LINC02085 Regulates Cell Growth and Inammatory Response in Rheumatoid Arthritis by Regulating PI3K/ AKT Signaling Pathway

Background: The present study explored the possible functions and the underlying mechanism of long Non-coding RNA LINC02085 in rheumatoid arthritis (RA). Methods: Primary broblast-like synoviocytes (FLS) were separated from synovial tissues and was established cell lines, then cultured for subsequent cell experiments by transfecting different vectors. Rat with AA were injected with sh-LINC02085. The progression of AA was explored by measuring arthritis score and histologic analysis. ELISA analysis was employed to detect the levels of inammatory 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. Results: The expression level of LINC02085 was signicant high in patients with RA, and positively associated with clinical indexes. 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 downexpression of LINC02085 received the opposite results. Knockdown of LINC02085 signicantly ameliorated the progression of AA reected by decreased arthritis score and cartilage destruction. Conclusion: The present study revealed that LINC02085 could regulate cell growth and inammatory response of RA-FLS by activating the PI3K/ AKT signaling pathway, subsequently playing important roles in promoting the occurrence and development of RA. for knockdown. promising therapeutic strategy for RA 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 veried 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 inammatory 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 inammatory response of RA-FLS by activating PI3K/AKT signals.

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).

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 x 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/ ), humidity (55%) and light/dark conditions (12/12 h). All the rats were allowed food and water ad libitum. All animal experiments were performed according to approved protocols by the Institutional Animal Care and Use Committee of the National Institutes of Health, USA. The adjuvant-induced arthritic (AA) rat model was established according to the protocol described in our previous study. Brie y, the AA model was induced in SD rats by subcutaneous injection into the right hind metatarsal footpad with 100 μL of Freund's complete adjuvant (FCA, Sigma). The rat of normal control group were also subcutaneously injected the saline solution with the same dosage.
For evaluating the therapeutic effects of LINC02085, when arthritis symptoms were appeared, the rats were randomly and evenly allocated into four groups, six rats in each group: normal control (NC), model control (MC), MC with the treatment of blank plasmid (sh-NC), MC with the treatment of LINC02085 plasmid (sh-LINC02085). Lentivirus stocks expressing shRNA for LINC02085 at a concentration of 5x108 virus particles per millilitre were injected via tail vein rats from the 4th day after the rst immunization.
The sh-NC group were injected with sh-NC to serve as an internal control. Arthritis scores (0-4 for each paw; maximum possible score 16) were determined every 5 days. Serum was collected for erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), TNF-α and IL-6 measurement. PBMCs were obtained to detect LINC02085 expression. Hematoxylin and eosin (HE) staining of the sections was performed to observe morphological changes in the synovial tissue.
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.

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 Tab 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; Tab 2). To explore the levels of LINC02085 in patients with RA, RT-qPCR was performed and the results showed signi cantly higher levels of LINC02085 in patients with RA compared with HC (p < 0.01; 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 (Tab 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 TNFαinduced 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. 8A-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.

LINC02085 treatment suppressed synovial in ammation and joint damage
To determine whether LINC02085 plays a crucial pathogenic role in the progression of RA, the lentivirus of LINC02085 shRNA or control shRNA was locally injected into in amed joints to knockdown the expression of LINC02085 in AA rat. As shown in Fig. 9A-B, we found that the arthritis index and paw swelling were signi cantly lower in the sh-LINC02085-treated group compared with that in the MC group. Our data from the RT-qPCR revealed that LINC02085 expression was signi cantly increased in MC group compared with NC group, while signi cantly decreased in sh-LINC02085 group (Fig. 9D). We also found that the serum level of ESR, CRP, RF, TNF-αand IL-6 were signi cantly decreased in the sh-LINC02085treated group (Fig. 9E-I). To further assess the effect of LINC02085 on developed arthritis, a histopathological evaluation was performed. The results suggested that the pathological features of RA were obviously observed in the ankle joints of MC group including in ammatory cell in ltration, synovial hyperplasia, and cartilage destruction. In contrast, sh-LINC02085 signi cantly attenuated the abovedescribed structural changes (Fig. 9C). Taken together, these ndings indicated that knockdown LINC02085 suppressed arthritic progression with signi cant amelior [1]ation of joint damage.

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. Furthermore, the in vivo results showed that knockdown of LINC02085 has an anti-in ammatory effect that can alleviate the progression of arthritis in AA rat, suggesting that LINC02085 knockdown could be potentially applied in the treatment of RA.
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.

Conclusion
Page 13 /18 In summary, the expression level of LINC02085 was signi cantly increased in RA. LINC02085 can in uence RA-FLS proliferation, invasion and migration, regulates in ammatory responses, which illustrates the pivotal role of LINC02085 in RA pathogenesis. Clinical studies have shown that there is a signi cant association between LINC02085 and other clinical parameters. In vitro assays suggested thatLINC02085 mediates cell biological functions and in ammatory response of RA-FLS by activating PI3K/AKT signals. In vivo experiment was conducted to further con rm that knockdown of LINC02085 has an anti-in ammatory effect that can alleviate the progression of arthritis in AA rat.

Declarations
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.