Application of m6A methyltransferase METTL14 in rheumatoid arthritis

Abstract

The invention discloses an application of m6A methyltransferase METTL14 in rheumatoid arthritis, which adopts real-time fluorescent quantitative PCR (polymerase chain reaction) (RT-qPCR) to detect m6A methylase gene mRNA (messenger ribonucleic acid) expression level and signal pathway in macrophage differential expression. The relationship between clinical immune inflammation markers, clinical symptoms, signal pathways and variables related to RA macrophage polarization and the m6A methyltransferase METTL14 level is also studied. Experimental results show that METTL14 is significantly increased in peripheral blood of RA patients and is related to immune inflammation markers and clinical symptoms. Meanwhile, METTL14 mediates macrophage polarization through an MAPK pathway, so that downstream inflammatory response is intensified. Therefore, the METTL14 has very strong diagnostic significance and is expected to become a novel RA marker and a novel treatment target.

Description

Technical Field

[0001] The invention belongs to the field of molecular biological detection of rheumatoid arthritis, and specifically relates to an application of m6A methyltransferase METTL14 in rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis is a common systemic autoimmune disease, which is mainly caused by an inflammatory response triggered by macrophage polarization. The inflammatory state of macrophages can be changed through epigenetic reprogramming. Macrophage polarization is obviously associated with RNA epigenetic changes known as n6-methyladenosine (M6A) RNA methylation. However, there are not many studies on the role of m6A methylation changes in RA treatment. The possible functions and underlying processes of m6A methylation changes in RA macrophage polarization changes are poorly understood. Summary of the invention

[0003] The present invention uses real-time fluorescence quantitative PCR (RT-qPCR) to detect the mRNA expression level and signaling pathway of m6A methylase gene in macrophage differential expression; enzyme-linked immunosorbent assay (ELISA) and flow cytometry are used to detect the expression level of specific cell secretory factors. The clinical importance and diagnostic usefulness of METTL14 for the occurrence of RA were examined.

[0004] The present invention also investigated the relationship between clinical immune-inflammatory markers, clinical symptoms, signaling pathways, and variables associated with RA macrophage polarization and the level of m6A methyltransferase METTL14.

[0005] Regarding the clinical manifestations and immune inflammatory markers of RA, the present invention found that increased expression of METTL14 was associated with joint discomfort and was positively correlated with CRP and RF levels. At the same time, METTL14 had a high sensitivity in predicting VAS. Proinflammatory factors (TNF-α) and m1 macrophage markers (CD68+CD86) were strongly correlated with the expression level of METTL14.

[0006] KEGG analysis showed that among the pathways related to inflammation, METTL14 had the strongest correlation with the MAPK signaling pathway. Further study of the relationship between MAPK pathway-related molecules and macrophage polarization revealed that JUN and EPK2 were positively correlated with the M1 macrophage marker CD68+CD86, and the proinflammatory factor TNF-a was positively correlated with these two markers. The expression levels of JUN and EPK2 in the METTL14 high expression group were significantly higher than those in the low expression group; there was no significant difference in the expression levels of p38 and EPK1.

[0007] The present invention discovered for the first time that the m6A methyltransferase METTL14 is significantly elevated in the peripheral blood of RA patients and is associated with immune inflammatory markers and clinical symptoms. At the same time, METTL14 mediates macrophage polarization through the MAPK pathway, thereby exacerbating downstream inflammatory responses. Therefore, METTL14 has a strong diagnostic significance and is expected to become a new RA marker and therapeutic target. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Screening of differentially expressed genes related to m6A methylase for RA macrophages. A: Venn diagram showing the intersection of m6A methylase genes and differentially expressed RA macrophage genes. B: Volcano plot of differentially expressed genes in RA macrophages. C: Heat map of differential expression of m6A methylase genes related to RA macrophages. D: Evaluation of m6A methylase gene mRNA expression levels in the two groups (***P<0.001).

[0009] Figure 2 This is a study on the correlation between immune-inflammatory markers, clinical symptoms and METTL14 in RA patients. A: Correlation analysis between METTL14 and immune-inflammatory markers in RA patients (*P<0.05; **P<0.01). B: Correlation analysis between METTL14 and clinical complaints of RA patients (*P<0.05). C: ROC curve.

[0010] Figure 3 Figure 2 shows the relationship between METTL14 expression and cytokines and macrophage polarization indices. A: Comparison of inflammatory factors TNF-α, IL-10 levels and macrophage markers CD68+CD86, CD68+CD206 between the two groups (***P<0.001). B: Correlation between TNF-α, IL-10 and CD68+CD86, CD68+CD206. C: Correlation between METTL14 and TNF-α, IL-10, CD68+CD86, CD68+CD206. D: Comparison of CD68+CD86, CD68+CD206, TNF-α, IL-10 levels between the METTL14 high expression group and the low expression group (*P<0.05; **P<0.01).

[0011] Figure 4 For KEGG and GO enrichment analysis.

[0012] Figure 5Correlation analysis between MAPK signaling pathway and METTL14. A: Comparison of mRNA expression levels of MAPK pathway-related molecules between the two groups (***P<0.001). B: Correlation between MAPK pathway-related chemicals and METTL14. C: Correlation between MAPK pathway-related molecules and macrophage polarization-related molecules (*P<0.05; **P<0.01). D: Evaluation of the expression levels of MAPK pathway-related molecules in the METTL14 low expression group and high expression group (*P<0.05). DETAILED DESCRIPTION

[0013] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the relevant principles and steps of the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention.

[0014] 1. Expression levels of m6A methylation modification enzyme genes associated with differential expression in RA macrophages

[0015] Using the G2R function, 5476 differentially expressed genes in RA macrophages were obtained by analyzing the dataset GSE97779. Then, the genes of m6A methylation modification enzymes were intersected with the differentially expressed genes in RA macrophages to obtain 4 genes. Figure 1 AB showed that these four genes (METTL14, CBLL1, RBM15 and KIAA1429) are all methyltransferases. The experiment determined the mRNA expression levels of each of these four genes. The results showed that the expression of METTL14 in the RA group was significantly higher than that in the healthy control group (P<0.001)( Figure 1 CD).

[0016] 2. Study on the relationship between METTL14 and immune inflammatory markers and clinical manifestations in patients with rheumatoid arthritis

[0017] The experiment found that METTL14 is expressed differently in RA participants compared to normal subjects. So, whether there is a relationship between immune inflammatory markers and the expression level of METTL14 mRNA in RA patients is unknown.

[0018] The present invention compares the expression of METTL14 mRNA with the degree of immune inflammation in RA patients and finds that METTL14 is significantly and favorably associated with RF and CRP ( Figure 2A). The expression level of METTL14 mRNA was compared between RA patient subgroups classified according to joint pain, morning stiffness, joint edema, joint mobility problems, and other clinical symptoms. The results showed that the expression level of METTL14 in the RA with joint pain group was significantly higher than that in the group without joint pain, and the difference was statistically significant (Z=2.883, P=0.004) ( Figure 2 B). According to the ROC curve results ( Figure 2 C), METTL14 is extremely sensitive to the visual analogue scale (VAS) pain index.

[0019] 3. Study on the relationship between METTL14 and cytokines and macrophage polarization markers in patients with rheumatoid arthritis

[0020] Flow cytometry was used to observe the expression of M1 macrophage markers CD68+CD86 and M2 macrophage markers CD68+CD206. In addition, ELISA was used to detect the expression of IL-10 and TNF-. Compared with the healthy control group, the expression of M1 macrophage markers CD68+CD86 and pro-inflammatory factor TNF- in the RA group was significantly increased, while the expression of M2 macrophage markers CD68+CD206 and anti-inflammatory factor IL-10 was significantly decreased ( Figure 3 A). CD68+CD86 was significantly positively correlated with TNF-α, and CD68+CD206 was significantly positively correlated with IL-10 ( Figure 3 B). These findings suggest that RA is associated with macrophage polarization and an unbalanced spectrum of inflammatory factors. Correlation analysis revealed that METTL14 was positively correlated with TNF-α and CD68+CD86 ( Figure 3 C). According to the median value of METTL14, RA patients were divided into METTL14 low expression group and METTL14 high expression group. Although there was no significant difference in the levels of CD68+CD206 and IL-10, the levels of TNF- and CD68+CD86 in the METTL14 high expression group were significantly higher than those in the low expression group ( Figure 3 D). These results suggest that high expression of METTL14 is closely related to M1 macrophage markers and pro-inflammatory factors.

[0021] 4. KEGG and GO enrichment analysis

[0022] The GSE97779 dataset was analyzed using the limit package in R to obtain the differentially expressed gene expression matrix. Correlation analysis was used to determine the genes that were significantly correlated with METTL14, and the screening criteria were p<0.01, r>0.6. For enrichment analysis, genes highly correlated with METTL14 were included in the DAVID database. GO analysis showed that BP was related to inflammatory response, endogenous immune response, positively regulated interleukin-6 synthesis, and cytokine-mediated signal transduction. On the other hand, CC and MF were related to peroxisomes and peroxisomal membrane processes. According to KEGG analysis, METTL14 is the most important pathway in the MAPK signaling pathway associated with inflammation ( Figure 4 AD). Therefore, in the following study, the MAPK signaling pathway was identified.

[0023] 5. Correlation analysis between MAPK signaling pathway and METTL14

[0024] Using RT-qPCR, we were able to measure the mRNA expression levels of MAPK pathway-related molecules in the serum of RA patients. We then studied the relationship between these compounds and METTL14. Comparing the RA group with the healthy control group, we found that the expression of c-Jun amino-terminal kinase (JUN), extracellular signal-regulated kinase 2 (EPK2), p38 mitogen-activated protein kinase (p38), and extracellular signal-regulated kinase 1 (EPK1) in the RA group was significantly increased ( Figure 5 A) Correlation Study Figure 5 B shows that METTL14 has no correlation with p38, but is significantly positively correlated with JUN, EPK1, and EPK2. Figure 5 C shows that there is a significant correlation between JUN and EPK2, the proinflammatory cytokine TNF-α, and the M1 macrophage marker CD68+CD86. RA patients were divided into two groups according to the median of METTL14: METTL14 high expression group and METTL14 low expression group. When the two groups were compared, it was found that the expression levels of JUN and EPK2 in the METTL14 high expression group were significantly higher than those in the low expression group, while the expression levels of p38 and EPK1 did not change significantly ( Figure 5 D) These findings suggest that METTL14 increases the subsequent inflammatory response via the MAPK signaling pathway.

[0025] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. An application of m6A methyltransferase METTL14 in rheumatoid arthritis, characterized in that: The preparation for detecting m6A methyltransferase METTL14 can be used for the preparation of diagnostic preparations for rheumatoid arthritis.

2. An application of m6A methyltransferase METTL14 in rheumatoid arthritis, characterized in that: Preparations for detecting m6A methyltransferase METTL14 can be used for the preparation of targeted therapeutic preparations for rheumatoid arthritis.

3. The use according to claim 1 or 2, characterized in that: The m6A methyltransferase METTL14 mediates macrophage polarization through the MAPK pathway, thereby aggravating downstream inflammatory responses.

Images