miR-330-3p在颞下颌关节骨关节炎软骨退变中的作用机制研究

doi:10.19438/j.cjoms.2022.04.002

中国口腔颌面外科杂志 ›› 2022, Vol. 20 ›› Issue (4): 320-327.doi: 10.19438/j.cjoms.2022.04.002

miR-330-3p在颞下颌关节骨关节炎软骨退变中的作用机制研究

王楚瑶^1,2, 邹璐芗¹, 陆川^1,*, 何冬梅^1,*

1.上海交通大学医学院附属第九人民医院口腔外科,上海交通大学口腔医学院,国家口腔医学中心, 国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海 200011;
2.青岛大学口腔医学院,山东青岛 266000

收稿日期:2022-04-25 修回日期:2022-06-01 出版日期:2022-07-20 发布日期:2022-07-20
通讯作者: 陆川,E-mail: 1041123231@qq.com;何冬梅,E-mail: lucyhe119@163.com。^*共同通信作者
作者简介:王楚瑶（1995-）,女,在读硕士研究生,E-mail: 1795507855@qq.com
基金资助:
国家重点研发计划(2017YFC1103900,2020YFC2002800); 国家自然科学基金(32071313); 上海市科委科研项目(20S31902500,20Y11903900)

Study on the mechanism of miR-330-3p in cartilage degeneration in temporomandibular joint osteoarthritis in rats

WANG Chu-yao^1,2, ZOU Lu-xiang¹, LU Chuan¹, HE Dong-mei¹

1. Department of Oral Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology. Shanghai 200011;
2. School of Stomatology, Qingdao University. Qingdao 266000, Shandong Province, China

Received:2022-04-25 Revised:2022-06-01 Online:2022-07-20 Published:2022-07-20

摘要/Abstract

摘要： 目的: 探讨miR-330-3p在颞下颌关节骨关节炎（temporomandibular joint osteoarthritis, TMJOA）软骨退变中的作用机制。方法: 对SW1353软骨细胞系进行炎症及加压刺激,建立大鼠单侧前牙反（unilateral anterior crossbite, UAC）TMJOA模型,通过RT-qPCR检测体外及体内软骨OA情况下miR-330-3p的表达变化。miR-330-3p过表达或抑制后,利用EDU荧光染色以及蛋白免疫印迹(WB)检测SW1353软骨细胞的增殖及合成分解代谢变化。生物信息学分析预测并筛选miR-330-3p的下游靶点,并在炎症及加压刺激下通过RT-qPCR和WB明确miR-330-3p对其调控作用。在miR-330-3p大鼠UAC TMJOA模型的关节腔内注射后,采用免疫组织化学方法检测靶分子的表达及软骨代谢的变化,明确miR-330-3p对TMJOA的治疗作用。采用SPSS 22.0软件包对数据进行统计学分析。结果: 在体外炎症和加压刺激下,以及大鼠UAC的TMJOA模型中,miR-330-3p在软骨中的表达均下降。抑制miR-330-3p可导致软骨细胞增殖能力下降,SOX9合成降低,MMP13表达增加,而过表达的结果则相反。生物信息学分析发现CTNNB1为可能的下游靶点之一,其在体外炎症和加压刺激后表达升高。过表达CTNNB1后,软骨细胞SOX9蛋白表达下降,MMP13蛋白表达升高,抑制CTNNB1的结果则相反。miR-330-3p与CTNNB1在mRNA及蛋白表达水平上存在负相关关系。大鼠UAC TMJOA模型关节腔内注射miR-330-3p模拟物后,CTNNB1的表达减少,软骨退变减轻。结论: miR-330-3p通过抑制CTNNB1的表达,减轻TMJOA的软骨退变。

关键词: 颞下颌骨关节炎, 软骨退变, miR-330-3p, CTNNB1

Abstract: PURPOSE: To study the mechanism of miR-330-3p in cartilage degeneration in temporomandibular joint osteoarthritis(TMJOA) in rats. METHODS: Human chondrocytes SW1353 was stimulated by inflammation and stress respectively and rat TMJOA model was established through unilateral anterior crossbite(UAC). The expression of miRNA-330-3p in vitro and in vivo was detected by RT-qPCR. The proliferation and metabolism changes of SW1353 were detected by EDU fluorescence staining and immunoblotting after miRNA-330-3p overexpression or inhibition. Furthermore, bioinformatics analysis was performed to predict and screen the downstream targets of miRNA-330-3p. RT-qPCR and immunoblotting were used to clarify the regulation of miRNA-330-3p on the target and the function of target. After intra-articular injection of miRNA-330-3p in TMJOA rats, the changes in cartilage metabolism were detected by immunohistochemistry to clarify the effect of miRNA-330-3p on TMJOA. SPSS 22.0 software package was used to analyze the data. RESULTS: The expression of miRNA-330-3p decreased in chondrocytes under stress or inflammatory stimuli in vitro and in TMJOA rats. Inhibition of miRNA-330-3p decreased chondrocyte proliferation, reduced SOX9 and increased MMP13 expression, while overexpression of miRNA-330-3p had opposite effects. Bioinformatics analysis revealed that CTNNB1 was one of the possible downstream targets, and its expression was elevated after inflammatory/stress stimuli in vitro and in TMJOA rats. SOX9 expression decreased, and MMP13 expression increased after overexpression of CTNNB1, with opposite results for inhibition of CTNNB1. There was a negative relationship between miRNA-330-3p and CTNNB1. Intra-articular injection of miRNA-330-3p in TMJOA rats alleviated cartilage degeneration. CONCLUSIONS: miRNA-330-3p attenuates cartilage degeneration in TMJOA by inhibiting the expression of CTNNB1.

Key words: Temporomandibular joint osteoarthritis, Cartilage degeneration, miR-330-3p, CTNNB1

中图分类号:

R782

王楚瑶, 邹璐芗, 陆川, 何冬梅. miR-330-3p在颞下颌关节骨关节炎软骨退变中的作用机制研究[J]. 中国口腔颌面外科杂志, 2022, 20(4): 320-327.

WANG Chu-yao, ZOU Lu-xiang, LU Chuan, HE Dong-mei. Study on the mechanism of miR-330-3p in cartilage degeneration in temporomandibular joint osteoarthritis in rats[J]. China J Oral Maxillofac Surg, 2022, 20(4): 320-327.

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miR-330-3p在颞下颌关节骨关节炎软骨退变中的作用机制研究

Study on the mechanism of miR-330-3p in cartilage degeneration in temporomandibular joint osteoarthritis in rats

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