lncRNA DLEU1 Modulates Proliferation, Inflammation, and Extracellular Matrix Degradation of Chondrocytes through Regulating miR-671-5p

Long noncoding RNAs (lncRNAs) have been shown to be involved in the development of osteoarthritis. However, the expression, function, and mechanism of DLEU1 in OA development remain largely unclear. The present reference demonstrates that DLEU1 is overexpressed in OA specimens compared to control cartilages. Inflammatory cytokines IL-1β, TNF-α, and IL-6 induce DLEU1 expression in chondrocytes. Ectopic expression of DLEU1 induces chondrocyte proliferation, degradation of ECM, and inflammation mediators such as IL-6, IL-8, and TNF-α secretion. Moreover, we demonstrated that DLEU1 targets miR-671-5p expression in chondrocytes. Overexpression of DLEU1 suppresses miR-671-5p expression in chondrocytes. The expression of miR-671-5p is decreased in OA specimens compared to control cartilages. There is a negative correlation between the expression of miR-671-5p and DLEU1 in OA specimens. Inflammatory mediators IL-1β, TNF-α, and IL-6 suppress miR-671-5p expression in OA specimens. Elevated expression of miR-671-5p suppresses chondrocyte proliferation, degradation of ECM, and secretion of inflammation mediators. DLEU1 overexpression promotes chondrocytes proliferation, degradation of ECM, and secretion of inflammation mediators via regulating miR-671-5p. These results suggested that DLEU1 acts as one destructive role in OA development via regulating miR-671-5p.

We studied the role of DLEU1 in OA. Firstly, we indicated that DLEU1 is overexpressed in OA specimens compared to control cartilages. Inflammatory cytokines IL-1β, TNF-α, and IL-6 induce DLEU1 expression in chondrocytes. Ectopic expression of DLEU1 induces chondrocyte proliferation, degradation of ECM, and secretion of inflammation mediators, such as IL-6, IL-8, and TNF-α.

Materials and Methods
2.1. Tissue Specimens. OA cartilages were collected from OA cases that underwent TKA (total knee arthroplasty), and control cartilages were obtained from cases due to the fracture of knee joint without rheumatoid arthritis or OA. Each case was given an informed consent, and our study was agreed by the Clinical Ethics Committee of our hospital. The demographic and clinical characteristics of OA patients are indicated in Table 1. 2.2. Chondrocyte Culture and Transfection. Human chondrocytes were obtained from Shanghai Chinese Academy of Sciences, and these cells were cultured in the RPMI-1640 medium containing antibiotics and fetal bovine serum (FBS). pcDNA-DLEU1 and control and miR-671-5p mimic and scramble mimic were collected from GenePharma. Cell transfection was carried out with using Lipofectamine 2000 (Invitrogen, USA) following the protocol.
2.4. Cell Viability Assay. Cell viability was detected using CCK-8 reagent (Dojindo, Japan) following the typical protocol. Cells were cultured in the 96-well dish and 10 μl CCK-8 fluids were added into each well. After incubation for additional 3 hours, the absorbance was read with a microplate reader at 450 nm.   2.6. ELISA. The culture supernatant was obtained after chondrocytes were treated. The IL-6, IL-8, and TNF-α concentration in the supernatant was detected with ELISA reagent (R&D Systems, UK) following the standard protocol.
2.7. Statistical Analysis. SPSS 18.0 software (Chicago, IL) was processed for statistical assay. Results were indicated as means ± SD. Student's t-test was applied to detect statistical difference between two groups. Statistically significance was set to p < 0:05.

Results
3.1. DLEU1 Is Overexpressed, and miR-671-5p Is Decreased in OA Specimens. The pathology of control cartilages and OA cartilages is shown in Figures 1(a) and 1(b). qRT-PCR assay was carried out to examine DLEU1 expression in control cartilages and OA cartilages. As indicated in Figure 2, DLEU1 is overexpressed in OA specimens compared to control cartilages. Moreover, qRT-PCR assay was carried out to examine miR-671-5p expression in control cartilages and OA cartilages. As indicated in Figure 3(a), miR-671-5p is decreased in OA specimens compared to control cartilages. There is a negative correlation between expression of miR-671-5p and DLEU1 in OA specimens (Figure 3(b)).

Discussion
Emerging reports have supported that lncRNAs are aberrantly expressed in many physiological and pathological procedures, including OA. In this research, we studied the role of DLEU1 in OA. Firstly, we indicated that DLEU1 is overexpressed in OA specimens compared to control cartilages. Inflammatory cytokines IL-1β, TNF-α, and IL-6 induce DLEU1 expression in chondrocytes. Ectopic expression of DLEU1 induces chondrocyte proliferation, degradation of ECM, and secretion of inflammation mediators such as IL-6, IL-8, and TNF-α. Moreover, we demonstrated that DLEU1 targets miR-671-5p in chondrocytes. Overexpression of DLEU1 suppresses miR-671-5p expression in chondrocytes. The expression of miR-671-5p is decreased in OA specimens compared to control cartilages. There is a negative correlation between expressions of miR-671-5p and DLEU1 in OA specimens. Inflammatory mediators IL-1β, TNF-α, and IL-6 suppresses miR-671-5p expression in OA specimens. Elevated expression of miR-671-5p suppresses chondrocyte proliferation, degradation of ECM, and secretion of inflammation mediators. DLEU1 overexpression promotes chondrocyte proliferation, degradation of ECM, and secretion of inflammation mediators via regulating miR-671-5p. These results suggested that DLEU1 acts one destructive role in OA development via regulating miR-671-5p.
Recently, studies have revealed that DLEU1 plays critical roles in the pathologic progression of renal cell carcinoma, glioma, osteosarcoma, cervical cancer, and bladder cancer [29][30][31][32][33]. For instance, DLEU1 was upregulated in glioblastoma and downregulated expression of DLEU1 suppressed glioblastoma cell growth and enhanced cell apoptosis partly via regulating miR-4429 [36]. Yue et al. [29] found that knockdown of DLEU1 suppressed renal cell carcinoma cell growth, invasion, and migration and impaired epithelial mesenchymal transition progression partly through modulating Akt. Chen et al. [31] demonstrated that DLEU1 was upregulated in osteosarcoma cells and specimens. DLEU1 downregulation decreased osteosarcoma cell migration, invasion, and migration. Moreover, DLEU1 was shown to be overexpressed in bladder cancer specimens and overexpression of DLEU1 promoted cell invasion and growth and cisplatin resistance via modulating miR-99b expression [32]. However, the expression, function, and mechanism of DLEU1 in OA development remain largely unclear. In the present reference, we firstly detected the expression of DLEU1 in OA specimens. Our results showed that DLEU1 is overexpressed in OA specimens compared to control cartilages. Inflammatory cytokines IL-1β, TNF-α, and IL-6 induce DLEU1 expression in chondrocytes. Ectopic expression of DLEU1 induces chondrocytes proliferation, degradation of ECM, and inflammation mediators such as IL-6, IL-8, and TNF-α secretion. Accumulating studies have proved that lncRNA performed as ceRNA to modulate disease development via sponging miRNA [37,38]. Wang and workmates showed that NEAT1 promoted chondrocyte inflammation and pro-liferation through regulating miR-181a [39]. Zhu and Jiang [40] showed that PART1 regulated cell apoptosis, proliferation, and degradation of extracellular matrix through modulating miR-373-3p expression. Chen et al. [41] demonstrated       Journal of Immunology Research that MEG3 alleviated extracellular matrix degradation in chondrocytes via targeting miR-93. Furthermore, DLEU1 regulated osteosarcoma cell migration, invasion, and migration through targeting miR-671-5p [31]. Following the software (http://starbase.sysu.edu.cn/index.php), miR-671-5p may be a potential combining target gene of DLEU1. Luciferase reporter analysis was carried out to study the relationship between miR-671-5p and DLEU1. The data indicated that cotransfection with miR-671-5p mimic and DLEU1-WT decreases luciferase activities when compared to miR-671-5p mimic and DLEU1-mut (Figure 7(b)). In addition, qRT-PCR assay data showed that overexpression of DLEU1 suppresses miR-671-5p expression in chondrocytes. In addition, we showed that the expression of miR-671-5p is decreased in OA specimens compared to control cartilages. There is a negative correlation between expression of miR-671-5p and DLEU1 in OA specimens. Inflammatory mediators IL-1β, TNF-α, and IL-6 suppress miR-671-5p expression in OA specimens. Elevated expression of miR-671-5p suppresses chondrocyte proliferation, degradation of ECM, and inflammation mediator secretion. DLEU1 overexpression promotes chondrocyte proliferation, degradation of ECM, and secretion of inflammation mediators via regulating miR-671-5p.
Our results defined that DLEU1 is overexpressed in OA specimens compared to control cartilages. DLEU1 overexpression promotes chondrocyte proliferation, degradation of ECM, and secretion of inflammation mediators via regulating miR-671-5p. These data suggested that DLEU1 acts a destructive role in OA development via regulating miR-671-5p expression.

Data Availability
No data were used to support this study.

Conflicts of Interest
There is no conflict of interest.