Abstract
Our previous study using RNA sequencing and reverse transcription quantitative polymerase chain reaction (RT-qPCR) validation identified a long non-coding RNA (lnc), lnc-AL928768.3, correlating with risk and disease activity of rheumatoid arthritis (RA), then the present study was conducted to further investigate the interaction of lnc-AL928768.3 with lymphotoxin beta (LTB) and their impact on proliferation, migration, invasion, and inflammation in RA-fibroblast-like synoviocytes (RA-FLS). Human RA-FLS was obtained and transfected with lnc-AL928768.3 overexpression, negative control overexpression, lnc-AL928768.3 short hairpin RNA (shRNA) and negative control shRNA plasmids. Then cell functions and inflammatory cytokine expressions were detected. Afterward, rescue experiments were conducted via transfecting lnc-AL928768.3 shRNA with or without LTB overexpression plasmids in RA-FLS. Lnc-AL928768.3 enhanced proliferation and invasion, inhibited apoptosis, while had little impact on migration in RA-FLS. In addition, lnc-AL928768.3 positively modulated interleukin-1β (IL-1β), IL-6 and IL-8 expressions in RA-FLS supernatant; moreover, it also positively regulated LTB mRNA expression, LTB protein expression, p-NF-κB protein expression, and p-IKB-α protein expression in RA-FLS. Furthermore, following experiment showed that lnc-AL928768.3 positively regulated LTB expression while LTB did not impact on lnc-AL928768.3 expression in RA-FLS. Furthermore, in rescue experiments, LTB overexpression curtailed the effect of lnc-AL928768.3 knock-down on regulating proliferation, invasion, apoptosis and inflammatory cytokine expressions in RA-FLS. Lnc-AL928768.3 promotes proliferation, invasion, and inflammation while inhibits apoptosis of RA-FLS via activating LTB mediated NF-κB signaling.
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INTRODUCTION
Similar to other autoimmune diseases, rheumatoid arthritis (RA) also has a heterogenous pathology, which mainly composes of synovitis, damage in cartilage and a systemic dysregulation of immune and inflammatory responses [1, 2]. Clinical presentations of RA result in a considerable disease burden in patients, these normally include the degeneration of joint functions that causes physical inactivity, disabling, severe complications, and increased psychological pressure, which all together contribute to a decrease in quality of life [1]. Reduction in disease activity and achieving clinical remission are the foundation of RA treatment, however, these goals are still hard to accomplish in many patients although the targeted medicine has already boosted in the last two decades [3,4,5]. Therefore, the dig in RA pathogenesis cannot stop until more valuable findings are uncovered to facilitate the diagnosis, treatment and quality of life improvement. Very fortunate, the improvement in gene sequencing technique and molecular experiments have revealed more potentials in this area.
Long non-coding RNA (lncRNA), a family of endogenous competing genes compose of more than 200 nucleotides, can barely function as protein coding genes but might be crucial in many biological and pathological process in human [6, 7]. Through years of investigations, lncRNAs have been brought to light in many disease conditions, such as cancers, neurological diseases, autoimmune diseases and inflammatory diseases [8,9,10,11]. Nonetheless, functional roles of lncRNAs in diseases are convoluted, which also accelerates the research in this area. Recently, a few lncRNAs have been reported in RA, such as lnc-HOTTIP and lnc-HOTAIR, these lncRNAs are revealed as potential regulators in RA pathogenesis; however, these investigations are far from enough [12, 13]. Previously, a lncRNA, lnc-AL928768.3, was identified by our study using RNA sequencing and subsequent reverse transcription quantitative polymerase chain reaction (RT-qPCR) validation, which discloses that this lncRNA is upregulated in synovium of RA patients compared to that of control, and it also positively correlates with inflammation level as well as disease activity in RA patients; moreover, our microarray analysis reveals a RA related gene as the target gene of lnc-AL928768.3, the lymphotoxin beta (LTB) gene [14]. However, more profound mechanistic role of lnc-AL928768.3 is not assessed in our previous study.
LTB is a cytokine belongs to the tumor necrosis factor (TNF) family, and the role of TNF is already well established in RA pathogenesis [15]. LTB is found to be involved in the regulation of many immune and inflammation related diseases, such as the colitis and autoimmune pancreatitis [16, 17]. Earlier, a few studies have also reported the role of LTB in RA. For instance, a study elucidates that LTB is upregulated in RA synovium and its expression is positively associated with the RA disease activity [18]. These indicate that LTB may be closely related to the RA pathogenesis. However, as one of the most crucial member of TNF family, the functional interactions of LTB with RA is yet to be deciphered. Herein, we presumed that LTB may also be involved in the modulation of RA progression.
Thus, based on the data of our previous study [14], we further investigated the interaction of lnc-AL928768.3 with LTB and their impact on cell proliferation, apoptosis, migration, invasion, and inflammatory cytokine expressions in rheumatoid arthritis-fibroblast-like synoviocytes (RA-FLS) in the present study.
MATERIALS AND METHODS
Cell Culture
Human RA-FLS (Cell Application, USA) and control-FLS (Ctrl-FLS; Procell, China) were cultured with 10% fetal bovine serum (FBS) (Gibco, USA) containing Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, USA) in a humidified incubator at 37 °C.
Plasmids Transfection
The lnc-AL928768.3 overexpression (Lnc( +)), negative control overexpression (NC( +)), lnc-AL928768.3 short hairpin RNA (shRNA) (Lnc (-)) and negative control shRNA (NC(-)) plasmids were constructed with pEXP-RB-Mam vector (Ribobio, China) and pRNAT-U6.1 vector (Ribobio, China), respectively. Meanwhile, the LTB overexpression plasmid (LTB( +)) were constructed with pEXP-RB-Mam vector (Ribobio, China) as well. To transfect the plasmids into RA-FLS, the HilyMax Transfection Reagent (Dojindo, Japan) was applied and the technical manual of reagent was followed. After the transfection, the RA-FLS were divided into Lnc( +), NC( +), Lnc(-) and NC(-) groups, respectively. And for the rescue experiment, the RA-FLS were termed as NC( ±), Lnc(-), LTB( +) and Lnc(-)/LTB( +) groups after transfection, respectively. The RA-FLS without transfection was used as normal control.
Cell Proliferation Assessment
At 0 h (h), 24 h, 48 h and 72 h after transfection, Cell Counting Kit-8 (Dojindo, Japan) was used to detect cell proliferation. In brief, 1 × 104 cells were seeded into 96-well plates and incubated. Then, 10 μl CCK-8 regent mixing with 100 μl DMEM was added into the plates and incubated with cells at 37 °C for 2 h. At last, the optical density (OD) value at 450 nm was measured with a microplate reader (BioTek, USA).
Cell Apoptosis Detection
At 48 h after transfection, the apoptosis of cells was evaluated by FITC Annexin V Apoptosis Detection Kit (BD, USA). After the collection of cells, the protocol of the kit’s was strictly followed. Then the cells were detected by a flow cytometer (BD, USA) and the data was analyzed by FlowJo 7.6 (BD, USA). The cells stained with Annexin V (with or without Propidium Iodide) were identified as apoptotic cells.
Migration and Invasion Assay
The wound healing and invasion assay were completed with the methods described in the previous study [19]. The photo was captured with an inverted fluorescence microscope (Olympus, Japan).
Enzyme-Linked Immunosorbent Assay (ELISA)
At 48 h after transfection, the supernatant was collected and the expression of Interleukin 1β (IL-1β), Interleukin 6 (IL-6), Interleukin 8 (IL-8), and tumor necrosis factor α (TNF-α) were evaluated with Human IL-1β ELISA Kit (mlbio, China), Human IL-6 ELISA Kit (mlbio, China) and Human IL-8 ELISA Kit (mlbio, China), respectively, according to the manufacturer’s instructions.
RT-qPCR
At 24 h after transfection, the cells were collected, then the total RNA extraction was performed with TRIzol™ Reagent (Invitrogen, USA). Afterwards, the transcription and qPCR were performed with PrimeScript™ RT reagent Kit (Takara, Japan) and TB Green Realtime PCR Master Mix (Takara, Japan). At last, the expressions of lnc-AL928768.3 and LTB were evaluated by 2−ΔΔCt method with the GAPDH served as internal reference. The primers were listed in Table 1.
Western Blot
After the cells were collected, the total protein extraction was performed with the application of RIPA Lysis Buffer (Sigma, USA). After quantified with BCA Protein Assay Kit (Beyotime, China), the protein was loaded into NuPAGE 4%-20% Bis–Tris Gel, then transferred to nitrocellulose filter membrane. After being blocked with 5% BSA (Beyotime, China), the membrane was incubated with primary antibodies at 4 °C overnight. Then, the incubation with secondary antibody was completed at 37 °C for 90 min. At last, the ECL Western Blotting Substrate (Thermo, USA) was adopted to visualized the protein bands. The antibodies applied were listed in Table 2.
Statistical Analysis
All the experiments were performed in triplicate. The data was expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 7.02 (GraphPad Software Inc., USA). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was used to detect difference among groups. P < 0.05 indicated statistically significant. P < 0.05, < 0.01 and < 0.001 were expressed as *, **, ***, respectively. Non-significant was marked as NS.
RESULTS
Regulation of lnc-AL928768.3 on Proliferation and Apoptosis of RA-FLS
Firstly, it was observed that lnc-AL928768.3 was upregulated in RA-FLS versus Ctrl-FLS (P < 0.01) (Fig. S1). Then, after transfections in RA-FLS, the relative expression of lnc-AL928768.3 was upregulated in Lnc( +) group compared to NC( +) group (P < 0.001) and was downregulated in Lnc(-) group compared to NC(-) group (P < 0.01), which suggested that the transfections were effective (Fig. 1A). In subsequent experiments, the OD value by CCK-8 was increased in Lnc( +) compared to NC( +) group at 48 h and 72 h, while was decreased in Lnc(-) group compared to NC(-) group at 72 h (all P < 0.05) (Fig. 1B). The apoptosis rate was reduced in Lnc( +) group compared to NC( +) group but was elevated in Lnc(-) group compared to NC(-) group (all P < 0.05) (Fig. 1C, D). These displayed that lnc-AL928768.3 promoted proliferation while inhibited apoptosis of RA-FLS.
Regulation of lnc-AL928768.3 on Migration and Invasion of RA-FLS
In terms of migration and invasion, the migration rate was of no difference between Lnc( +) group and NC( +) group (P > 0.05) while was slightly reduced in Lnc(-) group compared to NC(-) group (P < 0.05) (Fig. 2A, B). In addition, the invasive cell number was increased in Lnc( +) group compared to NC( +) (P < 0.01) while was decreased in Lnc(-) group compared to NC(-) group (P < 0.01) (Fig. 2C, D). These data indicated that lnc-AL928768.3 promoted invasion of RA-FLS.
Regulation of lnc-AL928768.3 on Inflammatory Cytokine Expressions in RA-FLS
The IL-1β (Fig. 3A) expression in RA-FLS supernatant was similar between Lnc( +) group and NC( +) group (P > 0.05), but was a little reduced in Lnc(-) group compared to NC(-) group (P < 0.05). Moreover, the IL-6 (Fig. 3B), IL-8 (Fig. 3C), and TNF-α (Fig. 3D) expressions in RA-FLS supernatant were both upregulated in Lnc( +) group compared to NC( +) group while were downregulated in Lnc(-) group compared to NC(-) group (all P < 0.05). These results disclosed that lnc-AL928768.3 elevated inflammation (reflected by pro-inflammatory cytokine expressions) in RA-FLS.
Regulation of lnc-AL928768.3 on LTB and NF-κB Signaling Pathway in RA-FLS
The LTB mRNA (Fig. 4A) and protein (Fig. 4B, C) expressions were enhanced in Lnc( +) group compared to NC( +) group, but were repressed in Lnc(-) group compared to NC(-) group (all P < 0.01). As for the NF-κB signaling pathway, the protein expressions of phosphorylated-NF-κB (p-NF-κB) and phosphorylated-nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor-α (p-IKB-α) were elevated in Lnc( +) group compared to NC( +) group (both P < 0.01), however, they were reduced in Lnc(-) group compared to NC(-) group (both P < 0.05). These implied that lnc-AL928768.3 positively regulated the LTB and NF-κB signaling in RA-FLS.
Effect of LTB Overexpression on Proliferation, Apoptosis, Migration, and Invasion of lnc-AL928768.3 Knocked Down RA-FLS
Subsequently, in the rescue experiments, the lnc-AL928768.3 mRNA expression was not impacted by LTB overexpression (all P > 0.05) (Fig. 5A); but LTB mRNA and protein expressions were downregulated by lnc-AL928768.3 knock-down in RA-FLS post transfections (all P < 0.05) (Fig. 5B-D). Furthermore, the OD value by CCK-8 at 48 h and 72 h was repressed by lnc-AL928768.3 knock-down in RA-FLS, while was enhanced by LTB overexpression in lnc-AL928768.3 knocked down RA-FLS (all P < 0.05) (Fig. 6A). In addition, the cell apoptosis rate was elevated by lnc-AL928768.3 knock-down in RA-FLS, while was reduced by LTB overexpression in lnc-AL928768.3 knocked down RA-FLS (all P < 0.05) (Fig. 6B, C). Similar to the cell proliferation (OD value by CCK-8), the cell migration rate (Fig. 7A, B) was downregulated by lnc-AL928768.3 knock-down in RA-FLS (P < 0.05), but was not impacted by LTB overexpression in lnc-AL928768.3 knocked down RA-FLS. As for cell invasion, the invasive cell number (Fig. 7C, D) was decreased by lnc-AL928768.3 knock-down in RA-FLS, while was increased by LTB overexpression in lnc-AL928768.3 knocked down RA-FLS. These indicated that lnc-AL928768.3 might regulate RA-FLS proliferation, apoptosis, and invasion via positively modulating LTB.
Effect of LTB Overexpression on Inflammatory Cytokine Expressions and NF-κB Signaling Pathway in lnc-AL928768.3 Knocked Down RA-FLS
The IL-1β (Fig. 8A), IL-6 (Fig. 8B), IL-8 (Fig. 8C), and TNF-α (Fig. 8D) expressions were decreased by lnc-AL928768.3 knock-down in RA-FLS supernatant, while were increased by LTB overexpression in supernatant of lnc-AL928768.3 knocked down RA-FLS (all P < 0.05). Besides, the p-NF-κB and p-IKB-α protein expressions were downregulated by lnc-AL928768.3 knock-down in RA-FLS (both P < 0.05), while were upregulated by LTB overexpression in lnc-AL928768.3 knocked down RA-FLS (both P < 0.05) (Fig. 9A, B). These data suggested that lnc-AL928768.3 might enhance inflammation and NF-κB signaling pathway via positively regulating LTB in RA-FLS.
DISCUSSION
RA patients not only suffer from a burden of physical inactivity but also bear an increased risk of many chronic diseases that may lead to a higher mortality risk, such as cardiovascular disease and pulmonary disease [20, 21]. Despite that the disease control of RA has achieved great improvement, thanks to the optimization of methotrexate use in practice, development in disease activity assessment tools, advancement in early diagnosis and prompt intervention, and the use of effective biologics, the present investigations still provide barely sufficient evidence for curing the disease [22,23,24,25,26,27]. Thus, experiments aiming at exploring more underlying pathology of RA has never stopped. Along with the boost in lncRNA studies, increasing researchers become to pay attention to the role of multiple specific lncRNAs in RA. Previously, we found that lnc-AL928768.3 is overexpressed in synovium of RA patients compared to that of controls, and it presents with good value for predicting RA risk; in addition, its expression in synovium shows positive correlation with C reactive protein (CRP) level and disease activity score 28 (DAS28) (ESR) in RA patients; furthermore, we also discovers that LTB is a target mRNA of lnc-AL928768.3 [14]. Therefore, in order to better investigate the mechanistic role of lnc-AL928768.3 and LTB in RA, the present study was conducted, which disclosed that: (1) lnc-AL928768.3 promoted proliferation, invasion, inflammation, but inhibited apoptosis in RA-FLS; (2) lnc-AL928786.3 positively regulated LTB expression and NF-κB signaling in RA-FLS; (3) LTB overexpression diminished the effect of lnc-AL928768.3 knock-down on proliferation, apoptosis, invasion, and inflammation in RA-FLS.
The mechanistic role of lnc-AL928768.3 in RA has yet to be revealed. Whereas, the other lncRNAs have been reported to regulate the RA pathogenesis in previous studies. For instance, a study displays that lnc-HIX003209 enhances inflammation through sponging microRNA-6089 (miR-6089) to regulate the TLR4/NF-κB signaling in PBMCs obtained from RA patients [28]. Another study reveals that in FLS obtained from RA patients, lnc-DILC enhances cell apoptosis and suppresses expression of IL-6, indicating it might be a regulator of RA inflammation [29]. In addition, another study illuminates that lnc-MEG3 represses proliferation and IL-23 expression through sponging miR-141 and positively regulating AKT/mTOR signaling in chondrocytes with damage induced by lipopolysaccharide (LPS) [30]. These previous studies present a role of some specific lncRNAs in regulating the pathological activities of RA just like ours. In regard to the lncRNA studied here, the lnc-AL928768.3, its mechanistic role in RA has yet to be reported and is elucidated for the first time by RNA sequencing in our previous study [14]. In the present functional study, we found that in RA-FLS, lnc-AL928768.3 promoted proliferation, invasion, inflammation, but inhibited apoptosis. These indicated that lnc-AL928768.3 was involved in the progression of RA through regulating FLS functions and inflammation. As for possible reasons for these results in the present study, we presumed that lnc-AL928768.3 probably regulated the RA-FLS functions and inflammation via interacting with some RA related factors, such as miRNAs, cytokines, chemokines or its target genes. For instance, our previous study found that many target genes of lnc-AL928768.3 are involved in the pathogenesis of RA, including SDC1, LTB, LAMA3, BTN2A2, etc., these genes are revealed to be abnormally expressed in RA patients or correlated with autoimmune dysregulation or inflammation [31,32,33,34]. However, these should be validated by further molecular experiments. In addition, as revealed by our subsequent rescue experiments, lnc-AL928768.3 might modulate cell functions and inflammation via positively mediating LTB and NF-κB signaling in RA-FLS.
In subsequent experiments, we found that lnc-AL928768.3 positively regulated the expression of LTB and NF-kB signaling in RA-FLS, indicating that lnc-AL928768.3 might regulate RA-FLS functions via these two items. LTB is one of the most studied cytokines that are closely related to autoimmunity, inflammation and cancers, and it is also involved in RA pathology [35,36,37]. As an example, a study illustrates that LTB producing lymphocytes interacts with responding FLS to promote the inflammatory cytokine expressions, metalloproteinase expressions, cell adhesion molecule expressions, and the expressions of chemokines C–C motif chemokine ligand 2 (CCL2) as well as CCL5 in FLS collected from RA patients [38]. And a clinical study shows that LTB gene expression in synovium is increased in RA patients compared to controls, and is correlated with increased inflamed level, pain visual analog score and Health Assessment Questionnaire Score in RA patients [18]. In terms of the NF-κB signaling pathway, it is even more established in RA pathology when compared with LTB. Most studies disclose the NF-κB signaling pathway as an essential factor promoting RA progression, for instance, a study reveals a regulatory network between seed genes and NF-κB protein family members, which was closely related to the inflammatory and immune signaling pathways related to the pathogenesis of RA [39]. Additionally, a study reports that receptor activator of nuclear factor-κB ligand (RANKL) inhibitors downregulates cell proliferation while upregulates cell apoptosis of RA-FLS via suppressing the NF-κB signaling [40]. These previous data suggest that LTB and NF-κB signaling both function as key promotors of the disease progression in RA.
Furthermore, the rescue experiments in our study uncovered that lnc-AL928768.3 possibly promoted proliferation, invasion, inflammation while inhibited apoptosis via activating LTB mediated NF-κB signaling in RA-FLS. To our best knowledge, the regulatory role of lnc-AL928768.3 for LTB and NF-κB signaling has not been reported. But the NF-κB could be regulated by other lncRNAs. For example, lnc-NEAT1 could promote the disease progression via sponging miR-204 and activating the NF-κB signaling in cell models of sepsis-induced acute kidney injury. In addition, lnc-HOTAIR participates in RA pathology as presented by reducing the inflammatory cytokine expressions in LPS treated chondrocytes via sponging miR-138 and downregulating the NF-κB signaling [12]. Besides, a study elucidates that lnc-Cox2 and lnc-AK170409 in macrophages present with NF-κB reporters as revealed by CRISPR/Cas-based screening, indicating these two lncRNAs participates in immune responses via regulating the NF-κB signaling [41]. However, in regard to the regulatory effect of any lncRNAs on LTB, no study has been reported. More importantly, the findings in our study shed light on the exploration of the mechanistic role of a novel lncRNA, lnc-AL928768.3, in RA, which provided more information on the fundamental etiology that could facilitate the search of targeted therapy for RA patients.
Conclusion
In conclusion, data in our study suggest that lnc-AL928768.3 promotes proliferation, invasion and inflammation while inhibits apoptosis of RA-FLS via activating LTB mediated NF-κB signaling.
Availability of Data And Materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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This study was supported by Natural Science Foundation of Zhejiang Province, China (Grant/Award Number: LY20H100002), Medical Science Research Foundation of Zhejiang Province, China (Grant/Award Number: 2020KY634) and Wenzhou Science and Technology Foundation (Grant/Award Number: Y2020922).
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JC contributed to the conception and design of the study. LS and LH contributed to the data acquisition. PC and YL contributed to the analysis and interpretation of data. JT contributed to drafting/revising of article. All authors read and approved the final manuscript.
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Sun, L., Hu, L., Chen, P. et al. Long Non-Coding RNA AL928768.3 Promotes Rheumatoid Arthritis Fibroblast-Like Synoviocytes Proliferation, Invasion and Inflammation, While Inhibits Apoptosis Via Activating Lymphotoxin Beta Mediated NF-κB Signaling Pathway. Inflammation 47, 543–556 (2024). https://doi.org/10.1007/s10753-023-01927-x
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DOI: https://doi.org/10.1007/s10753-023-01927-x