Long noncoding RNA LERFS negatively regulates rheumatoid synovial aggression and proliferation

Abstract

Fibroblast-like synoviocytes (FLSs) are critical to synovial aggression and joint destruction in rheumatoid arthritis (RA). The role of long noncoding RNAs (lncRNAs) in RA is largely unknown. Here, we identified a lncRNA, LERFS (lowly expressed in rheumatoid fibroblast-like synoviocytes), that negatively regulates the migration, invasion, and proliferation of FLSs through interaction with heterogeneous nuclear ribonucleoprotein Q (hnRNP Q). Under healthy conditions, by binding to the mRNA of RhoA, Rac1, and CDC42 - the small GTPase proteins that control the motility and proliferation of FLSs - the LERFS-hnRNP Q complex decreased the stability or translation of target mRNAs and downregulated their protein levels. But in RA FLSs, decreased LERFS levels induced a reduction of the LERFS-hnRNP Q complex, which reduced the binding of hnRNP Q to target mRNA and therefore increased the stability or translation of target mRNA. These findings suggest that a decrease in synovial LERFS may contribute to synovial aggression and joint destruction in RA and that targeting the lncRNA LERFS may have therapeutic potential in patients with RA.

Keywords: Immunology; Rheumatology.

Figure 1. Decreased levels of LERFS lncRNA in FLSs and STs from patients with RA.

(A) Total RNA harvested from RA FLSs (n = 3) and HC FLSs (n = 3) was screened by microarray analysis. Microarray heatmap of differentially expressed lncRNAs. (B) Volcano plot shows differentially expressed lncRNAs between RA FLSs and HC FLSs. P < 0.05, by Student’s t test. (C) The expression level of LERFS was validated by RT-qPCR in HC FLSs (n = 29) and RA FLSs (n = 34). Ct values were normalized to GAPDH. Data are presented as the mean ± SEM. **P < 0.01 versus HCs, by Student’s t test. (D) RA FLSs were stimulated with IL-1β (10 ng/ml), TNF-α (10 ng/ml), PDGF-BB (10 ng/ml), IL-17 (10 ng/ml), LPS (10 ng/ml), synovial fluid (SF), or synovial fluid containing IgG (SF + IgG) or a PDGF-neutralizing antibody (SF + anti-PDGF) (50 ng/ml) for 24 hours. (E and F) RA FLSs were treated with MTX (E) or DXM (F) for 24 hours. (D–F) *P < 0.05, **P < 0.01, and ***P < 0.001 versus untreated control, by 1-way ANOVA with Bonferroni’s post hoc comparison (n = 5). (G) Localization of LERFS was evaluated by RNA FISH assay. For silencing of LERFS, HC FLSs were transfected with a specific mixture of siRNA and ASO for LERFS (siLERFS). Shown are representative images of LERFS (green) and nuclei (blue). Graph shows the quantification of staining intensity for 5 different RA patients and HCs. Original magnification, ×630. **P < 0.01 versus HCs, by Student’s t test. (H) LERFS expression, detected by ISH staining, on STs from HCs and RA patients. Shown are representative images and quantification of the percentage of LERFS-positive cells for 5 different RA patients or HCs. Also shown is a representative image of RA in remission from 2 remitted patients treated with MTX and TNF-α inhibitor. A scrambled probe was used as a NC. Red arrows indicate LERFS-positive (blue) cells. Original magnification, ×400. ***P < 0.001 versus HCs, by Student’s t test. C, control. Data are presented as the mean ± SEM.

Figure 2. Inhibitory effects of LERFS overexpression on RA FLS migration and invasion.

(A and B) Chemotaxic migration of RA FLSs (A) or HC FLSs (B) was evaluated using a Transwell assay. Representative images (original magnification, ×100) are shown. Graphs indicate the relative migration rates. (C and D) The migration of RA FLSs (C) or HC FLSs (D) was analyzed using a wound-healing assay. Representative images are shown (original magnification, ×50). The relative migration rate represents the number of migrated cells normalized to the vector control. (E and F) In vitro invasion was determined using inserts coated with Matrigel Basement Membrane Matrix. The relative invasion rate was calculated by counting invaded cells and then normalized to the vector control. Representative images (original magnification, ×100) are shown. Graphs indicate the relative invasion rates. (G) LERFS overexpression impaired the formation of pseudopodium in RA FLSs. RA FLSs were wounded and stimulated with PDGF-BB (10 ng/ml) for 4 hours. Representative images are shown. Original magnification, ×400 (top); ×1,000 (bottom). Red arrow indicates lamellipodia formation; yellow arrow indicates filopodia formation. Graph indicates the number of RA FLSs with positive lamellipodia or filopodia. (H and I) Images show that LERFS knockdown promoted HC FLS migration (H) and invasion (I). Original magnification, ×100. Graphs indicate the relative migration (H) and invasion (I) rates. (J) Effect of LERFS overexpression on in vivo migration of RA FLSs. Representative images are shown (original magnification, ×400); red arrows indicate human FLSs. Graph indicates the number of migrated human FLSs stained with anti-human class I HLA antibody. (K) Effect of LERFS overexpression on the invasion of RA FLSs into human cartilage implants transferred under the skin of SCID mice. Arrows indicate RA FLS invasion into cartilage (Ca). Original magnification, ×200 (left); ×400 right (enlarged). Graph indicates the invasion scores. Data are shown as the mean ± SEM of 5 independent experiments involving 5 different RA patients or HCs. *P < 0.05, **P < 0.01, and ***P < 0.001 versus vector control, by Student’s t test.

Figure 3. Effect of LERFS overexpression on the proliferation and apoptosis of RA FLSs.

(A and B) An EdU incorporation assay was performed to evaluate cell proliferation. Representative images show proliferation of RA FLSs (A) and HC FLSs (C) labeled with EdU (red) and nuclei stained with Hoechst 33342 (blue) (original magnification, ×200). Graphs in A and C indicate the mean ± SEM of 5 independent experiments involving 5 different RA patients or HCs. (B) Detection of cell growth rates in vitro using an MTT assay at the indicated time points after lentivirus infection (D0 indicates the day of infection). Values are expressed relative to D0 as the mean ± SEM of 5 independent experiments. (D and E) Effects of LERFS overexpression on phases of the cell cycle. (D) Representative plots of cell-cycle distribution. (F) LERFS knockdown promotes proliferation by HC FLSs. Representative images are shown (original magnification, ×200). Data are shown as the mean ± SEM of 5 independent experiments involving 5 different RA patients (E) or HCs (F). (G) Effect of LERFS overexpression on apoptosis of RA FLSs. The cellular apoptosis rate was measured by annexin V and 7-AAD staining and detected by flow cytometry. Representative flow plots are shown. Total apoptosis represents the mean ± SEM percentage of 5 independent experiments involving 5 different RA patients. (H) Quantitative measurement of caspase 3/7 activity. Data are expressed relative to vector values and presented as the mean ± SEM of 5 independent experiments involving 5 different RA patients. (I) Effect of LERFS overexpression on FasL-induced apoptosis of RA FLSs. Cells were stimulated with or without 100 ng/ml recombinant human FasL for 24 hours. Total apoptosis represents the mean ± SEM of 3 independent experiments involving 3 different RA patients. *P < 0.05, **P < 0.01, and ***P < 0.001 versus vector, by Student’s t test.

Figure 4. LERFS functions by interacting with hnRNP Q.

(A) Experimental design for pulldown assays and identification of LERFS-associated cellular proteins. LERFS RNA was biotinylated by in vitro transcription, refolded, and incubated with lysates of RA FLSs. (B) Silver staining of biotinylated LERFS-associated proteins. A LERFS-specific band was excised and analyzed by MS, which identified hnRNP Q. (C) Western blot of proteins from LERFS-pulldown assays. (D) RIP evaluation of the interaction between hnRNP Q and LERFS within RA FLSs using an anti–hnRNP Q antibody (5 μg), with IgG (5 μg) as a NC. SnRNP70 was used as a positive control (right). **P < 0.01 and ***P < 0.001 versus IgG, by Student’s t test.U1, U1 small nuclear RNA (snRNA). (E) Comparison of LERFS binding with hnRNP Q between RA FLSs and HC FLSs. Data are shown as the mean ± SEM of 3 independent experiments involving 3 different RA patients and HCs. ***P < 0.001 versus HC FLSs, by Student’s t test. (F–H) Effect of hnRNP Q knockdown on the migration, invasion, and proliferation of RA FLSs. Representative images are shown (original magnification, ×200). Data for relative migration (F), invasion (G), and proliferation (H) are shown as the mean ± SEM of 5 independent experiments involving 5 different RA patients. *P < 0.05, **P < 0.01, and *** P < 0.001 versus siControl (siC) or vector, by Student’s t test. (I–K) Overexpression of hnRNP Q suppressed the migration (I), invasion (J), and proliferation (K) of RA FLSs. Representative images are shown. Original magnification, ×100 (I and J); ×200 (K). Data in I–K were normalized to the control group (vector) and are presented as the mean ± SEM of 5 independent experiments involving 5 different RA patients. **P < 0.01 and *** P < 0.001 versus siC or vector, by Student’s t test.

Figure 5. LERFS–hnRNP Q complex coregulates the expression of RhoA, Rac1, and CDC42.

(A and B) Effect of LERFS overexpression on mRNA (A) and protein (B) expression of RhoA, Rac1, and CDC42 in RA FLSs. (C) Effect of LERFS overexpression on the activation of RhoA, Rac1, and CDC42. RhoA, Rac1, and CDC42 activity was measured by G-LISA. (D–F) Effect of hnRNP Q knockdown on expression levels of mRNA (D) and protein (E) and activity (F) of RhoA, Rac1, and CDC42. (G) Effect of LERFS overexpression on protein expression of hnRNP Q. (A–G) *P < 0.05, **P < 0.01, and ***P < 0.001 versus vector or siC, by Student’s t test. (H) RIP detection of the combination of hnRNP Q and target mRNAs in RA FLSs. Values were normalized to the input. ***P < 0.001 versus IgG, by Student’s t test. (I) Effect of LERFS overexpression on the association between hnRNP Q and mRNA expression of RhoA, Rac1, and CDC42. Cell lysates from RA FLSs infected with control lentivirus (Vector) or LERFS OE were measured by RIP assay using antibodies against hnRNP Q or control IgG, followed by RT-qPCR assay of the indicated targets. Values were normalized to the input. *P < 0.05 versus vector, by Student’s t test. (J and K) Effect of hnRNP Q knockdown on LERFS overexpression–induced protein expression and activation of RhoA, Rac1, and CDC42. RA FLSs were transfected with hnRNP Q siRNA or siC for 24 hours, followed by infection of control lentivirus or LERFS OE. Three days later, cells were collected and subjected to Western blot analysis and G-LISA. Data shown are the quantification of protein levels (J) and activity (K) of RhoA, Rac1, and CDC42. Data are expressed as the mean ± SEM of 5 independent experiments involving 5 different RA patients. *P < 0.05 and ***P < 0.001, versus siC plus vector; ^#P < 0.05 and ^##P < 0.01, versus siC plus LERFS OE, by 1-way ANOVA.

Figure 6. Proposed model for LERFS-mediated regulation of the migration, invasion, and proliferation of FLSs.