Long Non-coding RNA LINC02085 Mediates Cell Growth and Inflammatory Response of Fibroblast-Like Synoviocytes by Activating PI3K/AKT Signals in Rheumatoid Arthritisis


 Background: The present study explored the possible functions and the underlying mechanism of Long Non-coding RNA LINC02085 in rheumatoid arthritis (RA). Methods: Peripheral blood mononuclear cells (PBMCs) were collected from patients with RA. Primary fibroblast-like synoviocytes (FLS) were separated from synovial tissues and was established cell lines, then cultured for subsequent cell experiments by transfecting different vectors. The RT-qPCR analysis was employed for evaluating the levels of LINC02085 in the PBMCs and RA-FLS. ELISA analysis was employed to detect the levels of inflammatory cytokines. CCK8 assay, migration and invasion assays were used to evaluate the proliferation, migration and invasion abilities of cells, respectively. Besides, the levels of the the PI3K/AKT pathway-related proteins were measured by WB and IF. Spearman correlation analysis, association rule analysis, logistic regression analysis were used to assess the correlation between LINC02085 and clinical parameters. Results: The expression level of LINC02085 was significant high in patients with RA, and positively associated with age, ESR, CRP, RF, CCP and DAS28. Moreover, logistic regression analysis indicated that ESR, CRP, RF and DAS28 were risk factors for LINC02085. We found that LINC02085 was upregulated in RA -FLS and TNF-αstimulated. And overexpression of LINC02085 could promote proliferation, migration and invasion induced by TNF-α, through upregulating the levels of TNF-αand TNFAIP2 and promoting the activation of PI3K/AKT pathway. Whereas knockdown of LINC02085 received the opposite results. ﻿Conclusion: In conclusion, the present study revealed that LINC02085 could regulate cell growth and inflammatory response of RA-FLS by activating the PI3K/ AKT signaling pathway, subsequently playing important roles in promoting the occurrence and development of RA.

various autoimmune diseases, such as ankylosing spondylitis (AS) [13], osteoarthritis (OA) [14]and systemic lupus erythematosus (SLE) [15]with the complete mechanism. LncRNAs can crosstalk with immune cells and mediate immunological and in ammatory response through phosphoinositide-3-kinase (PI3K) / protein kinase B (AKT) signaling pathway [16]. Recently, a number of studies indicated that a number of dysregulated lncRNAs contribute to the in ammatory response in RA [17]. Certain differentially expressed lncRNAs in RA have been reported to affect the disease activity [18]. In our previous study, many differentially expressed lncRNAs were screened out from a high-throughput sequencing analysis, including LINC02085 [19]. Nevertheless, the precise role and mechanisms of LINC02085 in RA pathogenesis remain unclear, particularly regarding its role in regulating in ammation.
In the present research, we selected LINC02085 as the subject and investigated its levels in the PBMCs of patients with RA. Next, we performed cellular experiments to verify the possible mechanism and assess the effects of the abnormally expressed LINC02085 in in ammatory responses and cell biological processes of RA-FLS. In addition, PI3K/AKT signal pathway aspect was found to be active under the LINC02085 condition. Based on these encouraging results, we believe that the present study could further elucidate a theoretical basis for the underlying mechanism of LINC02085 in RA.

Ethics approval
This study was approved by the Ethics Committee of the First A liated hospital of Anhui University of Traditional Chinese Medicine, and all patients signed a statement of informed consent.

Subjects and Samples
The present study recruited 30 patients with RA from May 2015 to July 2015. The inclusion criteria were (1) all the subjects ful lled the 2010 American College of Rheumatology (ACR) criteria for the diagnosis of RA [20]; (2) complete clinical data of all patients were available. The exclusion criteria were (1) patients below the age of 18 years and older than 75 years were also excluded; (2) pregnant women, people with severe mental illness, people with liver and kidney function injury were excluded; (3) people for biologic agents treatment were excluded. In addition, 30 healthy participants served as healthy controls (HC).
PBMC collection and total RNA extraction Peripheral blood (5 ml) was collected from all subjects in vacuum blood tubes containing EDTA-K2.
PBMCs were isolated by density gradient centrifugation (Histopaque-1077, Sigma). Total RNA was extracted from PBMCs using TRIzol reagent (Invitrogen; Thermo Fisher Scienti c, Inc.) according to the manufacturers protocol and and stored at -80˚C. The RNA concentration was determined with a NanoDrop (Thermo Scienti c), and RNA integrity was assessed by agarose gel electrophoresis.
Cell culture Primary FLS were separated from synovial tissues. Brie y, synovial tissue samples were obtained from RA patients who had undergone joint replacement surgery, which was cut into small pieces and then minced and digested with 4 mg/mL collagenase type I (Sigma) for 1 h in Dulbecco's modi ed Eagle's medium (DMEM; Sangon Biotech, Shanghai, China) at 37 °C for 1 h. The cells were then digested using 0.25% trypsin. After centrifugation at 800 g and further at 1000 g, the RA-FLS was maintained in RPMI-1640 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 100 mg/L streptomycin and 1 × 105 U/L penicillin. The separated RA-FLS were cultured to the third to sixth generations and were used for further study.

Cell Transfection
The coding sequence of LINC02085 was ampli ed and inserted into pcDNA3.1 (+) to produce the overexpressed vector of LINC02085 (pcDNA3.1-LINC02085). The silenced vectors si-LINC02085 and si-NC, and scrambled siRNA (si-NC) were obtained from GenePharma Co., Ltd. (Shanghai, China). The RA-FLS were transiently transfected with the above vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Next, the transfected RA-FLS were incubated for 24 h and the cells were treated with TNF-α (10 ng/mL) for 24 h.
Cell Counting Kit 8 (CCK-8) assay CCK-8 was utilized to determine the viability of both the treated and untreated RA-FLS. Transfected cells were placed in three replicates at a density of 3 × 10 4 cells per well in a 96-well plate with 100 µl of medium and incubated for 0, 24, 48, and 72 h after treatment with 10 ng/ml TNF-α. Then, the cells were incubated for 2 h in 10 µl of CCK-8 solution. Absorbance (optical density, OD) was assessed at 490 nm using an absorbance reader. The experiment was performed in triplicate.
Quantitative real-time PCR (RT-qPCR) Total cellular RNA was extracted using TRIzol reagent (Invitrogen, NY, USA). To measure the expression of LINC02085, cDNA was reversely transcribed from total RNA by Prime Script TM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, Liaoning, China). And then qPCR was performed using TB Green™ Premix Ex Taq ™ (Tli RNaseH Plus) (TaKaRa, Shiga, Japan) according to the manufacturer's protocol. β-actin was used as an internal control. The primers were synthesized by Sangon Biotech. The primer sequences are listed in Table 1. The data were analyzed using the 2 − ΔΔct method. Cell invasion assay Transwell invasion assay was carried out following a similar procedure as the Transwell migration assay. In brief, wells in a 96-well plate were pre-coated with 5 µg of Matrigel (BD Matrigel matrix, Matrigel basement membrane matrix, Biosciences). Matrigel was diluted 1:5 with DMEM. RA-FLS were transfected for 48 h and suspended in 100 µL serum-free medium at a nal concentration of 3 × 10 4 cells/ml were seeded in the upper well. Similarly, in each lower chamber, 600 ml of DMEM medium with 10% FBS was added. Microscopic visualization and cells counting were conducted as described with the migration assays.

Western blotting (WB)
The RA-FLS were lysed by RIPA lysis buffer (Sangon Biotech), following which cytoplasmic and nuclear proteins were extracted from RA-FLS using a commercial kit (Pierce, Rockford, IL, USA). Each sample (25 mg protein) was prepared for electrophoresis running on 10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore). The membrane was then blocked in a 5% nonfat dry milk/Trisbuffered solution and incubated at 4 °C with primary antibody overnight: rabbit anti-human anti-p-PI3K (dilution, 1:1000; cat. No. ab182651), rabbit anti-human anti-p-AKT (dilution, 1:2000; cat. No. 4060s). The membrane was then washed in PBST for three times, and incubated with the horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) secondary antibody (dilution, 1:20000, ab6721) at 37 °C for 1.5 h. Lastly, the proteins were detected by enhanced chemiluminescence (ECL, Millipore, USA). The densities of the bands were quanti ed by Image J Software (version 1.45, NIH, USA).

Immuno uorescence assay (IF)
The RA-FLS were xed with 4% paraformaldehyde at 48 h post transfection, washed three times with PBS and blocked with 2% BSA in PBS for 15 min. After blocking, cells were incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. The RA-FLS were washed three times with PBS and incubated for 1 h with uorophore-conjugated secondary antibodies (1:1000; Invitrogen) in blocking buffer. Cell nuclei were stained with DAPI (Invitrogen). Images were acquired using a Zeiss LSM710 confocal microscope. Association rule mining (ARM) The clincial indexes rise was set to "T", while the clincial indexes decline was set to "F". The Aprior module of SPSS Clementine 11.1 software was used to analyze the correlation between observation clincial indexes.The most famous association rule is the Apriori algorithm, which aims to nd out the relationship between items in a data set, also known as shopping blue analysis. In our data, each drug was treated as a variable [21]. The formulae were as follows: where X→Y is an association rule, X (left-hand side [LHS]) and Y (right-hand side [RHS]) represent the set of LINC02085, σ(X) is the frequency of itemset X, X∪Y is the union of itemset X and Y, σ(X∪Y) is the frequency with which itemset X and itemset Y appear together, support(X→Y) is the frequency with which X and Y appear together, and con dence(X→Y) is the probability that itemset Y appears in the presence of X. The lift is the ratio of the probability of itemset Y appearing in the presence of X to the frequency of Y. Support and con dence are often used to eliminate meaningless combinations; lift is the validity of the rules.

Statistical analysis
Statistical analyses were performed with GraphPad Prism software 8.0 (GraphPad). Data are represented as the mean ± SD or median (interquartile ranges) and analyzed using Students t-test or one-way ANOVA. A Chi square test was used to compare categorical variables. Spearman correlation analysis was introduced to evaluate the correlations between the LINC02085 with the items of ESR, RF, CCP, DAS28, et al. Logistic regression analysis was used to identify the independent risk factors of LINC02085. A statistically signi cant difference was de ned as p < 0.05.

Results
Characteristics of the study subjects An independent cohort consisting of 30 RA patients and 30 HC were enrolled in the validation set for evaluation of abnormal LINC02085. The characteristics of the study subjects are summarized in Table 2. There were no signi cant differences between RA patients and HC regarding age or sex. However, patients with RA had higher ESR, CRP, RF, CCP, IGA, IGG, IGM, C4, C4, DAS28, VAS, SAS and SDS than those in HC (p < 0.05; Table 2).  Fig. 1A). To evaluate the diagnostic value of LINC02085, receiver operating characteristic (ROC) curve analysis was performed. The AUC of LINC02085 was 0.8861 (95% CI 0.7958-0.9765), which suggested that LINC02085 had potential diagnostic value for RA patients (Fig. 1B).
Association rule analysis of LINC02085 with clinical characteristics of RA patients Then we conducted an association rule analysis to determine the con dence, support, and lift value of LINC02085 and clinical characteristics of RA patients. The results are shown that the con dence and support value of LINC02085 and clinical characteristics both higher than 80%, and the degree of lift was more than 1 and P 0.05 through Aprior module analysis (Table 3). To assess risk factors for LINC02085, logistic regression analysis was carried out. Signi cant differences in LINC02085 was found between RA patients with ESR (p = 0.023), RF (p = 0.000), CCP (p = 0.013) and DAS28 (p = 0.002), indicating that ESR, CRP, RF and DAS28 were risk factors for LINC02085, the higher expression of ESR, CRP, RF and DAS28, the higher expression of LINC02085 (Fig. 3).
The expression of LINC02085 in RA-FLS To evaluate the expression level of LINC02085 in RA-FLS, we detected LINC02085 by RT-qPCR analysis in RA-FLS. A signi cant upregulation in the expression level of LINC02085 was observed in the RA-FLS were stimulated with TNF-αcompared with RA-FLS (Fig. 4). Also, the e ciency of overexpression and knockdown was assessed by RT-qPCR. The results suggested that LINC02085 was signi cantly elevated by the transfection of LINC02085 overexpression vector, and the knockdown of LINC02085 resulted in signi cantly reduced LINC02085 expression (Fig. 4).
Effects of LINC02085 aberrant expression on cell proliferation Furthermore, the cell viability of RA-FLS was remarkably increased by the TNF-α level relative to that in RA-FLS (Fig. 5). Consistently, TNF-αinduced cells with pc-DNA3.1 LINC02085 showed higher cell viability relative to the TNF-αinduced cells (Fig. 5). In addition, the cell viability was dramatically decreased in TNF-αinduced cells with si-LINC02085 relative to TNF-αinduced cells (Fig. 5).

Effects of LINC02085 aberrant expression on cell migration and invasion
To explore the effects of LINC02085 on RA-FLS migration and invasion ability, we performed a migration assay and an invasion assay using Boyden chamber. Transwell migration and invasion assays showed that TNF-α increased the ability of the migration and invasion of RA -FLS signi cantly, overexpression of LINC02085 effectively increased the migration and invasion ability of the RA-FLS, while knockdown of LINC02085 inhibited the migration and invasion of the RA-FLS (Fig. 6A-B).
Effects of LINC02085 aberrant expression PI3K/AKT pathway To further nd the possible correlation of LINC02085 abnormal expression and the pathway involved in RA, we performed experiments to detect the expression of PI3K/AKT pathway-related proteins. In relation to the untreated cells, the proteins expression of p-PI3K and p-AKT were signi cantly upregulated in TNFαinduced cells (Fig. 8A-C). The TNF-αinduced cells transfected with pcDNA31.-LINC02085 showed dramatically enhanced relative protein expression of p-PI3K and p-AKT compared with those in TNFαinduced cells ( Fig. 8A-C), whereas the protein levels were signi cantly reversed in the TNF-αinduced cells transfected with si-LINC02085 compared with those in the TNF-αinduced cells (Fig. A-C). Similar results were also illustrated in the hippocampus by immuno uorescence analysis (Fig. 8E-F). These ndings indicated that the PI3K/ AKT signal pathway could be activated by the highly expressed LINC02085 in RA.

Discussion
Expanding numbers of studies have documented that lncRNAs play critical roles in physiological and pathological responses in different human disease including RA [22; 23]. The current study rstly provided evidence that LINC02085 was overexpressed in PBMCs of patients with RA compared with the healthy participants. The LINC02085 levels in RA blood samples positively correlated with those of age, ESR, CRP, RF, CCP and DAS28. Additionally, there are high con dence, support and lift value between LINC02085 and clinical characteristics. Meanwhile, ESR, RF, CCP and DAS28 are independent risk factors for LINC02085. Further analysis showed that overexpression of LINC02085 promoted RA-FLS proliferation, migration and invasion ability, enhanced the in ammatory response by increasing the levels of TNF-αand TNFAIP2. Furthermore, upregulated LINC02085 remarkably increased the expression of p-PI3K and p-AKT. However, the opposite results results were observed for LINC02085 knockdown. LINC02085 offers promising therapeutic strategy for RA patients.
An increasing number of studies have indicated that abnormal lncRNAs expression is involved in various immune-mediated diseases and serves an important role in regulating the in ammatory response and cell growth [24]. A study revealed that lncRNA PVT1 can regulate the proliferation and in ammatory responses of RA-FLS by targeting microRNA-145-5p [25]. Another additional study demonstrated that inhibiting role of LINC01197 in in ammation in RA through the microRNA-150/THBS2 axis [26]. The present study found the upregulation of LINC02085 in RA. A recent study further demonstrated that abnormal expression levels of lncRNA in RA closely related to the severity of symptoms [27; 28]. Similarly, our study revealed a positive correlation of LINC02085 levels with those of ESR, CRP, RF and DAS28, indicating LINC02085 levels were associated with the severity of RA. In addition, the current study found that upregulated LINC02085 enhanced proliferation, migration, and invasion ability of cells. RA-FLS proliferation, migration and invasion are the most important pathologic features of RA, which together with in ammatory responses affect and promote each other, and involved in the pathogenesis of RA [29].
Our results suggested that overexpression of LINC02085 could promote RA-FLS cell growth. The previous studies have con rmed that RA-FLS could secrete various kinds of in ammatory cytokines, thereby directly aggravating the in ammatory response [30; 31]. Our ndings showed that in ammatory cytokines of TNF-αand TNFAIP2 were dramatically increased in TNF-α-treated RA-FLS transfected with pcDNA3.1-LINC02085; however, the levels were reversed when LINC02085 was suppressed, implying that the reduction in LINC02085 expression could suppress cell in ammatory response. Thus, we speculated that LINC02085 might participate in the occurrence and development of RA by involving in cell growth and in ammatory response of FLS.
To further nd out the potential correlation and mechanism of LINC02085 at the level of the signal pathway in RA-FLS, we selected the PI3K/AKT signaling pathway based on the following aspects. First, the PI3K/AKT signaling pathway has been showed to articipate in the pathogenesis of RA and may serve as an important target in RA therapies. For example, Huang et al. demonstrated that miR-26a-5p enhances cells proliferation, invasion, and apoptosis resistance of FLS in RA by regulating PTEN/PI3K/AKT pathway [32]. Similarly, Li reported that cinnamaldehyde attenuates the progression of RA through down-regulation of PI3K/AKT signaling pathway [33]. Second, lncRNA THRIL mediates cell growth and in ammatory response of FLS by activating PI3K/AKT signals in RA [16]. Based on these ndings, we speculated the existence of a possible direct or indirect regulatory correlation between LINC02085 and the PI3K/ AKT pathway. Our ndings showed that overexpression and knockdown of LINC02085 retrained the protein expression of p-PI3K and p-AKT, which veri ed our speculation. It can be said that PI3K/AKT not merely involved in immune-mediated responses, but also mediates important signaling transduction in cell biological progression. The activation of p-PI3K could rst induce the downstream p-AKT, followed by inducing high expression levels of in ammatory factors TNF-αand TNFAIP2, subsequently increased proliferation, migration and invasion ability. This study concluded that LINC02085 could induce the PI3K/ AKT signaling pathway activation. Therefore, we speculated that LINC02085 mediates cell growth and in ammatory response of RA-FLS by activating PI3K/AKT signals.
In summary, the present study revealed that LINC02085 was highly expressed in RA and its abnormal expression could regulate the cell growth and in ammatory response of RA-FLS by activating the PI3K/AKT signaling pathway through affecting proliferation, migration and invasion ability of RA-FLS, thereby promoting the occurrence and development of RA. Additionally, the model of sponge which lncRNA interacts with other miRNA may be one of the important regulatory mechanisms in cells. Based on the the current ndings, we have great interests in this research, our research team is attempting to explore the possible ceRNA molecular mechanism of LINC02085 in RA in our future work.    The expression of LINC02085 in RA-FLS. The LINC02085 level was detected in control RA-FLS, the cells were stimulated with TNF-α(10 ng/ml), the cells with pcDNA3.1-NC and TNF-α, pcDNA3.1-LINC02085 and TNF-α, si-NC and TNF-α, and si-LINC02085 and TNF-αby qRT-PCR. All experiments were repeated three times and data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 compared with RA-FLS, ▲p < 0.05, ▲▲p < 0.01, ▲▲▲p < 0.001 compared with TNF-α+RA-FLS.