Corrigendum: Exploring the shared molecular mechanisms between systemic lupus erythematosus and primary Sjögren's syndrome based on integrated bioinformatics and single-cell RNA-seq analysis.

[This corrects the article DOI: 10.3389/fimmu.2023.1212330.].

Exploring the shared molecular mechanisms between systemic lupus erythematosus and primary Sjögren's syndrome based on integrated bioinformatics and single-cell RNA-seq analysis.

Background: Systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) are common systemic autoimmune diseases that share a wide range of clinical manifestations and serological features. This study investigates genes, signaling pathways, and transcription factors (TFs) shared between SLE and pSS. Methods: Gene expression profiles of SLE and pSS were obtained from the Gene Expression Omnibus (GEO). Weighted gene co-expression network analysis (WGCNA) and differentially expressed gene (DEG) analysis were conducted to identify shared genes related to SLE and pSS. Overlapping genes were then subject to Gene Ontology (GO) and protein-protein interaction (PPI) network analyses. Cytoscape plugins cytoHubba and iRegulon were subsequently used to screen shared hub genes and predict TFs. In addition, gene set variation analysis (GSVA) and CIBERSORTx were used to calculate the correlations between hub genes and immune cells as well as related pathways. To confirm these results, hub genes and TFs were verified in microarray and single-cell RNA sequencing (scRNA-seq) datasets. Results: Following WGCNA and limma analysis, 152 shared genes were identified. These genes were involved in interferon (IFN) response and cytokine-mediated signaling pathway. Moreover, we screened six shared genes, namely IFI44L, ISG15, IFIT1, USP18, RSAD2 and ITGB2, out of which three genes, namely IFI44L, ISG15 and ITGB2 were found to be highly expressed in both microarray and scRNA-seq datasets. IFN response and ITGB2 signaling pathway were identified as potentially relevant pathways. In addition, STAT1 and IRF7 were identified as common TFs in both diseases. Conclusion: This study revealed IFI44L, ISG15 and ITGB2 as the shared genes and identified STAT1 and IRF7 as the common TFs of SLE and pSS. Notably, the IFN response and ITGB2 signaling pathway played vital roles in both diseases. Our study revealed common pathogenetic characteristics of SLE and pSS. The particular roles of these pivotal genes and mutually overlapping pathways may provide a basis for further mechanistic research.

Serum Uric Acid as a Diagnostic Biomarker for Rheumatoid Arthritis-Associated Interstitial Lung Disease.

Previous studies have suggested a correlation between uric acid (UA) and lung lesion in some diseases. However, it remains unknown whether UA contributes to the lung injury in rheumatoid arthritis (RA). Our study aimed to investigate the clinical value of the UA level in the severity of rheumatoid arthritis-associated interstitial lung disease (RA-ILD). We measured UA in serum and bronchoalveolar lavage fluid (BALF), and UA levels of subjects were compared. As for the role of UA on ILD, we incubated A549 cells with UA and the expression of EMT markers was measured by immunofluorescence staining. The concentrations and messenger RNA expression of IL-1, IL-6, and transforming growth factor-ß (TGF-ß) were measured by ELISA and RT-PCR, respectively. We observed that serum UA levels in RA were significantly higher than those in controls. And, higher UA was measured in both serum and BALF of patients with RA-ILD, particularly those with interstitial pneumonia (UIP) pattern. Additionally, the correlation of the serum and BALF UA levels with serum KL-6, a biomarker of ILDs, in RA was significant (r = 0.44, p < 0.01; r = 0.43, p < 0.01). And, the negative correlations of UA, in both serum and BALF, with forced vital capacity (r = -0.61, p < 0.01; r = -0.34, p < 0.01) and diffusing capacity for carbon monoxide (r = -0.43, p < 0.01; r = -0.30, p < 0.01) were measured in patients. In the ROC curve analysis, the AUC value of UA for RA-ILD was 0.744 (95% CI: 0.69-0.80; p < 0.01), and the AUC of serum UA for predicting UIP pattern of patients with RA-ILD was 0.845 (95% CI: 0.78-0.91; p < 0.01), which showed the significance of the UA in clinical settings. Also, the in vitro experiment showed that UA induced epithelial-to-mesenchymal transition (EMT) and production of IL-1, IL-6, and TGF-ß in A549 cells. Therefore, the elevated UA levels may be a diagnostic marker in RA-ILD, particularly RA-UIP.

Mesenchymal stem cells negatively regulate CD4+ T cell activation in patients with primary Sjögren syndrome through the miRNA­125b and miRNA­155 TCR pathway.

Treatment with mesenchymal stem cells (MSCs) has been revealed to suppress CD4+ T cells and autoimmunity in both mouse models and patients with primary Sjögren syndrome (pSS); however, the underlying mechanism remains unclear. MicroRNAs (miRNAs or miRs) mediate CD4+ T cell activation, but the mechanism is not understood, particularly for CD4+ T cells treated with MSCs. Characterization of miRNAs may reveal pSS pathogenesis, guide MSC treatment and provide more personalized management options. The present study aimed to perform an miRNome analysis of quiescent and T cell receptor (TCR)­activated CD4+ T cells treated with MSCs via miRNA profiles and bioinformatics. Following 72 h of co­culture, MSCs inhibited TCR­induced CD4+ T cell activation and decreased IFN­Î³ levels. The numbers of aberrant miRNAs in pSS naïve (vs. healthy naïve), pSS activation (vs. pSS naïve), MSC treatment and pre­IFN­Î³ MSC treatment (vs. pSS activation) groups were 42, 55, 27 and 32, respectively. Gene enrichment analysis revealed that 259 pathways were associated with CD4+ T cell stimulation, and 240 pathways were associated with MSC treatment. Increased miRNA­7150 and miRNA­5096 and decreased miRNA­125b­5p and miRNA­22­3p levels in activated CD4+ T cells from patients with pSS were reversed by MSC treatment. Notably, the proliferation of CD4+ T cells and CD4+ IFN­Î³+ cells, expression levels of miRNA­125b­5p and miRNA­155 in CD4+ T cells and supernatant IFN­Î³ secretion were associated with disease activity. miRNA may play a vital role in MSC treatment for activated CD4+ T cells. The results indicated that the expression levels of miRNA­125b­5p and miRNA­155 in TCR­activated CD4+ T cells from patients with pSS may provide insight regarding autoimmune diseases and offer a novel target for prospective treatment. Therefore, these results may be crucial in providing MSC treatment for pSS.

Murine embryonic mesenchymal stem cells attenuated xerostomia in Sjögren-like mice via improving salivary gland epithelial cell structure and secretory function.

BACKGROUND: Xerostomia is the main manifestation from patients with Sjögren syndrome (SS). However, traditional immunosuppressive agents are nearly invalid due to complicated etiopathogenesis in salivary glands, including aberrant immune dysregulation, epithelial structure destruction, and diminished secretory function. OBJECTIVE: To investigate the therapeutic effect of murine embryonic mesenchymal stem cells (ME-MSCs) on salivary glandular epithelium structure and secretory function in Sjögren-like mice. METHODS: Salivary flow rate (SFR), blood glucose, and body weight was weekly monitored among treatment group, disease group, and health control group. ME-MSCs were used to treat NOD mice via tail vein injection. HE staining and transmission electron microscope was used to evaluate the structure of salivary gland epithelial cells (SGEC). TUNEL fluorescence staining and PCNA immumohistochemical staining was used to evaluate the SGEC apoptosis and proliferation. The SGEC secretory function was tested by PAS staining and amylase immumohistochemical staining. RESULTS: ME-MSC treatment could elevate SFR, restore the acini and micromorphologies, promote the SGEC proliferation, and suppress the SGEC apoptosis in NOD mice, but not restore to that in health control group. The SGEC structure was more intact in treatment group. Mucopolysaccharide and amylase of salivary acinar cells in treatment group was better than that in disease group, although transmission electron microscopy showed secretory granules were lower than those in healthy control. CONCLUSION: ME-MSCs demonstrated its potential as a candidate treatment for xerostomia due to some effects on salivary flow rate in NOD mice by restoring the SGEC impairment and secretory function.

Effect of mesenchymal stem cells on Sjögren-like mice and the microRNA expression profiles of splenic CD4+ T cells.

Mesenchymal stem cells (MSCs) serve immuno-regulatory functions and offer a promising novel treatment for certain autoimmune diseases. The present study investigated the therapeutic effect of mice bone marrow (BM)-MSCs on mice with relatively late stage of Sjögren-like disease and the impact of BM-MSCs on the microRNA (miRNA) expression profiles of splenic CD4+ T cells. Female NOD/Ltj mice were randomized into two groups: The disease group (n=8) and the MSC-treated group (n=8). Female ICR mice served as the healthy control group (n=8). The MSC-treated group received an injection of MSCs when they were 26 weeks old. Water intake, blood glucose and salivary flow rate were measured and submandibular glands were resected and stained with hematoxylin and eosin to calculate the focus score. The concentrations of interleukin (IL)-2, IL-6, hepatocyte growth factor, interferon γ, IL-10, prostaglandin E2, transforming growth factor ß1 and tumor necrosis factor-α in serum were measured using ELISA. The expression of miRNAs in splenic CD4+ T cells were measured using deep sequencing. The results demonstrated that treatment with BM-MSCs prevented a decline in the salivary flow rate and lymphocyte infiltration in the salivary glands of NOD mice, indicating that MSC-treatment had a therapeutic effect on NOD mice with relatively late stage of Sjögren-like disease. ELISA and deep sequencing results showed that the three groups of mice had different serum concentrations of cytokines/growth factors and different miRNA expression profiles of splenic CD4+ T cells. This implies that the alteration in serum levels of cytokines/growth factors and miRNA expression profiles of splenic CD4+ T cells may explain the therapeutic effect MSCs have on Sjögren's syndrome.

miR-15b and miR-16 regulate TNF mediated hepatocyte apoptosis via BCL2 in acute liver failure.

Acute liver failure (ALF) still has an unacceptable high mortality rate, despite substantial improvements with multidisciplinary care. The precise underlying mechanism of ALF remains to be explored. It has been reported that microRNAs (miRNAs) are novel regulators in a number of liver diseases, but the role of miRNAs in the development of ALF is not fully understood. An ALF murine model was generated by ip injection of D: -GalN/LPS, which was confirmed with histopathology and biochemistry. The hepatic miRNA expression profile in ALF was determined by microarray and verified by qRT-PCR. The functions and signal pathways of the targeted genes of these deregulated miRNAs were predicted, using bioinformatics analysis. The possible underlying mechanism was investigated by exploring the relationship between miRNA modification and hepatocyte apoptosis. There were a total of 95 significantly changed miRNAs in ALF compared to mock-treated (P < 0.01). Among these 95 miRNAs, 20 were up-regulated and 26 were down-regulated at both 5 and 7 h time points. Bioinformatics analysis predicted that some of these 46 miRNAs were involved in apoptosis. Among the up-regulated miRNAs involved in apoptosis, miR-15b and miR-16 showed the highest enrichment and targeted the common anti-apoptotic gene, BCL2. Our in vitro data demonstrated that miR-15b and/or miR-16 regulated BCL2 at the protein level. Inhibition of miR-15b and/or miR-16 reduced hepatic apoptosis and TNF production. These data suggest that miR-15b and miR-16 regulate TNF mediated hepatic apoptosis via BCL2 during ALF, and may shed light on the development of a therapeutic strategy for treatment of ALF.

[The role of miRNA-122 expression during the acute liver failure in mice induced by D-GalN/LPS].

OBJECTIVE: To investigate the expression of miR-122 and its relationship with progression and development of acute liver failure in mice induced by D-GalN/LPS, and to explore new biomarker(s) for early diagnosis of acute liver failure. METHODS: BALB/C mice were randomly divided into four groups: the mice were given D-GalN (900 mg/kg body weight) and LPS (10 micog/kg body weight) intraperitoneally (i.p.) to construct the acute liver model; whereas the control groups were given D-GalN (900 mg/kg), LPS (10 microg/kg) and normal saline respectively. All biochemical and histological indexes were determined at 0, 1, 3, 5, 7 and 9 h respectively after administration. Real-time RT-PCR were used to detect the expression of miR-122 and pro-inflammatory cytokines, furthermore, the expression of miR-122 was verified by LNA (lock nucleic acid)-Northern-blot. ALT and AST levels were tested by biochemistry analyzer. Serum pro-inflammatory cytokine levels were tested by ELISA. RESULTS: The mortality rate was about 80% at 24h after D-GalN/LPS treatment, but no mortality was observed in the other three control groups. Liver special miRNA miR-122 was highly expressed in liver tissue of normal mice (ct is approximately equal to 14), it was up-regulated significantly (P = 0.013) at first hour after treatment then down-regulated according to the development of acute liver failure, the change was more obvious at 9 h (ct is approximately equal to 15, P = 0.002). ALT and AST levels increased obviously at 3h after treatment and reached peak at 7 hours then they were declined sharply. It was found that the expression of miR-122 was faster and more durable than ALT. Pro-inflammatory cytokines related to acute liver failure including TNFa and IL-6 were all up-regulated in serum as well as liver tissue (P less than 0.05). Correlation analysis showed that miR-122 had a negative correlation with ALT (correlation coefficients -0.505) and positive correlations with TNFa and IL-6 (correlation coefficients were 0.493 and 0.674 respectively). CONCLUSIONS: Liver-specific miR-122 supposed be a new marker molecule for early diagnosis of liver cells injury in the acute liver failure.

Effect of ribavirin and interferon beta on miRNA profile in the hepatitis C virus subgenomic replicon-bearing Huh7 cells.

Hepatitis C virus (HCV) infection is still a major global health issue despite decades of research. The liver-specific microRNA-122 (miR-122) can stimulate HCV replication/translation in vitro, indicating that miR-122 contributes to pathogenesis of HCV. However, it remains controversial whether interferon (IFN) inhibits HCV via modulating miR-122 expression. The underlying mechanism of ribavirin (RBV) in enhancing IFN treatment for HCV patients has yet to be explored. We investigated the relationship between miR-122 expression and anti-HCV activity of IFN beta in combination with RBV in vitro, due to difficulty accessing an HCV animal model. Upregulation of ISG54 mRNA or cytostatic effect was detected in Huh7 and HCV replicon cell lines in response to IFN beta or RBV stimulation, respectively. It was found that IFN beta and/or RBV suppressed miR-122 expression marginally, with a synergetic anti-HCV effect between IFN beta and RBV. Marginal modification of other miRNAs was also observed in these cell lines, using miRNA array following IFN beta and RBV treatment. Taken together, our data suggest that miRNAs are not crucial in anti-HCV action, following IFN beta and/or RBV stimulation in vitro.

MicroRNA155 is induced in activated CD4(+) T cells of TNBS-induced colitis in mice.

AIM: To investigate the expression of microRNA155 (miRNA155) in trinitrobenzene sulphonic acid (TNBS)-induced colitis and the relationship between miRNA155 and tumor necrosis factor (TNF) expressions. METHODS: In TNBS colitis mice, miRNA155 and TNF mRNA expressions were measured in colons and CD4(+) T cells of draining lymph nodes (LNs). CD4(+) T cells were cultured in vitro with or without anti-CD3/CD28 antibody, and the expressions of miRNA155 and TNF mRNA in cells and TNF concentration in culture media were examined. RESULTS: miRNA155 and TNF mRNA expressions in colons and in cells of LNs were significantly increased in TNBS colitis compared with controls. In TNBS colitis, miRNA155 and TNF mRNA expressions in CD4(+) T cells of LNs and TNF concentration in CD4(+) T cells culture media increased compared with controls. When cultured with anti-CD3/CD28 antibody, miRNA155 and TNF mRNA expressions in CD4(+) T cells and TNF concentration in the CD4(+) T cells culture media were significantly higher than those cultured without anti-CD3/CD28 antibody. Following analysis using the Pearson's correlation coefficient, miRNA155 expression had a significant positive correlation with either TNF mRNA expression in CD4(+) T cells (r = 0.860, P < 0.05) or TNF concentration in CD4(+) T cells culture media (r = 0.892, P < 0.05). CONCLUSION: miRNA155 is induced in colons and activated CD4(+) T cells in TNBS colitis, and the levels of miRNA155 and TNF expressions have a significant positive correlation.

[Real-time quantification of microRNAs in Huh7 cells by stem-loop reverse transcriptase polymerase chain reaction].

OBJECTIVE: To establish a convenient realtime PCR which can detect microRNAs in the human hepatoma cell line, Huh7 cells. METHODS: Total RNAs in Huh7 cells were extracted. MicroRNA 122, 24 and 146a were assayed by microRNA array, and then verified by Northern blot. Stem-loop RT-PCR and poly(A)-tailed RT-PCR were used to detect the above microRNAs. Data were analyzed with Quantity One software and 7500 system software. RESULTS: Microarray signal intensity of microRNA 122, 24 and 146a in Huh7 cells was 2201.49, 410.20 and 4.70, whose relative expression was confirmed as 0.0383, 0.0249, 0.0001 through Northern blot. While the poly(A)-tailed RT-PCR might only measure microRNA 122, Stem-loop RT-PCR could detect microRNA 122, 24 and 146a, whose average dCt was 2.5, 5.8 and 12.1 in accordance with microRNA array and Northern blot. CONCLUSION: Stem-loop RT-PCR can specifically and sensitively quantity microRNA levels, regardless of their abundance.

Expression of angiotensin-converting enzyme 2 in CCL4-induced rat liver fibrosis.

The renin angiotensin system (RAS) plays a major role in liver fibrosis. A novel homologue of angiotensin converting enzyme, ACE2, was identified as a negative regulator of RAS as it degrades Ang II to Ang1-7. We investigated in vivo the expression of ACE2 in liver fibrosis. We evaluated the relationship between biochemical variables and liver tissue expression of ACE2, the correlation between a histological assessment of liver fibrosis and liver tissue expression of ACE2. Male SD rats were randomly divided into a CCL4 group which received injections of CCL4 and the control group which received injections of olive oil. Liver pathology was examined by H&E and Sirius red staining, and real-time PCR was performed to determine the gene expression levels of ACE2 and ACE. Real-time PCR analysis revealed that ACE2 mRNA was higher at the two-, four-, and six-week time points, respectively (p<0.01). Similarly, hepatic ACE mRNA was significantly increased after CCL4 injection. There was a significant correlation between ACE and ACE2 gene expression (r=0.750, P<0.001). ACE2 gene expression strongly correlated with ALT (r=0.669, P<0.0001) and AST levels (r=0.815, P<0.0001). There was a significant correlation between circulating ACE2 and histological scores of liver fibrosis. ACE2 and ACE gene expression correlated with the ISHAK score (r=0.850, P<0.001; r=0.806, P<0.001). There was a significant relationship between ACE2 gene expression and the degree of liver fibrosis. ACE2 plays a crucial role in liver fibrogenesis.

Inhibitory effects of antisense oligodeoxynucleotide on expression of vascular endothelia growth factor by human hepatocarcinoma cells.

BACKGROUND: It is widely recognized that the growth of solid tumor depends on angiogenesis. Vascular endothelia growth factor (VEGF) is an endothelial cell-specific mitogen that promotes angiogenesis in solid tumor. Inhibition of angiogenesis is considered a promising approach for cancer therapy, and treatments including administration of antisense drugs and RNA interference for the VEGF gene are geared to the suppression of tumor angiogenesis. METHODS: As a new approach for gene therapy of hepatocellular carcinoma (HCC), four groups of antisense oligodeoxynucleotide (ASODN) (A-Cap, A-AUG, A-UGA and A-Exon-3) were used to block the expression of VEGF, then VEGF mRNA and protein were detected by RT-PCR and Western blot. RESULTS: After treatment with ASODN, the relative VEGF mRNA levels of A-Cap, A-AUG, A-UGA, and A-Exon-3 were decreased significantly to (32+/-9)%, (63+/-1)%, (86+/-3)%, and (70+/-5)%, respectively(F=64.18, P<0.001). The relative VEGF protein levels of A-Cap, A-AUG, A-UGA and A-Exon-3 were decreased significantly to (41+/-5)%, (59+/-3)%, (88+/-7)%, and (79+/-9)% respectively (F=60.64, P<0.001). CONCLUSIONS: Among the four ASODNs, the ASODN for Cap structure showed the strongest inhibitory effect and that for A-UGA, the least (P<0.05 ). The inhibitory effect of ASODN on the expression of VEGF proteins was similar to that of VEGF mRNA expression.

Methylation profile of the promoter CpG islands of 14 "drug-resistance" genes in hepatocellular carcinoma.

AIM: To establish the DNA methylation patterns of the promoter CpG islands of 14 "drug-resistance" genes in hepatocellular carcinoma (HCC). METHODS: The methylation specific polymerase chain reaction in conjunction with sequencing verification was used to establish the methylation patterns of the 14 genes in the liver tissues of four healthy liver donors, as well as tumor and the paired non-cancerous tissues of 30 HCC patients. RESULTS: While 11 genes (ATP-binding cassette, sub-family G (WHITE), member 2(ABCG2), activating transcription factor (ATF2), beta-2-microglobulin (B2M), deoxycytidine kinase (DCK), occludin (OCLN), v-raf-1 murine leukemia viral oncogene homolog (RAF1), ralA binding protein 1 (RALBP1), splicing factor (45 kD) (SPF45), S-phase kinase-associated protein 2 (p45) (SKP2), tumor protein p53 (Li-Fraumeni syndrome) (TP53) and topoisomerase (DNA) II beta (TOP2B)) maintained the unmethylated patterns, three genes displayed to various extents the hypermethylation state in tumor tissues in comparison with the normal counterparts. The catalase (CAT) was hypermethylated in tumor and the neighboring non-cancerous tissue of one case (3.3%). Both glutathione S-transferase pi (GSTpi) (80%, 24/30 in tumor and 56.7%, 17/30 in the paired non-cancerous tissues) and cystic fibrosis transmembrane conductance regulator, ATP-binding cassette (sub-family C, member 7) (CFTR) (77%, 23/30 in tumor and 50%, 15/30 in the paired non-cancerous tissues) genes were prevalently hypermethylated in HCC as well as their neighboring non-cancerous tissues. No significant difference in the hypermethylation occurrence was observed between the HCC and its neighboring non-cancerous tissues. CONCLUSION: Hypermethylation of promoter CpG islands of both CFTR and GSTpi genes occurs prevalently in HCC, which may correlate with the low expression of these two genes at the mRNA level and has the profound etiological and clinical implications. It is likely to be specific to the early phase of HCC carcinogenesis.

[Inhibitory effects of antisense oligonucleotides on VEGF gene expression by human hepatocellular carcinoma cells].

OBJECTIVE: To investigate the inhibitory effects of antisense oligonucleotides to different sequences on VEGF gene expression by human hepatoma cells. METHODS: SMMC7721 cells were cultured under normoxic or hypoxic conditions for 24 h, followed by being transfected with different antisense oligonucleotides (A06513 to cap structure, A06514 to translation initiation, A06515 to Exon-3 and A06516 to translation terminal). The total RNAs from the cells were extracted and the VEGF expression were examined with RT-PCR. The relative concentrations of VEGF transcripts in SMMC772 cells from different groups were determined using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA as internal standard. RESULTS: In response to the hypoxic challenge, SMMC7721 cells upregulated VEGF mRNA; Comparative to the control (no oligonucleotides), A06513, A06514, A06515, and A06516 had obvious sequence-specific inhibitory effect on VEGF gene expression, with the ratio of VEGF over GAPDH of 0.49+/-0.08, 0.71+/-0.12, 0.72+/-0.11 and 0.86+/-0.12, respectively (F=12.21, P< 0.05). A06513 showed the strongest inhibitory effect (P<0.01). CONCLUSION: The antisense oligonucleotides complementary to VEGF cap structure, may become a potential alternative for antisense gene therapy of HCC.