Long noncoding RNA LINC00461 induced osteoarthritis progression by inhibiting miR-30a-5p

Mounting studies have shown that long noncoding RNAs (lncRNAs) play important roles in the development and occurrence of several human diseases. However, the role of LINC00461 in osteoarthritis (OA) remains obscure. A CCK-8 assay was performed to detect cell viability, and qRT-PCR analysis was used to measure mRNA expression. The targeting by miR-30a-5p of the LINC00461 3’UTR was detected using a luciferase reporter assay. Our data indicated that the inflammatory mediators IL-6 and TNF-α induced LINC00461 expression in chondrocytes and that the expression of LINC00461 was upregulated in OA tissues. Furthermore, we showed that TNF-α and IL-6 suppressed the expression of miR-30a-5p and that miR-30a-5p expression was lower in OA tissues than in normal samples. The expression level of miR-30a-5p in OA tissues was negatively related to LINC00461 expression. In addition, we showed that LINC00461 directly interacted with miR-30a-5p in chondrocytes. Elevated expression of LINC00461 induced chondrocyte proliferation, cell cycle progression, inflammation, and extracellular matrix (ECM) degradation. However, we demonstrated that ectopic expression of miR-30a-5p suppressed cell growth, cell cycle progression, inflammation and ECM degradation. Finally, we found that overexpression of LINC00461 enhanced chondrocyte proliferation, cell cycle progression, inflammation, and ECM degradation by downregulating miR-30a-5p. These data demonstrated that LINC00461 may modulate the development of OA by suppressing miR-30a-5p expression in chondrocytes. We propose that LINC00461 and miR-30a-5p may be potential therapeutic and diagnostic targets for OA.


INTRODUCTION
Osteoarthritis (OA) is characterized by inflammatory and degenerative processes that affect articular cartilage and joints and is the 4 th leading cause of disability and pain worldwide [1][2][3][4].Approximately 18% of females and 10% of males over sixty years old are diagnosed with OA [5][6][7].The pathology and mechanism of OA could be regulated through the processing of both environmental and genetic information [8][9][10][11].Chondrocytes are activated via growth factors and cytokines to induce abnormal differentiation and catabolism, which results in extracellular matrix (ECM) degradation [6,[12][13][14][15][16].For example, IL-6 was found to be involved in the development of OA [17,18].Thus, it is imperative to explore the molecular mechanism of OA.
Long noncoding RNAs (lncRNAs) are novel noncoding RNAs (ncRNAs) that are longer than 200 nucleotides in length with no or limited proteincoding potential [19][20][21][22].A growing number of studies have revealed that lncRNAs are deregulated in several diseases, such as tumors, intervertebral disc degeneration, infection and OA [23][24][25][26][27]. LncRNAs have been shown to participate in a number of cell biological processes, including apoptosis, stem cell AGING differentiation, proliferation, ECM degradation and invasion [28][29][30][31].Recently, the novel lncRNA LINC00461 was found to play important roles in the progression of several tumors, including glioma, multiple myeloma and breast cancer [32][33][34].For instance, Yang et al. [32] showed that LINC00461 was overexpressed in glioma samples and that knockdown of LINC00461 inhibited the expression of cyclin D1/A/E and cell growth in glioma cells partly by regulating the PI3K/AKT and MAPK/ERK signaling pathways.In addition, LINC00461 knockdown suppressed miR-9 expression and the related genes TMEM161B and MEF2C.However, the functional role of LINC00461 in OA development remains unknown.
In our research, we aimed to study the role of LINC00461 in OA development.We first confirmed that the inflammatory mediators IL-6 and TNF-α induced LINC00461 expression in chondrocytes and that the expression of LINC00461 was upregulated in OA tissues.Elevated expression of LINC00461 induced chondrocyte proliferation, cell cycle progression, inflammation, and extracellular matrix (ECM) degradation.

TNF-α and IL-6 induced the expression of LINC00461 in chondrocytes
To explore the effect of TNF-α and IL-6 on LINC00461 expression in OA, we treated chondrocytes with TNF-α and IL-6.We first confirmed that IL-6 could significantly induce chondrocyte proliferation by using a CCK-8 assay (Figure 1A).We also found that TNF-α significantly promoted chondrocyte growth using CCK-8 analysis (Figure 1B).In addition, we indicated that IL-6 enhanced the expression of LINC00461 in chondrocytes (Figure 1C).We demonstrated that TNF-α induced LINC00461 expression in chondrocytes (Figure 1D).

The expression of LINC00461 was upregulated in OA tissues
Furthermore, we tested whether the expression of LINC00461 was deregulated in OA and whether LINC00461 expression was decreased in OA cases and normal control patients.We demonstrated that LINC00461 expression was higher in OA tissues than in normal samples (Figure 2, ***p<0.001).

Elevated expression of LINC00461 induced chondrocyte proliferation, cell cycle progression, inflammation, and extracellular matrix (ECM) degradation
Next, we showed that overexpression of LINC00461 significantly enhanced cell proliferation in chondrocytes (Figure 5A).We also demonstrated that elevated expression of LINC00461 increased cell cycle progression in chondrocytes (Figure 5B).Interestingly, we found that IL-6 expression was upregulated in chondrocytes after LINC00461 treatment (Figure 5C).Moreover, we showed that LINC00461 overexpression enhanced IL-10 expression in chondrocytes (Figure 5D).Ectopic expression of LINC00461 promoted MMP-2 expression in chondrocytes (Figure 5E).The expression level of MMP-3 was increased in chondrocytes after LINC00461 treatment (Figure 5F).Furthermore, we showed that elevated expression of LINC00461 enhanced MMP-13 expression (Figure 5G) and decreased type II collagen expression (Figure 5H) in chondrocytes.

DISCUSSION
Emerging reports have demonstrated that lncRNAs are involved in the development of OA [2,35,36].In our study, we demonstrated that the inflammatory mediators IL-6 and TNF-α induced LINC00461 expression in chondrocytes and that the expression of LINC00461 was upregulated in OA tissues.Furthermore, we showed that TNF-α and IL-6 suppressed the expression of miR-30a-5p and that miR-30a-5p expression was lower in OA tissues than in normal samples.The expression level of miR-30a-5p in OA tissues was negatively correlated with LINC00461 expression.In addition, we showed that LINC00461 directly interacted with miR-30a-5p in chondrocytes.Elevated expression of LINC00461 induced chondrocyte proliferation, cell cycle progression, inflammation, and extracellular matrix (ECM) degradation.However, we demonstrated that ectopic expression of miR-30a-5p suppressed cell growth, cell cycle progression, inflammation and ECM degradation.Finally, we found that overexpression of LINC00461 enhanced chondrocyte proliferation, cell cycle progression, inflammation, and ECM degradation by downregulating miR-30a-5p.These data demonstrated that LINC00461 may modulate the development of OA by suppressing miR-30a-5p expression in chondrocytes.
Accumulating studies have revealed that LINC00461 plays important roles in the progression of several tumors, including glioma, multiple myeloma and breast cancer [32,33].For instance, Yang et al. [32] found that LINC00461 was overexpressed in glioma samples and that knockdown of LINC00461 inhibited the expression of cyclin D1/A/E and cell growth in glioma cells partly by regulating the PI3K/AKT and MAPK/ERK signaling pathways.In addition, LINC00461 knockdown suppressed the expression of miR-9 and the related genes TMEM161B and MEF2C.Deng et al. [33] indicated that LINC00461 expression was upregulated in multiple myeloma and that LINC00461 knockdown reduced multiple myeloma cell proliferation and promoted cell apoptosis by regulating miR-15a and miR-16.Dong et al. [34] demonstrated that LINC00461 was overexpressed in breast cancer cell lines and samples and that LINC00461 overexpression induced breast cancer cell invasion and migration, enhanced ZEB1 and vimentin expression and inhibited E-cadherin expression.However, the functional role of LINC00461 in OA development remains unknown.In our research, we first confirmed that the inflammatory mediators IL-6 and TNF-α induced LINC00461 expression in chondrocytes and that the expression of LINC00461 was upregulated in OA tissues.Elevated expression of LINC00461 induced chondrocyte proliferation, cell cycle progression, inflammation, and extracellular matrix (ECM) degradation.These data suggested that LINC00461 induced the progression of OA.
Previous studies have suggested that miRNAs are modulated by lncRNAs and play critical roles in the functional activity of lncRNAs [37][38][39].For example, LINC00461 plays an oncogenic role in breast cancer by regulating miR-30a-5p expression [34].Liu et al. found that lncRNA THRIL enhanced lipopolysaccharideinduced inflammatory injury by inhibiting miR-125b in ATDC5 cells.Xiao et al. [40] showed that lncRNA HOTAIRM1 variant 1 downregulation contributes to OA by modulating miR-125b/BMPR2 expression and promoting the JNK/MAPK/ERK signaling pathway.Hu et al. [41] showed that lncRNA HOTAIR induced OA development by regulating miR-17-5p and FUT2/βcatenin axis expression.In line with these findings, we also demonstrated that LINC00461 directly interacted with miR-30a-5p in chondrocytes.Moreover, we showed that TNF-α and IL-6 suppressed the expression of miR-30a-5p and that miR-30a-5p expression was lower in OA tissues than in normal samples.The expression level of miR-30a-5p in OA tissues was negatively correlated with LINC00461 expression.Ectopic expression of miR-30a-5p decreased cell growth, cell cycle progression, inflammation and ECM degradation.Furthermore, we found that overexpression of LINC00461 enhanced chondrocyte proliferation, cell cycle progression, inflammation, and ECM degradation by downregulating miR-30a-5p.We will study the mechanism/pathway of how LINC00461 exerts its potential effects in OA chondrocytes in our next work.
In summary, we demonstrated that LINC00461 was overexpressed in OA tissues compared to normal samples and that IL-6 and TNF-α induced LINC00461 expression in chondrocytes.Overexpression of LINC00461 enhanced chondrocyte proliferation, cell cycle progression, inflammation, and ECM degradation by downregulating miR-30a-5p.These findings suggested that lncRNA LINC00461 promoted the progression of OA partly by regulating miR-30a-5p expression.

Tissues
OA cartilage samples were obtained from OA cases (n=25, age 61.04 ± 4.809 years; 18 female, 7 male) that underwent total knee arthroplasty, and control cartilage

Figure 2 .
Figure 2. The expression of LINC00461 was upregulated in OA tissues.LINC00461 expression was detected in OA cases and normal control patients.***p<0.001.

Figure 5 .Figure 6 .
Figure 5. Elevated expression of LINC00461 induced chondrocyte proliferation, cell cycle progression, inflammation, and extracellular matrix (ECM) degradation.(A) Overexpression of LINC00461 enhanced cell proliferation in chondrocytes.(B) Elevated expression of LINC00461 increased cell cycle progression in chondrocytes.(C) IL-6 expression was upregulated in chondrocytes after LINC00461 treatment.(D) LINC00461 overexpression enhanced IL-10 expression in chondrocytes.(E) Ectopic expression of LINC00461 promoted MMP-2 expression in chondrocytes.(F) The expression level of MMP-3 was increased in chondrocytes after LINC00461 treatment.(G) Elevated expression of LINC00461 enhanced MMP-13 expression.(H) The expression of type II collagen was measured by qRT-PCR assay.*p<0.05 and **p<0.01.