研究背景

骨关节炎（Osteoarthritis,OA）是世界上最常见的关节疾病，中老年人多发，是一种以关节软骨破坏、软骨下骨硬化、骨质增生和滑膜炎性病变为病理特征的常见关节疾病，临床表现为关节疼痛、关节肿胀、关节僵硬和关节活动受限等，最好发于膝关节、髋关节和指间关节。骨关节炎主要由创伤、代谢、衰老和遗传等因素引起。骨关节炎最常见的病因是衰老，因此骨关节炎是一种老年退变性疾病。随着人口老龄化、肥胖症患病率的增加、医疗技术水平的提升以及人类寿命的增长，骨关节炎的患病率逐年增高，特别是在欧洲、美国和其他发达国家，预计到2030年，35%的人最终将因骨关节炎而残疾，而且这一数字预计还将进一步扩大。近期研究表明，骨关节炎是一种复杂的疾病，人口老龄化以及人类寿命延长是骨关节炎高患病率的重要原因。目前骨关节炎的治疗方法为阶梯治疗，第一层为教育、运动疗法等，第二层为消炎镇痛药、关节腔注射玻璃酸钠等，第三层为关节镜、软骨修复术等，第四层为关节置换、截骨术等关节重建手术。

目前，由于骨关节炎的发病机制尚不清楚，因此缺乏有效的治疗策略来预防、减缓、甚至逆转骨关节炎的进展，因此有必要进一步研究骨关节炎发生、发展的分子机制，以减轻或减缓患病人群的关节疼痛和功能障碍进程。深入了解骨关节炎的分子机制和其病因学，有利于建立有效的治疗方法。长链非编码RNA（Long noncoding RNA，lncRNA）是新近发现的一类在骨关节炎发生发展（炎症反应、细胞外基质稳态以及细胞凋亡和增殖）中起关键调节作用的非编码RNA。目前，越来越多的证据表明，lncRNA在骨关节炎的多种病理生理过程发挥重要作用。

研究方法

以本研究前期筛选的在早期和晚期骨关节炎软骨组织中差异表达的lncRNA作为本实验的研究对象。在全膝关节置换术中，搜集了10例诊断为膝关节内侧单间室（内侧软骨磨损，外侧软骨相对完好）骨关节炎的受试者股骨截骨块的软骨组织，将获得的内侧关节软骨作为实验组，外侧关节软骨作为对照组，筛选差异表达的lncRNA，采用定量逆转录聚合酶链反应（Quantitative reverse transcription polymerase chain reaction，RT-qPCR）验证在早期骨关节炎发生发展为晚期骨关节炎中可能发挥重要调节作用的lncRNA，并将验证后的差异表达lncRNA作为本实验的研究对象。

基于IL-1β诱导C28/I2细胞形成的骨关节炎软骨细胞模型，阐明了lncRNA THUMPD3-AS1增强骨关节炎软骨细胞的增殖、抑制细胞凋亡和增强炎症反应的作用机制。使用IL-1β诱导C28/I2细胞成为骨关节炎软骨细胞，转染lncRNA THUMPD3-AS1 质粒或THUMPD3-AS1 siRNA（Small interfering RNA，小干扰RNA）分别使C28/I2细胞高或低表达THUMPD3-AS1。5-溴-2'-脱氧尿苷(5- bromo -2'-deoxyuridine，BrdU) 法检测各组细胞增殖率，流式细胞术检测各组细胞的凋亡情况和细胞周期，此外，蛋白质印迹法（Western blot，WB）检测细胞周期相关和凋亡相关蛋白cyclin E2、CDK4、caspase-3和Bcl-2，揭示lncRNA THUMPD3-AS1对骨关节炎软骨细胞增殖和凋亡的影响及其机制；酶联免疫吸附实验（Enzyme linked immune sorbent assay，ELISA）检测细胞培养上清液中白介素-6（Interleukin-6，IL-6）、肿瘤坏死因子α（Tumor necrosis factor-α，TNF-α）、一氧化氮（Nitric oxide，NO）的表达水平来观察骨关节炎软骨细胞中lncRNA THUMPD3-AS1过表达和沉默时的炎症反应程度。Western blot检测了p38和p65这些信号蛋白在骨关节炎软骨细胞中的激活情况，探究lncRNA THUMPD3-AS1调控骨关节炎炎症反应的信号通路。

研究结果

研究结果表明与早期骨关节炎相比，晚期骨关节炎软骨细胞中lncRNA THUMPD3-AS1、HAS-AS1和SNHG5的表达水平明显降低，GAS5的表达未发现显著统计学差异，DANCR则高表达。既往文献已经报道了lncRNA SNHG5、GAS5和DANCR在骨关节炎发生发展中的作用机制研究。THUMPD3-AS1的差异倍数大于HAS-AS1，且有文献报道了THUMPD3-AS1在非小细胞肺癌中高表达，因此选择lncRNA THUMPD3-AS1作为本研究的研究对象。

结果表明，THUMPD3-AS1 质粒或THUMPD3-AS1 siRNA可以成功使骨关节炎软骨细胞过表达或下调THUMPD3-AS1。lncRNA THUMPD3-AS1过表达的骨关节炎软骨细胞增殖率接近于正常C28/I2细胞，显著高于IL-1β诱导的骨关节炎软骨细胞。流式细胞术的实验结果与BrdU实验结果一致，两种检测结果均显示lncRNA THUMPD3-AS1有促进骨关节炎软骨细胞增殖、减少细胞凋亡的作用。过表达lncRNA THUMPD3-AS1增加了cyclin E2、CDK4、Bcl-2的表达，但降低了caspase-3的表达。ELISA结果显示lncRNA THUMPD3-AS1的表达水平与IL-6、TNF-α和NO的表达水平呈正相关。Western Blot检测p38和p65这些信号蛋白在骨关节炎软骨细胞中的磷酸化情况，结果显示lncRNA THUMPD3-AS1的表达水平与P-p38/p38和P-p65/p65的比值呈正相关，p38丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）和核因子κB（Nuclear Factor κB，NF-κB）p65 信号通路的激活促进了IL-6、TNF-α和NO等炎症因子的表达，进一步验证了lncRNA THUMPD3-AS1在骨关节炎炎症反应的信号通路中发挥重要作用。

研究结论

本研究结果发现了lncRNA THUMPD3-AS1在促进骨关节炎软骨细胞增殖、抑制骨关节炎软骨细胞凋亡和促进骨关节炎软骨细胞炎症反应过程中的多重作用，这扩展了对lncRNA在骨关节炎发生发展中具有多种功能的认识。

Background

Osteoarthritis (OA), the most common joint disease in the world, often occurs in middle-aged and elderly people, the clinical manifestations of which are joint pain, joint swelling, joint stiffness, and joint movement limitation, etc., preferably in the knee, hip, and interphalangeal joints. OA is mainly caused by factors such as trauma, metabolism, aging, and genetics. The most common cause of OA is aging. Accordingly, OA is a degenerative disease of old age. With the aging of the population and the increase in the prevalence of obesity, and with the improvement of medical technology and the increase in human lifespan, the prevalence of OA is increasing year by year, especially in Europe, the United States, and other developed countries. It is expected, 35% of people will eventually be disabled by OA in 2030, and this number is expected to expand further. Recent studies have shown that OA is a complex disease and an aging population/extended lifespan is one of the reasons for the high prevalence of OA. At present, the treatment method for OA is stepped therapy. The first layer is education, exercise therapy, etc.; the second layer is anti-inflammatory analgesics, intra-articular injection of sodium hyaluronate, etc.; the third layer is arthroscopy, cartilage repair, etc.; the fourth layer is joint reconstruction surgery such as joint replacement and osteotomy.

At present, there is a lack of effective therapeutic strategies to prevent, slow down, and even reverse the progression of OA since the pathogenesis of OA is remain unclear. Therefore, it is necessary to further study the molecular mechanisms of the occurrence and development of OA to relieve or slow down the pain and dysfunctional processes of joints of the diseased population. An in-depth understanding of the molecular mechanisms of OA and its etiology will facilitate the establishment of effective treatments. Long noncoding RNA (lncRNA) is a newly discovered noncoding RNA that plays a key regulatory role in the occurrence and development of OA. Currently, increasing evidence suggests that lncRNA plays important roles in various pathophysiological processes of OA.

Methods

The preliminary work of the current study screened the differentially expressed lncRNAs between the early and late osteoartiritis articular cartilage that play important roles in OA as the research object of this experiment. During total knee arthroplasty, the cartilage tissues of femur from 10 subjects diagnosed with OA of single compartment of knee joint (the medial cartilage wear and the lateral cartilage is relatively intact) was collected, and the obtained medial cartilage was used as the experimental group, and the lateral cartilage was used as the control group. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to verify the expression level of lncRNAs that may play important regulatory roles in the occurrence and development of OA. Differentially expressed lncRNA was used as the research object of this experiment.

Subsequently, this study mainly clarified the mechanism of lncRNA THUMPD3-AS1 enhancing the proliferation, reducing the cell apoptosis, and enhancing the inflammatory response of OA chondrocytes. IL-1β was used to induce C28/I2 cells to become OA chondrocytes, and lncRNA THUMPD3-AS1 plasmid or THUMPD3-AS1 siRNA (Small interfering RNA) was transfected to make C28/I2 cells express THUMPD3-AS1 at high or low levels, respectively. The proliferation rate of chondrocytes in each group was detected by BrdU assay, the apoptosis of chondrocytes and cell cycle in each group was detected by flow cytometry assay. In addition, Western blot (WB) was used to detect the expression level of cell cycle-related and apoptosis-related proteins cyclin E2, CDK4, caspase-3, and Bcl-2, to reveal the effect and mechanism of lncRNA THUMPD3-AS1 on the proliferation and apoptosis of OA chondrocytes. Enzyme linked immune sorbent assay (ELISA) was used to demonstrate the degree of inflammatory response by detecting the levels of IL-6 (Interleukin-6), TNF-α (Tumor necrosis factor-α), and NO (Nitric oxide) in the groups of overexpression and silencing of THUMPD3-AS1 in OA chondrocytes. Western blotting detected the activation of these signaling proteins p38 and p65 in OA chondrocytes and explored the signaling pathway of  lncRNA THUMPD3-AS1 regulating OA inflammatory response.

Results

The results showed that the expression levels of lncRNA THUMPD3-AS1, HAS-AS1, and SNHG5 in the experimental group were significantly lower than those in the control group, and there was no significant difference for the expression of GAS5.While DANCR was highly expressed in the OA group compared with the control group. Previous literature has reported the role and mechanism of SNHG5, GAS5, and DANCR in the occurrence and development of OA. The fold change of THUMPD3-AS1 is greater than that of HAS-AS1, and it has been reported that THUMPD3-AS1 is highly expressed in non-small cell lung cancer, so lncRNA THUMPD3-AS1 was selected as the research object of this study.

The results showed that THUMPD3-AS1 plasmid or THUMPD3-AS1 siRNA could successfully overexpress or downregulate the expression level of THUMPD3-AS1 in OA chondrocytes. The proliferation rate of OA chondrocytes overexpressing lncRNA THUMPD3-AS1 was close to that of normal C28/I2 cells and was significantly higher than that of OA chondrocytes. The experimental results of flow cytometry assay were consistent with the results of BrdU experimental. Both results of BrdU and flow cytometry assays showed that lncRNA THUMPD3-AS1 could promote the proliferation of OA chondrocytes and reduce apoptosis. Moreover, overexpression of lncRNA THUMPD3-AS1 increased the expression of cyclin E2, CDK4, and Bcl-2, but decreased the expression of caspase-3. The results of ELISA showed that the expression level of lncRNA THUMPD3-AS1 was positively correlated with the expression levels of IL-6, TNF-α, and NO. The phosphorylation of these signaling proteins p38 and p65 in OA chondrocytes was detected by western blot assay, and the results of which showed that the expression level of lncRNA THUMPD3-AS1 was positively correlated with the ratio of P-p38/p38 and P-p65/p65. Furthermore, p38 mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) p65 signaling pathways promote the expression of inflammatory factors such as IL-6, TNF-α, and NO, which further verified that lncRNA THUMPD3- AS1 plays an important role in the signaling pathway of OA inflammatory response.

Conclusions

Taken together, our findings highlight the differential effects of lncRNA THUMPD3-AS1 on cell proliferation, cell apoptosis, and inflammatory responses of OA chondrocytes, which extended the understanding of the diverse functions of lncRNA in the occurrence and development of OA.