Toll-like receptor 3 gene regulates cataract-related mechanisms via the Jagged-1/Notch signaling pathway

Abstract

Epithelial-melancholy transition (EMT) is the main cause of organ fibrosis and a common pathogenetic mechanism in most cataracts. This study aimed to explore the molecular mechanism of Toll-like receptor (TLR)-3 in the occurrence and development of post-cataract EMT and to provide new ideas for the prevention and treatment of posterior capsule opacification (PCO). In the presence or absence of TLR3, the human lens epithelial cell (LEC) line, SRA01/04, was treated with the transforming growth factor (TGF)-β2. Cell counting kit-8 (CCK-8) and Transwell assays were used to analyze the cell proliferation, migration, and invasion. The expression levels of proteins and RNAs were detected by western blotting and quantitative polymerase chain reaction (qPCR) experiments. Functional gain and loss studies showed that TLR3 regulates the proliferation, migration, and invasion of LECs and EMT induced by TGF-β2. Moreover, TLR3 regulates the expression of Jagged-1, Notch-1, and Notch-3 These findings indicate that TLR3 prevents the progression of lens fibrosis by targeting the Jagged-1/Notch signaling pathway to regulate the proliferation, migration, and invasion of LECs, and TGF-β2-induced EMT. Therefore, the TLR3-Jagged-1/Notch signaling axis may be a potential therapeutic target for the treatment of fibrotic cataracts.

Keywords: EMT; Jagged-1/Notch signaling pathway; TLR3; fibrotic cataract.

Graphical abstract

Figure 1.

FN, α-SMA, Col I, Acan, and TLR3 were upregulated in fibrotic lens tissues. (a) Gene expression levels in normal lens epithelium and fibrotic cataract tissues were detected by quantitative polymerase chain reaction (qPCR). (b-c) Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is used to detect the Toll-like receptor (TLR)-3 and transforming growth factor (TGF)-β2 expression levels in normal lens epithelium and fibrotic cataract. **P< 0.01, ***P< 0.001 vs. Normal group.

Figure 2.

TLR3 is upregulated in the lens epithelial cell (LEC) line, SRA01/04. (a, b) Gene expression levels in LECs treated with or without the transforming growth factor (TGF)-β2 (5 ng/mL) for 48 h by qPCR analysis. (c) Comparison of TLR3 gene expression levels in LECs treated with or without TGF-β2 (5 ng/mL) for 48 h by qPCR analysis. **P< 0.01, ***P< 0.001 vs. Control group.

Figure 3.

TLR3 knockdown inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of human LECs. (a) After transfection with si-TLR3, qPCR was used to detect the levels of TLR3 in the cells. (b) Analysis of the gene expression levels of fibronectin (FN), α-smooth muscle actin (α-SMA), collagen type I (Col I), and E-cadherin (E-cad) after transfection of si-TLR3 in the LECs was performed using qPCR. (c) The cell counting kit-8 (CCK-8) method was used to detect the cell viability after transfection of si-TLR3 in the lens epithelial fibrosis model induced by TGF-β2. (d-e) Transwell method is used to detect the cell migration and invasion abilities after transfection of si-TLR3 in the lens epithelial fibrosis model induced by TGF-β2. (f) Western blotting was used to detect the expression levels of EMT-related proteins in cells transfected with si-TLR3 in the lens epithelial fibrosis model induced by TGF-β2. **P< 0.01, ***P< 0.001 vs. Control group. ^##P< 0.01, ^###P< 0.001 vs. TGF-β2+ si-nc group.

Figure 4.

TLR3 overexpression promoted the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of human LECs. (a) After transfection with oe-TLR3, qPCR was used to detect the levels of TLR3 in the cells. (b) The cell counting kit-8 (CCK-8) method was used to detect the cell viability after transfection of oe-TLR3 in the lens epithelial fibrosis model induced by TGF-β2. (c-d) Transwell method is used to detect the cell migration and invasion abilities after transfection of oe-TLR3 in the lens epithelial fibrosis model induced by TGF-β2. (e) Western blotting was used to detect the expression levels of EMT-related proteins in cells transfected with oe-TLR3 in the lens epithelial fibrosis model induced by TGF-β2. **P< 0.01, ***P< 0.001 vs. Control group. ^#P < 0.05, ^##P< 0.01 vs. TGF-β2+ oe-nc group.

Figure 5.

TLR3 directly acts on the Jagged-1/Notch signaling pathway. (a) Western blotting was used to detect the expression levels of the Jagged-1/Notch pathway proteins in LECs treated with or without TGF-β2 (5 ng/mL) for 48 h. (b) After transfection with si-TLR3 and overexpressed TLR3, LECs were treated with TGF-β2 (5 ng/mL). The expression levels of the Jagged-1/Notch pathway proteins were detected by western blotting. **P< 0.01, ***P< 0.001 vs. Control or oe-NC group. ^##P< 0.01, ^###P< 0.001 vs. si-nc group.

Figure 6.

TLR3 promotes the proliferation, migration, invasion, and EMT of human LECs by directly acting on the Jagged-1/Notch signaling pathway. The TGF-β2 treated SRA01/04 cells were treated with si-TLR3, Notch pathway activator valproic acid (VA) and inhibitor DAPT. Next, (a) qRT-PCR is used to detect the gene expression levels of FN, α-SMA, Col I, and E-cad in each group. (b) CCK-8 method is used to detect the cell viability in each group. (c-d) Transwell method is used to detect the cell migration and invasion abilities of LECs in each group. (e) Western blotting is used to detect the expression levels of EMT-related proteins and Jagged-1/Notch signaling pathway-related proteins in each group. **P< 0.01, ***P< 0.001 vs Control. ^##P< 0.01, ^###P< 0.001 vs. TGF-β2+ si-nc, ^&P< 0.05, ^&&P< 0.01 vs TGF-β2+ si-TLR3 group.