Osteoarthritis, a major cause of disability in the elderly, is still lacking effective methods for treatment. In recent years, a growing number of targeted drugs have shown outstanding therapeutic effects in clinical treatment; finding potential therapeutic targets has become one of the important directions of current research. The occurrence of OA is the result of multiple factors, among which a significant element is a decline in chondrocyte numbers. A variety of lncRNAs are involved in the OA development via regulating the biological behavior of chondrocytes, thus lncRNA has received extensive attention for the past few years[10, 17]. The level of PVT1 increases in chondrocytes of patients, hence knocking down PVT1 in IL-1β treated chondrocytes can not only promote cell activity and autophagy, but also inhibit cell apoptosis and inflammatory response. Herein, elevated PVT1 was found in OA cartilage tissue and chondrocytes induced by IL-1β, whereas knocking down PVT1 could promote proliferation and reduce cell apoptosis in these chondrocytes. Cartilage ECM plays a crucial role in maintaining cartilage structure and function. Interleukin-1β can reduce the synthesis of anabolic genes (aggrecan and collagen Type II) and increase the level of catabolic factors (MMP-9), thus intensifying the degradation of chondrocyte ECM[18, 19]. We found that aggrecan and collagen Type II levels lowered, whereas those of MMP-9 elevated in IL-1β-induced chondrocytes, while knocking down PVT1 reduced the impacts of IL-1β on these three proteins. Consequently, PVT1 knockdown may be one of the main methods to treat OA.
Growing evidence shows that lncRNA can function as a molecular sponge of miRNA by combining with it, thereby participating in biological behaviors. For example, PVT1 promotes the proliferation and migration of pancreatic cancer cells through regulating miR-448 as a molecular sponge[20, 21]. Also, MALAT1 can promote the malignant growth of triple negative breast cancer cells through regulating miR-129-5p. Furthermore, PVT1 knockdown inhibits IL-1β-induced chondrocyte injury by regulating the miR-27b-3P/TRAF3 axis. Here, we used biological prediction software and acquired binding sites between PVT1 and miR-497 to investigate the promising mechanism of PVT1 function involved in OA progression; PVT1 knockdown was able to increase miR-497 in IL-1β-induced chondrocytes. The dual luciferase report also showed that an interaction between miR-497 and PVT1 occurred. The RNAs miRNA and lncRNA are also commonly found in the human body, and can be involved in the regulation of a third of all human genes. The imbalance of miRNA, a key regulator of gene expression, is considered to be a major cause of disease. Previous reports have shown that miR-497, which belongs to the miR-15/16/195/424/497 family, is downregulated in IL-1β treated chondrocytes. In the present study, the presence of this downregulation was also confirmed. In addition, the effects of IL-1β on chondrocyte proliferation and ECM were weakened upon increasing miR-497 expression in IL-1β treated chondrocytes, which indicates that miR-497 and PVT1 are potential therapeutic targets for OA.
Target genes can be regulated by miR by cutting their mRNA and inhibiting protein synthesis, thus preventing gene function. Biological prediction software was used again for prediction to understand how miR-497 participates in OA progression, with targeted binding sites between miR-497 and AKT3. The expression of AKT3 was detected in chondrocytes transfected with miR-497-mimics and miR-NC, while dual luciferase reporter analysis revealed that AKT3 can be negatively regulated by miR-497. The AKT3 protein comes from the AKT serine/threonine kinase family, which can regulate cell signaling and participate in multiple processes in the cell, such as growth, proliferation, differentiation and apoptosis[26, 27]. It is elevated in many inflammatory diseases including OA[28, 29]. Previous studies have revealed that the AKT3 gene can be used as target for miR, enabling miR to indirectly participate in disease progression. For example, miR-29a can act as a tumor suppressor in papillary thyroid carcinoma by the targeted regulation of AKT3. Also, miR-384 can inhibit the proliferation of colorectal cancer cells through the negative regulation of AKT(Wang, et al., 2018). In this study, we transfected IL-1β-induced chondrocytes with miR-497 mimics and then with sh-AKT3. The impacts of miR-497 mimics on the proliferation, apoptosis and ECM related molecules of IL-1β-induced chondrocytes were reversed by sh-AKT3, suggesting that miR-497 can function through the negative regulation of AKT3. At the end of this study, we conducted rescue experiments to further investigate the regulatory mechanism between PVT1 and miR-497/AKT3. It was found that transfection by miR-497 inhibitor or sh-AKT3 could reverse the effects of transfection by si-PVT1 on proliferation, apoptosis and ECM-related molecules of OA chondrocytes. Therefore, we believe that the regulation of PVT1 in chondrocytes depends on the regulation of the miR-497/AKT3 axis.
Although this study confirmed that the knockdown of PVT1 could decrease the effects of IL-1β on proliferation, apoptosis and degradation of chondrocyte ECM through the miR-497/AKT3 axis, it still has some deficiencies. Firstly, for example, no animal experiments have been conducted to analyze the effects of PVT1 on subchondral osteosclerosis and osteophyte formation. Secondly, neither the expression levels of miR-493 and AKT3 in OA cartilage tissue were measured, nor the clinical effects of PVT1, miR-497 and AKT3 in OA were investigated. Moreover, potential mechanisms of PVT1 function other than the miR-497/AKT3 axis have not been explored. The above limitations are expected to be corrected by further experiments in follow-up studies.