lncRNA IGHCγ1 Acts as a ceRNA to Regulate Macrophage Inflammation via the miR-6891-3p/TLR4 Axis in Osteoarthritis

Accumulating data have implicated that long noncoding RNA (lncRNA) plays an important role in osteoarthritis (OA), which may function as a competitive endogenous RNA (ceRNA) of microRNAs (miRNAs). lncRNA IGHCγ1 has been demonstrated to regulate inflammation and autoimmunity. Nonetheless, the altering effect of IGHCγ1 in OA remains unclear. This study is aimed at investigating the mechanism and function of lncRNA IGHCγ1 in OA. CCK-8, EdU, and transwell assays were used to estimate macrophage proliferation and migration. Fluorescence in situ hybridization (FISH) was performed to estimate the local expression of lncRNA IGHCγ1 in macrophages. Luciferase reporter assay was adopted to validate the ceRNA role of IGHCγ1 as miRNA sponge. lncRNA IGHCγ1 was primarily localized in macrophage cytoplasm and upregulated in OA. miR-6891-3p inhibited macrophage proliferation, migration, and inflammatory response by targeting TLR4, while lncRNA IGHCγ1 promoted TLR4 expression by functioning as a ceRNA for miR-6891-3p through the NF-κB signal in macrophages. This study strongly supports that lncRNA IGHCγ1 regulates inflammatory response via regulating the miR-6891-3p/TLR4/NF-κB axis in macrophages.


Introduction
Osteoarthritis (OA) is one of the most common degenerative diseases and a major cause of disability in older adults. It leads to a great burden on society and economy. Apart from damages in cartilage and subchondral bone, OA also causes synovitis [1,2]. The etiology and pathogenesis of OA remain not fully understood up till now. It has been well documented that age, obesity, abnormal anatomical structure, joint trauma history, and excessive use of joints are closely related to OA [3]. Accumulated data have suggested many inflammatory cells and their produced inflammatory mediators contribute to OA pathogenesis, such as IL-6 and TNF-α [4,5]. IL-6 and TNF-α can be produced by macrophages, synoviocytes, or articular cartilage itself, which contribute to OA by inducing the expression of metalloproteinases (MMP). In addition, the dysfunction of macrophages has been suggested to play a key role in OA pathogenesis [6]. Therefore, besides tissue engineering, biological therapies by targeting inflammation-associated genes or cells are potential therapeutic strategies for OA patients. Moreover, there are still many challenges for the regeneration of articular cartilage in OA treatment particularly under inflammatory microenvironment.
Emerging studies have shown noncoding RNAs (ncRNAs), for instance, microRNAs (miRNAs), circular RNAs (circRNAs), and long noncoding RNAs (lncRNAs), are involved in OA development and progression [7][8][9]. A large body of data has demonstrated lncRNA and circRNA can act as competitive endogenous RNAs (ceRNAs) via miRNAs sponge, leading to suppression of miRNAs [10][11][12]. miRNAs are common ncRNAs involved in regulating autoimmunity and inflammation, which can decrease the expression of targeted mRNAs. Available studies have revealed a variety of miRNAs are aberrantly expressed in OA patients [13,14]. Our previous study has shown that lncRNA IGHCγ1 (also called IGHCgamma1) is involved in regulating arthritis [15], but the altering effect of lncRNA IGHCγ1 in OA is unknown. With development in bioinformatics and molecular biology techniques, the ceRNA mechanism based on interactions among lncRNA, miRNA, and mRNA has been extensively elucidated [16,17]. However, whether lncRNA IGHCγ1 functions as a ceRNA in regulating OA needs to be investigated. The current study is aimed at elucidating the role of lncRNA IGHCγ1 and its mechanism in OA.
2.6. Enzyme-Linked Immunosorbent Assay (ELISA). In brief, lncRNA IGHCγ1 or TLR4 overexpressed cells (1 × 10 5 /ml) are seeded into 96-well cell culture plate in serum-free medium overnight and then treated by miR-6891-3p mimics or not for 24 hrs. The supernatant was acquired for cytokine determination by use of the ELISA kits. IL-6 and TNF-α ELISA kits (R&D, Minnesota, USA) are adopted based on the protocol as previously described [18].

Fluorescence
In Situ Hybridization (FISH). We carry out FISH assay to estimate the local expression of lncRNA IGHCγ1 in pTHP-1 cells. 5 × 10 5 /ml cells are fixed with paraformaldehyde (4%). After protease-K incubation, pTHP-1 cells are dehydrated in a gradient manner with diverse concentrations of ethanol and then hybridized by IGHCγ1 probe labeled by fluorescence. DAPI (Life Technologies, Carlsbad, USA) is used to stain the nucleus. The fluorescence is scanned by a microscope. 2.10. NF-κB Activity Assay. The activity of transcriptional factor NF-κB is detected in a whole cell extract based on the protocol of the ELISA-based kit (Active Motif, CA, USA). Briefly, lncRNA IGHCγ1 overexpressed and the control plasmids were used to transfect macrophages. After that, 1 × 10 5 /ml macrophages were cultured in serum-free medium and administrated into miR-6891-3p mimics and the control vectors for 24 hours. Lastly, cells were harvested and used for the detection of NF-κB activity according to the kit instructions mentioned above. Individual experiments were repeated for at least three times.
2.11. RNA Binding Protein Immunoprecipitation (RIP) Assay. The Magna RIP Kit (Millipore, Bedford, USA) is used for analysis. Cell lysates were immunoprecipitated using Ago2 antibody or IgG in the buffer. The expression of IGHCγ1 in immunoprecipitates is detected by real-time PCR.
2.12. Statistical Analysis. Data are presented as mean ± SE. ANOVA or unpaired Student's t-test is adopted for comparisons. GraphPad Prism (San Diego, CA, USA) and SPSS software (Chicago, IL, USA) are applied in this study.

lncRNA IGHCγ1 Was Elevated in OA and Macrophage
Cell Lines. lncRNA IGHCγ1 was found to be highly expressed in PBMCs of OA patients (Figure 1(a)). Besides, it was demonstrated that the expression of lncRNA IGHCγ1 was upregulated in a time-dependent manner in PMAinduced THP-1 (pTHP-1) macrophages, which were activated by LPS for 12, 24, and 48 hours (Figure 1(b)). It was well known that macrophages are major immune cells involved in OA. Accordingly, we hypothesized that lncRNA IGHCγ1 might affect macrophage-mediated autoimmunity and inflammation regulation in OA. , which suggested lncRNA IGHCγ1 might confer its effect by combining with other RNAs or proteins in the cytoplasm of macrophages. High lncRNA IGHCγ1 expression in OA PBMCs was partly due to increased copy number gains as shown in Figure 3(c). LPS could also increase the copy number of lncRNA IGHCγ1 relative to miR-6891-3p in pTHP-1 macrophages (Figure 3(d)). Thirdly, lncRNA IGHCγ1 was demonstrated to be negatively related to miR-6891-3p regarding their expression in macrophages determined by real-time PCR (Figure 3(e)). In order to further investigate whether the two noncoding RNAs can interact with each other by base complementary binding, we performed bioinformatics analysis. lncRNA IGHCγ1 was predicted to interact with miR-6891-3p after screening in database of starBase (http://starbase.sysu.edu.cn/). It could specifically recognize the seed sequence of miR-6891-3p (Figure 3(f)).

lncRNA
We hypothesized lncRNA IGHCγ1 could act as a ceRNA of miR-6891-3p in macrophages. The dual-luciferase reporter assay revealed that lncRNA IGHCγ1 could target miR-6891-3p (Figure 3(g)). Moreover, RIP assay showed IGHCγ1 could be detected in the immunoprecipitates as demonstrated by real-time PCR. Taken together, lncRNA IGHCγ1 could function as a ceRNA of miR-6891-3p in macrophages.   has been reported to be a potential regulator in inflammation and immunity [19]. Significantly reduced miR-6891-3p was also demonstrated in OA PBMCs and pTHP-1 cells under stimulation of LPS (Figures 4(a) and 4(b)).

Mediators of Inflammation
To elucidate its functions in OA, we evaluated the influence of miR-6891-3p on macrophage proliferation and migration by use of inhibitors of miR-6891-3p. The realtime PCR showed inhibitors of miR-6891-3p could efficiently inhibit miR-6891-3p expression in macrophages (Figure 4(c)). After downregulation of miR-6891-3p, pTHP-1 cell proliferation was significantly promoted as demonstrated by CCK-8 analysis (Figure 4(d)). Taken together, downregulation of miR-6891-3p promoted macrophage proliferation in vitro.
3.7. lncRNA IGHCγ1 Acted as a ceRNA for miR-6891-3p via NF-κB. NF-κB is a downstream signaling molecule and a key transcriptional factor involved in regulating TLR4mediated autoimmune and inflammation. Therefore, the effect of lncRNA IGHCγ1 on the downstream signaling pathway of TLR4 was investigated. lncRNA IGHCγ1 enhanced NF-κB phosphorylation by sponging miR-6891-3p, while miR-6891-3p mimics could inhibit NF-κB activation by rescuing the proinflammatory effect of lncRNA IGHCγ1 in macrophages (Figures 7(a) and 7(b)). In addition, the activity of NF-κB in macrophages was estimated. As shown in Figure 7(c), NF-κB activity was significantly enhanced in pTHP-1 cells with lncRNA IGHCγ1 upregulation, whereas miR-6891-3p mimics could reduce the activation of NF-κB. Moreover, lncRNA IGHCγ1 promoted the generation of IL-6 and TNF-α cytokines in the downstream of the NF-κB pathway in macrophages (Figures 7(d) and 7(e)). As a result, it could be concluded that lncRNA IGHCγ1 acted as a ceRNA for miR-6891-3p and thus induced NF-κB activation and downstream cytokine production in macrophages.

Discussion
lncRNAs belong to noncoding RNAs, which do not encode proteins but possess important biological activity. Accumulating data have suggested lncRNAs are crucial noncoding RNAs involved in regulating cancer, autoimmunity, and inflammation, such as lincRNA-Cox2 and lncRNA-Dreh [20][21][22]. During the past few years, lncRNA has been demonstrated to be involved in OA with abnormal expression and/or dysregulated functions, particularly in inflammatory cells such as macrophages [23,24]. Those differentially expressed lncRNAs are specific in OA and may be used as diagnostic or therapeutic targets in the future. lncRNA IGHCγ1 is an aberrantly expressed lncRNA in inflammatory arthritis [15]. However, the precise role of lncRNA IGHCγ1 in OA is not fully elucidated. In the current study, lncRNA IGHCγ1 has been found to be upregulated in OA. It enhances the proliferation of macrophages in vitro. Most interestingly, lncRNA IGHCγ1 is capable of acting as a ceRNA for miR-6891-3p in macrophages. Furthermore, lncRNA IGHCγ1 aggravates inflammation via regulating the miR-6891-3p/TLR4/NF-κB axis in macrophages. These findings are useful for investigating biological markers for OA treatment.
As a kind of noncoding RNA, lncRNA can function as ceRNA and restrain miRNA by lncRNA-miRNA sponge in cancer, cardiovascular disease, and so on [25,26]. Several functional lncRNAs have been demonstrated to influence osteoblast differentiation and OA pathogenesis through lncRNA-miRNA-mRNA ceRNA mechanism [27][28][29][30]. Some lncRNAs can affect the degradation of the extracellular matrix of chondrocytes by functioning as ceRNAs of specific miRNAs and thus participate in the development and progression of OA, such as lncRNAs of HOTTIP, MEG3, and XIST [28,29,31]. Some lncRNAs have been well documented to regulate the differentiation of osteoblasts via the lncRNA-miRNA ceRNA network [27]. Besides, Fan et al. have found that DANCR can regulate the progression of OA via targeting miR-577 through ceRNA mechanism [32]. Moreover, the study by Mao et al. shows the evidence that lncRNA HOTAIR is involved in OA pathogenesis by regulating synovial inflammation and proliferation and apoptosis of synoviocytes [33]. However, whether lncRNAs regulate inflammation in OA based on the lncRNA-miRNA ceRNA network warrants to be investigated. In this study, lncRNA IGHCγ1 has been firstly revealed to be dysregulated in OA, which is capable of acting as a ceRNA for miR-6891-3p and promoting inflammation in macrophages. As a result, all  Mediators of Inflammation findings strongly support that the lncRNA-miRNA network plays a critical role in OA.
Accumulating evidence has suggested miRNAs exert significant effects on the development of OA by regulating targeted genes at the posttranscriptional level. miRNAs are involved in articular cartilage damage and repair, extracellular matrix degradation, arthritis, and maintenance of bone homeostasis [34][35][36]. Some established miRNAs dysregulated in OA have been documented to regulate in a targeted manner certain toll-like receptors (TLRs), the well-known pattern recognition receptors (PRRs) participating in innate immunity [37][38][39]. miR-6891-3p has been implicated to regulate in a targeted manner TLRs in inflammatory arthritis [19]. Here, we hypothesize miR-6891-3p is involved in OA  It has been well documented that macrophages play a critical role in inflammatory response and autoimmunity against virus infection and tumorigenesis, which are also major cells involved in OA pathogenesis [37]. TLRs are critical PRRs mainly expressed on the membrane or in macrophages. A number of studies have implicated that TLRmediated biological effects in macrophages can maintain or expand inflammatory Th1 and Th17 cell response, such as TLR2, TLR3, TLR4, and TLR9 [40,41]. Moreover, TLRmediated inflammation and immune regulation in macrophages are involved in OA development and progression, and TLR4 is one of the most important ones [42][43][44]. TLR4-mediated inflammatory response in macrophages induces high inflammatory state characteristic of increased inflammatory cytokines in bone microenvironment, such as IL-6 and TNF-α. Those inflammatory cytokines can lead to bone inflammation and bone injuries in OA. We have further elucidated the critical role of TLR4 in macrophage inflammatory response in this study. It shows evidence that TLR4 is a target of miR-6891-3p, while lncRNA IGHCγ1 can regulate TLR4 via lncRNA-miRNA sponge in macrophages. We have demonstrated a lncRNA IGHCγ1-miR-6891-3p-TLR4 axis in OA pathogenesis, which serves as target for the treatment of OA patients.
Although lncRNA IGHCγ1 has been documented to aggravate TLR4-mediated inflammation in macrophages via sponging miR-6891-3p in vitro, the effect of lncRNA IGHCγ1 as a ceRNA warrants being confirmed in in vivo studies including animal models of OA. In addition, although an IGHCγ1/miR-6891-3p/TLR4/NF-κB axis has been demonstrated in OA pathogenesis, more studies are needed to explore the downstream signaling points of the IGHCγ1/miR-6891-3p/TLR4 axis in regulating macrophage inflammation.
In summary, findings in this study will help to understand OA pathogenesis. The lncRNA IGHCγ1/miR-6891-3p/TLR4/NF-κB axis will offer potential biomarkers for OA treatment. Nevertheless, more studies are needed to fully elucidate the molecular mechanism of lncRNA IGHCγ1 in OA, especially studies in vivo.