15±0.04,0.31±0.08,0.72±0.12,F=142.932,P=0.000; 0.11±0.03,0.20±0.04,0.22±0.04,0.36±0.05,1.13±0.21,F=170.345,P=0.000; 1.32±0.34,0.95±0.13,0.95±0.12,0.21±0.04,0.11±0.03,F=91.486,P=0.000)。miRNA-214高表达组、阴性对照组、模型组及miRNA-214低表达组大鼠软骨组织聚集蛋白聚糖、ColⅡ蛋白表达量均低于假手术组(P=0.000,P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000,P=0.000),MMP-13蛋白表达量高于假手术组(P=0.000,P=0.000,P=0.000,P=0.000); miRNA-214高表达组大鼠软骨组织聚集蛋白聚糖、ColⅡ蛋白表达量均低于阴性对照组、模型组及miRNA-214低表达组(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),MMP-13蛋白表达量高于阴性对照组、模型组及miRNA-214低表达组(P=0.005,P=0.004,P=0.000); miRNA-214低表达组大鼠软骨组织聚集蛋白聚糖、ColⅡ蛋白表达量均高于阴性对照组和模型组(P=0.000,P=0.000; P=0.000,P=0.000),MMP-13蛋白表达量低于阴性对照组和模型组(P=0.000,P=0.000); 阴性对照组大鼠软骨组织聚集蛋白聚糖、ColⅡ、MMP-13蛋白表达量与模型组比较,组间差异均无统计学意义(P=0.535,P=0.278,P=1.000)。⑤miRNA-214靶向基因验证结果。在转染ATF4-WT-psiCHECK2质粒的293T细胞中,miRNA-214模拟物组的荧光酶相对活性小于miRNA-214阴性对照组(0.45±0.07,1.02±0.23,t=5.301,P=0.001); 在转染ATF4-MUT-psiCHECK2质粒的293T细胞中,miRNA-214模拟物组的荧光酶相对活性与miRNA-214阴性对照组比较,差异无统计学意义(1.05±0.19,1.03±0.18,t=0.171,P=0.869)。⑥大鼠软骨组织中ATF4蛋白表达量检测结果。5组大鼠软骨组织ATF4蛋白表达量比较,组间差异有统计学意义(0.11±0.03,0.17±0.03,0.18±0.04,0.31±0.05,0.79±0.11,F=213.111,P=0.000)。miRNA-214高表达组、阴性对照组、模型组及miRNA-214低表达组大鼠软骨组织ATF4蛋白表达量均低于假手术组(P=0.000,P=0.000,P=0.000,P=0.000); miRNA-214高表达组大鼠软骨组织ATF4蛋白表达量低于阴性对照组、模型组及miRNA-214低表达组(P=0.000,P=0.000,P=0.000); miRNA-214低表达组大鼠软骨组织ATF4蛋白表达量高于阴性对照组和模型组(P=0.000,P=0.000); 阴性对照组大鼠软骨组织ATF4蛋白表达量与模型组比较,组间差异无统计学意义(P=0.535)。结论:OA患者膝关节软骨损伤与软骨组织中miRNA-214的表达有关,抑制miRNA-214表达能够减轻大鼠膝关节炎症反应、减少软骨基质降解、促进软骨修复,miRNA-214的作用机制与其抑制IL-17、IL-23、MMP-13表达和促进聚集蛋白聚糖、ColⅡ、ATF4表达有关,ATF4可能是与OA软骨损伤相关的miRNA-214的靶基因之一。
Abstract:
Objective:To investigate the mechanism and targeted genes of micro RNA(miRNA)-214 in osteoarthritis(OA)-induced cartilage injury.Methods:①Detection of miRNA-214 expression in injured cartilage of the knee joint in patients with OA.The knee cartilage tissues of OA patients were graded according to the Outerbridge classification for cartilage injury.The total RNA of each cartilage tissue was extracted,and the expression of miRNA-214 in the injured cartilage was detected by real-time quantitative PCR after reverse transcription of cDNA.②Analysis of mechanism of miRNA-214 in cartilage injury in OA rats.The OA model was established in rats by the surgical cutting of the anterior cruciate ligament.Forty OA model rats were randomly divided into a miRNA-214 high expression group,a miRNA-214 low expression group,a negative control group,and a model group.Another 10 rats undergoing surgery without cutting the anterior cruciate ligament were assigned into the sham operation group.The sequences of miRNA-214 mimics,miRNA-214 inhibitors,and miRNA-214 negative control were designed and synthesized,and lentivirus expression vector was constructed,followed by lentiviral packaging.Rats in the miRNA-214 high expression group,the miRNA-214 low expression group,the negative control group,the model group,and the sham operation group were injected with 100 μL of miRNA-214 mimic lentivirus suspension,100 μL of miRNA-214 inhibitor lentivirus suspension,100 μL of miRNA-214 negative control lentivirus suspension,100 μL of normal saline,and 100 μL of normal saline,respectively,at the right knee cavity.At four weeks after the intervention,blood in the abdominal aorta of all rats was sampled and serum interleukin(IL)-17 and IL-23 levels were measured.Paraffin sections of knee cartilage tissues were prepared,and the pathological changes in cartilage tissues were observed following HE staining.The relative protein expression levels of aggrecan,collagenⅡ(ColⅡ),and matrix metalloproteinase(MMP)-13 in the knee cartilage tissues of rats in each group were determined by Western blot.③Analysis of miRNA-214 targeted genes associated with OA-induced cartilage injury.The targeted genes of miRNA-214 were retrieved from the miRNA targeted gene database,and the human miRNA-214 targeted genes related to bone metabolism were screened according to the literature.The dual luciferase assay was employed to verify the targeting of miRNA-214 on activating transcription factor 4(ATF4).The relative protein expression of ATF4 in the right knee cartilage tissues of rats in each group was detected by Western blot.Results:①Detection results of miRNA-214 expression in injured cartilage tissues of knee joint in OA patients.Thirty-eight patients with OA were enrolled,including 28 with gradeⅢand 10 with gradeⅣ.The relative expression level of miRNA-214 in gradeⅣinjured cartilage tissues was higher than that in gradeⅢinjured cartilage tissues(1.67±0.40 vs 0.51±0.16,t=12.941,P=0.000).②Detection results of serum inflammatory factors in rats.The serum levels of IL-17 and IL-23 in the five groups were statistically significant(62.94±8.12,45.19±5.34,44.63±6.67,24.74±3.90,18.15±2.33 pg/mL,F=94.153,P=0.000; 72.39±10.26,48.70±5.87,47.59±5.76,27.54±4.05,18.13±3.05 pg/mL,F=111.701,P=0.000).Serum levels of IL-17 and IL-23 in the miRNA-214 high expression group,the negative control group,the model group,and the miRNA-214 low expression group were higher than those in the sham operation group(P=0.000,P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000,P=0.000).Serum levels of IL-17 and IL-23 in the miRNA-214 high expression group were higher than those in the negative control group,the model group,and the miRNA-214 low expression group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000).Serum levels of IL-17 and IL-23 in the miRNA-214 low expression group were lower than those in the negative control group and the model group(P=0.000,P=0.000; P=0.000,P=0.000).There was no significant difference in serum levels of IL-17 and IL-23 between the negative control group and the model group(P=0.838,P=0.675).③Pathological examination results of cartilage tissues.As revealed by HE staining,the sham operation group showed clear tide mark of cartilage tissues with intact structure and uniform distribution of chondrocytes,and the negative control group and the model group displayed blurred tide mark of cartilage tissues with seriously damaged structure and aggregated chondrocytes.The cartilage injury in the miRNA-214 high expression group was severer than that in the negative control group and the model group.The miRNA-214 low expression group exhibited distinguishable tide mark of cartilage tissues with comparatively intact structure,and chondrocyte aggregation was improved compared with the conditions in the negative control group,the model group,and the miRNA-214 high expression group.④Detection results of protein expression levels of aggrecan,ColⅡ,and MMP-13 in rat cartilage tissues.There were significant differences in protein expression levels of aggrecan,ColⅡ,and MMP-13 in cartilage tissues of rats between the five groups(0.08±0.02,0.14±0.03,0.15±0.04,0.31±0.08,0.72±0.12,F=142.932,P=0.000; 0.11±0.03,0.20±0.04,0.22±0.04,0.36±0.05,1.13±0.21,F=170.345,P=0.000; 1.32±0.34,0.95±0.13,0.95±0.12,0.21±0.04,0.11±0.03,F=91.486,P=0.000).The protein expression levels of aggrecan and ColⅡin the miRNA-214 high expression group,the negative control group,the model group,and the miRNA-214 low expression group were lower than those in the sham operation group(P=0.000,P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000,P=0.000),and the protein expression levels of MMP-13 were higher than that in the sham operation group(P=0.000,P=0.000,P=0.000,P=0.000).The protein expression levels of aggrecan and ColⅡin the miRNA-214 high expression group were lower than those in the negative control group,the model group,and the miRNA-214 low expression group(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),and the protein expression level of MMP-13 was higher than that in the negative control group,the model group,and the miRNA-214 low expression group(P=0.005,P=0.004,P=0.000).The protein expression levels of aggrecan and ColⅡin the miRNA-214 low expression group were higher than those in the negative control group and the model group(P=0.000,P=0.000; P=0.000,P=0.000),and the protein expression level of MMP-13 was lower than that in the negative control group and the model group(P=0.000,P=0.000).There was no significant difference in the protein expression levels of aggrecan,ColⅡ,and MMP-13 in cartilage tissues of rats between the negative control group and the model group(P=0.535,P=0.278,P=1.000).⑤Results of miRNA-214 targeted gene verification.In 293T cells transfected with ATF4-WT-psiCHECK2 plasmid,the relative activity of luciferase in the miRNA-214 mimics group was lower than that in the miRNA-214 negative control group(0.45±0.07 vs 1.02±0.23,t=5.301,P=0.001).In 293T cells transfected with ATF4-MUT-psiCHECK2 plasmid,there was no significant difference in the relative activity of luciferase between the miRNA-214 mimics group and the miRNA-214 negative control group(1.05±0.19 vs 1.03±0.18,t=0.171,P=0.869).⑥Detection results of ATF4 protein expression in rat cartilage tissues.The protein expression of ATF4 in the cartilage tissues of rats in the five groups was statistically significant(0.11±0.03,0.17±0.03,0.18±0.04,0.31±0.05,0.79±0.11,F=213.111,P=0.000).The protein expression level of ATF4 in cartilage tissues of rats in the miRNA-214 high expression group,the negative control group,the model group,and the miRNA-214 low expression group were lower than that in the sham operation group(P=0.000,P=0.000,P=0.000,P=0.000).The protein expression level of ATF4 in cartilage tissues of rats in the miRNA-214 high expression group was lower than that in the negative control group,the model group,and the miRNA-214 low expression group(P=0.000,P=0.000,P=0.000).The protein expression level of ATF4 in cartilage tissues of rats in the miRNA-214 low expression group was higher than that in the negative control group and the model group(P=0.000,P=0.000).There was no significant difference in ATF4 protein expression between the negative control group and the model group(P=0.535).Conclusion:Cartilage injury of knee joint in OA patients is related to the expression of miRNA-214 in cartilage tissues,and the inhibition of miRNA-214 expression can relieve knee inflammatory response,reduce cartilage stromal breakdown,and promote cartilage repair in rats.The underlying mechanism of miRNA-214 is attributed to the inhibition of the expression of IL-17,IL-23,and MMP-13 and the promotion of the expression of aggrecan,ColⅡ,and ATF4.ATF4 is potentially one of the targeted genes of miRNA-214 associated with OA-induced cartilage injury.

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更新日期/Last Update: 1900-01-01