Excessive MALAT1 promotes the immunologic process of neuromyelitis optica spectrum disorder by upregulating BAFF expression

2.1 Participants

Between September 2017 and September 2021, a total of 38 patients were enrolled in this study, all from the First Affiliated Hospital of Fujian Medical University, following 2015 NMOSD diagnostic criteria developed by the international NMO Diagnostic Group (IPND) [21], including 35 patients who were AQP4-IgG positive and three patients were MOG-IgG positive (CBA assay, in house). Clinical data such as duration of disease, frequency of attack, treatment plan, and EDSS score were presented in Table 1. All patients were in the acute stage. Thirty-eight healthy control groups were matched by sex and age. According to recent studies, MOG antibody-associated disease (MOG-AD) has been separated from NMOSD disease as a new disease entity [22–24]; therefore, the NMOSD patients referred to in this research only include AQP4 antibody-positive patients. Total transcriptome sequencing was performed in three pairs of untreated NMOSD patients and matched healthy controls. All of the above patients were from the clinical cohort (NCT: 04386018).

2.2 CD14⁺ cells isolation

From NMOSD patients and controls, 10 mL of blood was collected in vacuette containing ethylenediaminetetraacetic acid and immediately processed according to biosafety rules. Peripheral blood mononuclear cells (PBMCs) were isolated on a Ficoll density gradient (TBDscience, China). All PBMC samples were studied fresh (operative time ≤6 h), which were classified into CD14⁺ cells bead by positive magnetic separation (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. The sorted cells were determined to have purity greater than 90% by flow cytometry.

2.3 Whole transcriptome sequencing

Three untreated NMOSD patients in the acute stage of the disease and three age–gender matched healthy controls were included. After extracting the total RNA, double-stranded cDNA was synthesized and purified. After terminal repair, a ring connector was connected at both ends of the double-stranded cDNA under the action of DNA ligase, and then the ring was opened by USER enzyme and the second strand of cDNA was disconnected. AMPure XP Beads (Beckman Coulter, Inc.) are used to screen the fragment length to make the target fragment length in the range of 300–400 bp. High-fidelity DNA polymerase is used for PCR amplification and AMPure XP Beads were purified. After quality inspection, the purified library was sequenced by Illumina HiSeq sequencing platform. Clean data were obtained by filtering the sequencing data and compared with the specified reference genome for analysis of transcription/gene expression and differential expression of transcription/gene. The databases involved include Ensemble, GO, KEGG, and STRING. Application software included FastQC, Cutadapt, HISat2, SAMtools, Stringtie, DESeq2, BCFTools, MISA, rMATS, CIRCexplorer, CPAT, R, and Cytoscape.

2.4 Dual-luciferase assay

The promoter validity and miRNA target genes were verified by luciferase double reporter assay. Since miRNA mainly acts on the 3′-UTR of the target gene, the MALAT1 3′-UTR region was constructed behind the reporter gene Luciferase in the vector (Table S1). After comparing the overexpression or interference of miR-30b-5p, changed in reporter gene expression (monitoring changes in luciferase activity) could quantitatively reflect the interaction ability between miR-30b-5p and MALAT1. Combined with site-directed mutation, their action sites were further identified. Plasmid construction: vector: GV272 (Jikai), XbaI/XbaI digestion (Jikai).

HEK293T cells were cultured to logarithmic growth stage, made cell suspension, counted, inoculated in a 24-well culture plate (about 105 cells), and cultured in 5% CO₂ incubator at 37°C until the cell fusion degree reached about 60%. The constructed plasmid was transfected with X Tremegene HP (ROCHE) transfection reagent. The expression of fluorescent-labeled genes on the plasmid was observed 24–48 h after transfection to determine the transfection efficiency. Luciferase (Dual-Luciferase^® Reporter Assay System, Promega) was detected 48 h after transfection. Firefly luminescence and Renilla luminescence were measured with a microplate tester.

The experimental data in this study were presented as the mean ± standard deviation (SD) of three independent experiments. The statistical significance of the differences in the experimental data was valued by ANOVA followed by Tukey’s post hoc test. The statistical significance level was set as *P ＜ 0.05, **P ＜ 0.01, and ***P ＜ 0.001.

2.5 Quantitative PCR of participant whole blood cells and monocytes

To further confirm the expression levels of miR-30b-5p, MALAT1, and BAFF, total RNA including non-coding RNAs was extracted from the whole blood cells and sorted CD14⁺ cell of NMOSD patients and matched with the healthy controls by miRNeasy extraction kit (Qiagen, Valenica, CA) using QIAzollysis reagent according to the manufacturer’s instructions. Concentration of RNA was determined using NanoDrop2000 which is very accurate to measure even the small quantities of RNA (Thermo Scientific, USA). Reverse transcription was carried out on extracted RNA in a final volume 20 μL reactions using RT2 first strand kit (Qiagen, Valenica, CA) according to the manufacturer’s instructions. Quantitative PCR was performed with the TaqMan Gene Expression Master Mix (Applied Biosystems) using primers against miR-30b-5p, MALAT1, and BAFF (Table S2). mRNA levels were standardized to the reference human β-actin and GAPDH Endogenous Control (Applied Biosystems). Real-time PCR was done on 20 μL reaction mixture using Rotor geneQ System (Qiagen) with the following conditions: 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 60°C for 60 s. Gene expression relative to internal control (2^−ΔΔCt) was calculated. Fold change was calculated using 2^−ΔΔCt for relative quantification. At the same time, we also analyzed the correlation between the three.

2.6 Construction of miR-30b-5p cell lines overexpression and knockdown

THP-1 cells were cultured in medium containing 10% fetal bovine serum at 37°C under 5% CO₂, and transfected with plasmid during logarithmic growth phase. THP-1 cells were divided into miR-30b-5p Nor group (transfected with empty vector), miR-30b-5p KD group (transfected with LV-has-mir-30b-5p-inhibition[43448-1]), and miR-30b-5p OE group (transfected with LV-has-mir-30b-5p[41827-3]). The cells were inoculated at a density of 5 × 10⁵/well and cultured in a cell incubator containing 5% CO₂ at 37°C. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used to transfect cells (4 Ig per well) and the cell adhesion integration rate was 80%. Cells were collected 24 h after transfection, and the expression of miR-30b-5p was detected to determine whether the overexpressed and knockdown miR-30b-5p cell lines were successfully constructed. Quantitative PCR method of miR-30b-5p, MALAT1, and BAFF was the same as the method mentioned above. The difference had statistical significance (P < 0.01).

2.7 Western blot analysis

The above THP-1 cells were lysed in protein lysis buffer containing a proteinase inhibitor (Thermo Fisher Scientific). Lysates were then centrifuged for 15 min at 14,000g at 4°C, and the protein concentration was determined using the Bradford Protein Assay (Thermo Fisher Scientific). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 10% polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 buffer and immunoblotted with primary antibodies, including anti-BAFF (Abcam) and anti-β-actin (Cell Signaling, BSN, USA). The band intensity was then quantified using Quantity One software (Bio-Rad, CA, USA).

2.8 Statistical analysis

The data were expressed as mean ± SD. Means of two continuous normally distributed variables were compared by independent samples Student’s t-test. Mann–Whitney U-test was used, respectively, to compare means of two and three or more groups of variables not normally distributed. Correlations were analyzed using Pearson’s correlation coefficient. Significance was set as P < 0.05. All analyses were performed using the SPSS 26.0 software and GraphPad Prism 9. A probability (P) value of <0.05 was considered to be significant.

1. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee. This study was approved by the ethics commission of the first affiliated hospital of Fujian Medical University.

2. Informed consent: Informed consent has been obtained from all individuals included in this study.

3.1 MALAT1 was aberrantly upregulated expression in patients with NMOSD

In order to search for the upstream regulatory sites of miR-30b-5p, we performed whole transcriptome sequencing based blood in three pairs of patients with NMOSD and matched healthy control. Using the criteria of >2-fold change, P < 0.05, and the threshold of fragments per kilobase of transcript per million (FPKM) mapped reads >50, we identified a total of 247,574 IncRNAs that were differentially expressed between patients and healthy control (Figure 1(a)). The results showed that the expression level of MALAT1 in NMOSD patients was significantly higher than that in healthy controls after difference analysis on IncRNA level (Figure 1(c)).

Figure 1

MALAT1 was overexpressed in NMOSD patients and had binding site with miR-30b-5p. (a) 247574 IncRNAs were differentially expressed in the volcano plot of differentially expressed gene, including MALAT1 (top 10). (b) RNAInter and Starbase v2.0 databases were used to predict the target IncRNA of miR-30b-5p, and MALAT1 was one of them. (c) Columnar statistics of FPKM values of MALAT1 in NMOSD and healthy controls (***P < 0.001). (d) Starbase2.0 database showed that MALAT1 (uplink) had a binding site with miR-30b-5p (downlink). The results of the double luciferase reporter gene experiment showed that miR-30b-5p could bind to the 3′-UTR region of MALAT1 and lost its binding ability after site-directed mutation. In positive control, TRAF6 gene 3′-UTR plasmid was used to overexpress hSA-miR-146b vector plasmid (***P < 0.001).

3.2 MALAT1 was interacted with miR-30b-5p

MALAT1 screened by whole transcriptome sequencing and miR-30b-5p noted in previous studies were predicted in starbase2.0 database and the RNAInter database, and both were found to have binding sites (Figure 1(b)). At the same time, Luciferase reporter gene assay was carried out. The sequence of MALAT1 3′-UTR region was constructed into Luciferase gene of GV272 vector, and then transfected into cells. After overexpression or interference of miR-30b-5p, the expression of Luciferase reporter gene was detected. The interaction between miR-30b-5p and MALAT1 could be quantitatively reflected by using sea kidney luciferase as an internal reference gene. Meanwhile, the sites of action were further identified by site-directed mutation. Experimental results confirmed that MALAT1 could bind to miR-30b-5p and had an interaction relationship (Figure 1(d)).

3.3 Increased accumulation of MALAT1 upregulated BAFF expression by the signaling pathway of miR-30b-5p in the whole blood cells and CD14⁺ monocytes of NMOSD patients

To further understand the mechanism of action of MALAT1 and miR-30b-5p in the disease process of NMOSD, expression in whole blood cells of 35 NMOSD patients and healthy controls were recruited for preliminary evaluation. Interestingly, MALAT1 and BAFF was overexpressed (Figure 2(a) and (c)), while miR-30b-5p expression was decreased (Figure 2(b)). Combined with functional annotation and signaling pathway prediction by whole transcriptome sequencing, the results showed that the relevant target genes were mainly enriched in pathways similar to systemic lupus erythematosus (SLE) immune inflammation, chronic myeloid cytopathy, and viral infection cancerous lesions (Figure 3(a) and (b)), which was why we focused more on inflammatory alterations in NMOSD myeloid immune cells, and in connection with previous literature reports, CD14⁺ monocytes became the breakthrough point for the next study.

Figure 2

Expression and relationship of MALAT1, miR-30b-5p, and BAFF in the whole blood cells and CD14⁺ monocytes of NMOSD patients in qPCR assay. (a–c) 2^−△△CT values of MALAT1, miR-30b-5p, and BAFF in the whole blood cells in control group and NMOSD group, respectively (*P < 0.05, ***P < 0.001, ****P < 0.0001). (d–f) 2^−△△CT values of MALAT1, miR-30b-5p, and BAFF in CD14⁺ monocytes in control group and NMOSD group, respectively (*P < 0.05, **P < 0.01, ****P < 0.0001). (g) Correlation between the expression of MALAT1 and miR-30b-5p in CD14⁺ monocytes. (h) Correlation between the expression of BAFF and miR-30b-5p in CD14⁺ monocytes.

Figure 3

Bioinformatic analysis of whole transcriptome sequencing, then quantitative PCR assay and western blot analysis for BAFF in miR-30b-5p OE group, miR-30b-5p KD group, and miR-30b-5p Nor group. (a) KEGG analysis and (b) GO analysis. (c) The respective bar graphs showed that the expression of MALAT1 gene was increased in miR-30b-5p KD group, and the expression of MALAT1 gene was decreased in miR-30b-5p OE group (**P < 0.01, ****P < 0.0001). (d) The respective bar graphs showed that the expression of BAFF gene was increased in miR-30b-5p KD group, and the expression of BAFF gene was decreased in miR-30b-5p OE group (**P < 0.01, ***P < 0.001, ****P < 0.0001). The cellular status after transfection with LV-has-miR-30b-5p-inhibition[43448-1] and LV-has-miR-30b-5p[41827-3] for 72 h. (e) Representative western blot for BAFF among three group (miR-30b-5p Nor, miR-30b-5p KD, and miR-30b-5p OE).

The relationship between MALAT1 and miR-30b-5p was extended to monocyte validation in patients, then we studied whether BAFF was identified as a downstream molecular target of both miR-30b-5p. Real time PCR results showed that MALAT1 and BAFF were significantly increased in CD14⁺ cells of NMOSD patients (Figure 2(d) and (f)), while miR-30b-5p was significantly decreased (Figure 2(e)). Meanwhile, the expression of MALAT1 was negatively correlated with miR-30b-5p (r = −0.41, P = 0.02, Figure 2(g)), and the expression of BAFF was negatively correlated with miR-30b-5p (r = −0.68, P = 0.0001, Figure 2(h)). Experimental evidence suggested MALAT1/miR-30b-5p/BAFF signal axis participated in immunity process of NMOSD monocyte. In this experiment, the purity of CD14⁺ cells sorted by flow-verified magnetic beads can reach over 90% (Figure S1).

In addition, comparing NMOSD patients, in CD14⁺ monocytes from three MOG-AD patients, MALAT1 expression was reduced and miR-30b-5p expression was elevated, while BAFF mRNA overexpression was not significant (P = 0.7459, Figure S2).

3.4 MALAT1 bound to miR-30b-5p to upregulate BAFF expression and trigger immune inflammatory responses

THP-1 was transfected with different plasmids to construct three cell lines: overexpression of miR-30b-5p THP-1 cell group (miR-30b-5p OE), knockdown of miR-30b-5p THP-1 cell group (miR-30b-5p KD), and empty viral THP-1 cells (miR-30b-5p Nor). The correlation between miR-30b-5p and BAFF was further verified in the above three types of cells. At the RNA level, real time PCR detection showed that the expression of MALAT1 was upregulated in THP-1 cells down-regulated miR-30b-5p, while the expression of MALAT1 was down-regulated in THP-1 cells overexpressing miR-30b-5p (Figure 3(c)). In addition, BAFFmRNA expression was up-regulated in THP-1 cells that down-regulated miR-30b-5p and down-regulated in THP-1 cells that overexpressed miR-30b-5p (Figure 3(d)). At the protein level, down-regulation of BAFF protein expression was found in THP-1 cells that overexpressed miR-30b-5p, while the down-regulation of BAFF protein expression was found in THP-1 cells that knocked down miR-30b-5p (Figure 3(e)).