Type I Interferon Dependent hsa-miR-145-5p Downregulation Modulates MUC1 and TLR4 Overexpression in Salivary Glands From Sjögren's Syndrome Patients

Abstract

Sjögren's syndrome (SS) is an autoimmune disease that mainly affects salivary glands (SG) and is characterized by overactivation of the type I interferon (IFN) pathway. Type I IFNs can decrease the levels of hsa-miR-145-5p, a miRNA with anti-inflammatory roles that is downregulated in SG from SS-patients. Two relevant targets of hsa-miR-145-5p, mucin 1 (MUC1) and toll-like receptor 4 (TLR4) are overexpressed in SS-patients and contribute to SG inflammation and dysfunction. This study aimed to evaluate if hsa-miR-145-5p modulates MUC1 and TLR4 overexpression in SG from SS-patients in a type I IFN dependent manner. Labial SG (LSG) biopsies from 9 SS-patients and 6 controls were analyzed. We determined hsa-miR-145-5p levels by TaqMan assays and the mRNA levels of MUC1, TLR4, IFN-α, IFN-β, and IFN-stimulated genes (MX1, IFIT1, IFI44, and IFI44L) by real time-PCR. We also performed in vitro assays using type I IFNs and chemically synthesized hsa-miR-145-5p mimics and inhibitors. We validated the decreased hsa-miR-145-5p levels in LSG from SS-patients, which inversely correlated with the type I IFN score, mRNA levels of IFN-β, MUC1, TLR4, and clinical parameters of SS-patients (Ro/La autoantibodies and focus score). IFN-α or IFN-β stimulation downregulated hsa-miR-145-5p and increased MUC1 and TLR4 mRNA levels. Hsa-miR-145-5p overexpression decreased MUC1 and TLR4 mRNA levels, while transfection with a hsa-miR-145-5p inhibitor increased mRNA levels. Our findings show that type I IFNs decrease hsa-miR-145-5p expression leading to upregulation of MUC1 and TLR4. Together, this suggests that type I interferon-dependent hsa-miR-145-5p downregulation contributes to the perpetuation of inflammation in LSG from SS-patients.

Keywords: Mucin 1; Sjögren’s syndrome; Toll-like receptor 4; Type I interferons; hsa-miR-145-5p.

Figure 1

hsa-miR-145-5p is downregulated in LSG from SS-patients and inversely correlates with IFN-β mRNA levels. (A) hsa-miR-145-5p levels in SS-patients (n=9) and control subjects (n=6). U48 snRNA was used as a housekeeping. (B) IFN-α transcript levels in SS-patients and control subjects. h18S was used as a housekeeping gene. (C) Spearman correlation between hsa-miR-145-5p and IFN-α mRNA levels in SS-patients and controls. (D) IFN-β transcript levels in SS-patients and control subjects. h18S was used as a housekeeping gene. (E) Spearman correlation between hsa-miR-145-5p and IFN-β mRNA levels in SS-patients and controls. Data are representative of at least three independent measurements. (*) p-value ≤ 0.05 was considered significant.

Figure 2

Overexpression of MX1, IFIT1, IFI44, and IFI44L and increased type I IFN score in LSG from SS-patients. Relative expression ratios of MX1 (A), IFIT1 (B), IFI44 (C) and IFI44L (D) in 9 SS-patients and 6 controls. h18S was used as a housekeeping gene. (E) Type I IFN score was calculated by summing the MX1, IFIT1, IFI44, and IFI44L standarized expression levels. (F) Spearman correlation between hsa-miR-145-5p levels and type I IFN score in SS-patients and controls. Data are representative of at least three independent measurements. (*) p-value ≤ 0.05 was considered significant.

Figure 3

MUC1 and TLR4 are overexpressed in LSG from SS-patients and inversely correlate with hsa-miR-145-5p. (A) MUC1 transcript levels in SS-patients and control subjects. h18S was used as a housekeeping gene. (B) Spearman correlation between hsa-miR-145-5p and MUC1 mRNA levels in SS-patients and controls. (C) TLR4 transcript levels in SS-patients and control subjects. h18S was used as a housekeeping gene. (D) Spearman correlation between hsa-miR-145-5p and TLR4 mRNA levels in SS-patients and controls. Data are representative of at least three independent measurements. (*) p-value ≤ 0.05 was considered significant.

Figure 4

Overexpression of MX1, IFIT1, IFI44, and IFI44L in IFN-α or IFN-β-stimulated HSG cells. Relative expression ratios of MX1 (A), IFIT1 (B), IFI44 (C) and IFI44L (D) in HSG cells stimulated with or without 10 ng/mL human recombinant IFN-α for 24 h. h18S was used as a housekeeping gene. Relative expression ratios of MX1 (E), IFIT1 (F), IFI44 (G) and IFI44L (H) in HSG cells stimulated with or without 10 ng/mL human recombinant IFN-β for 24 h. h18S was used as a housekeeping gene. Data are representative of at least three independent experiments. (*) p-value ≤ 0.05 was considered significant.

Figure 5

hsa-miR-145-5p is downregulated and MUC1 and TLR4 are overexpressed in type I IFNs-stimulated HSG cells. (A) hsa-miR-145-5p levels in HSG cells stimulated with or without 10 ng/mL human recombinant IFN-α for 24 h. U48 was used as a housekeeping gene. Transcript levels of MUC1 (B) and TLR4 (C) in HSG cells stimulated with IFN-α. Spearman correlation between hsa-miR-145-5p and MUC1 (D) or TLR4 (E) transcript levels in IFN-α stimulated HSG cells. (F) hsa-miR-145-5p levels in HSG cells stimulated with or without 10 ng/mL human recombinant IFN-β for 24 h. U48 was used as a housekeeping gene. Transcript levels of MUC1 (G) and TLR4 (H) in HSG cells stimulated with IFN-β. Spearman correlation between hsa-miR-145-5p and MUC1 (I) or TLR4 (J) transcript levels in IFN-β stimulated HSG cells. Data are representative of at least three independent experiments. (*) p-value ≤ 0.05 was considered significant.

Figure 6

IFN-α and IFN-β increase MUC1 and TLR4 protein levels in HSG cells. (A, C) representative MUC1 western blot (90-300 kDa) in HSG cells stimulated with 10 ng/mL IFN-α or IFN-β for 24 h. Relative protein levels were normalized to the control condition. β-actin was used as a loading control. (B, D) representative TLR4 western blot (95 kDa) in HSG cells stimulated with 10 ng/mL IFN-α or IFN-β for 24 h. Relative protein levels were normalized to the control condition. β-actin was used as a loading control. Data are representative of at least three independent experiments. (*) p-value ≤ 0.05 was considered significant.

Figure 7

Expression of MUC1 and TLR4 after hsa-miR-145-5p overexpression or inhibition. (A) Increased hsa-miR-145-5p levels in HSG cells transfected with the mimic miRNA. (B) Decreased hsa-miR-145-5p levels in HSG cells transfected with inhibitor miRNA. (C) Relative expression ratio of MUC1 transcripts in HSG cells transfected with mimic hsa-miR-145-5p or inhibitor (D) Relative expression ratio of TLR4 transcripts in HSG cells transfected with mimic hsa-miR-145-5p or inhibitor. Data are representative of at least three independent experiments. (*) p-value ≤ 0.05 was considered significant.