VOPP1 as a Novel Susceptibility Gene in Rheumatoid Arthritis: Insights Into Its Mechanisms From Mendelian Randomization and Experimental Validation

Abstract

Background: Genetic factors are key determinants of vulnerability to rheumatoid arthritis (RA), a systemic inflammatory disease that causes inflammation, pain, swelling, and destruction of the joints. Expression quantitative trait loci (eQTLs) have been shown to detect novel disease-risk loci in previous studies. In this paper, we identified new susceptibility genes in RA and investigated their underlying mechanisms using integrated Mendelian randomization (MR) analysis.

Methods: Two-sample MR analyses were used to determine the causative links among eQTLs, metabolites, and RA risk. The study was conducted between January 2023 and June 2024. Synovial tissue samples were collected from patients undergoing joint surgery at the Affiliated Hospital of Nantong University. Functional validation of the candidate gene vesicular overexpressed in cancer pro-survival protein 1 (VOPP1) was performed in vitro using rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs), and in vivo in a collagen-induced arthritis (CIA) rat model. Expression levels of VOPP1 were evaluated by quantitative real-time PCR and Western blot. Additional assays assessed cell proliferation, inflammatory cytokine expression, and activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway.

Results: Our findings offer the first evidence that RA risk is increased by the VOPP1 eQTL. Furthermore, we discovered that the VOPP1 eQTL positively modulates the X-23,587 metabolite's levels, and raising this metabolite may make RA risk worse. Moreover, we demonstrate that VOPP1 is highly expressed in RA synovial tissues and RA-FLSs. VOPP1 stimulates the proliferation of RA-FLSs and the inflammatory response through the p38 MAPK signaling pathway according to functional experiments. We showed that VOPP1 knockdown reduced articular damage and synovial inflammation in vivo using a CIA rat model.

Conclusion: This study identifies VOPP1 as a novel gene associated with rheumatoid arthritis susceptibility. VOPP1 may contribute to disease progression by elevating X-23,587 metabolite levels and activating the p38 MAPK signaling pathway.

Keywords: Mendelian randomization; VOPP1; expression quantitative trait loci; fibroblast-like synoviocytes; p38 MAPK signaling pathway; rheumatoid arthritis; vesicular overexpressed in cancer pro-survival protein 1.

Figure 1

The whole Mendelian randomization (MR) study design.

Figure 2

VOPP1 was discovered to be a novel RA susceptibility gene by MR analysis.

Figure 3

VOPP1 is upregulated in RA synovial tissue and RA-FLSs. (A) The GSE55457 database analyses validated the VOPP1 expressions in RA. (B) q-PCR was used to measure VOPP1 mRNA levels in synovial tissues from NC (n=5) and RA patients (n=5). (C) Western blot analysis was performed to measure VOPP1 protein levels in synovial tissues from NC and RA patients. (D) Western blot analysis was performed to detect VOPP1 protein levels in NC-FLSs and RA-FLSs. *** p < 0.001 compared with the NC group (A); *p < 0.05 compared with the NC group (B–D).

Figure 4

MR analysis demonstrated that genetic susceptibility to VOPP1 eQTL could increase the risk of elevated X-23,587 metabolite levels.

Figure 5

MR analysis revealed that genetic susceptibility to X-23,587 metabolite levels could increase the risk of RA.

Figure 6

VOPP1 promotes cell proliferation and inflammatory response in RA-FLSs through activation of the p38 MAPK signaling pathway. (A) Western blot analysis was used to measure the efficiency of VOPP1 silencing in RA-FLSs. (B) The protein expression levels of VOPP1, p-p38, and p38 were assessed via Western blot after transfecting cells with VOPP1 siRNA or a negative control siRNA, followed by stimulation with LPS. (C) The protein expression levels of p-p38 and p38 were assessed via Western blot after transfecting cells with VOPP1 siRNA and/or then stimulated by anisomycin. (D) Cell cycles were analyzed by flow cytometry in RA-FLSs. (E) Cell proliferation of RA-FLSs was analyzed by EdU incorporation. (F) TNF-α and IL-6 levels in the supernatant of RA-FLSs were analyzed by ELISA. *, # p < 0.05 * compare with Con group (B) or NC group (C–F), # compare with LPS+NC group (B) or siRNA group (C–F).

Figure 7

Knowdown of VOPP1 reduced arthritis severity in CIA rats. (A) The arthritis index for different groups. (B) The paw thickness for different groups. (C) Representative images of H&E staining of ankle joint sections. Scale bar, 200 µM. (D) Representative images of Safranin O/Fast Green staining of ankle joint sections. Scale bar, 200 µM. (E) TNF-α and IL-6 levels in the serum of the different groups were analyzed by ELISA. *, # p < 0.05 * compare with Con group, # compare with CIA+siCtrl group.

Figure 8

Sketch map of the potential mechanism of VOPP1 in RA.