Transcriptome exploration of ferroptosis-related genes in TGFß- induced lens epithelial to mesenchymal transition during posterior capsular opacification development.

BACKGROUND: Posterior capsular opacification (PCO) is the main reason affecting the long-term postoperative result of cataract patient, and it is well accepted that fibrotic PCO is driven by transforming growth factor beta (TGFß) signaling. Ferroptosis, closely related to various ocular diseases, but has not been explored in PCO. METHODS: RNA sequencing (RNA-seq) was performed on both TGF-ß2 treated and untreated primary lens epithelial cells (pLECs). Differentially expressed genes (DEGs) associated with ferroptosis were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to investigate their biological function. Additionally, protein-to-protein interactions among selected ferroptosis-related genes by PPI network and the top 10 genes with the highest score (MCC algorithm) were selected as the hub genes. The top 20 genes with significant fold change values were validated using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Our analysis revealed 1253 DEGs between TGF-ß2 treated and untreated pLECs, uncovering 38 ferroptosis-related genes between two groups. Among these 38 ferroptosis-related genes,the most prominent GO enrichment analysis process involved in the response to oxidative stress (BPs), apical part of cell (CCs),antioxidant activity (MFs). KEGG were mainly concentrated in fluid shear stress and atherosclerosis, IL-17 and TNF signaling pathways, and validation of top 20 genes with significant fold change value were consistent with RNA-seq. CONCLUSIONS: Our RNA-Seq data identified 38 ferroptosis-related genes in TGF-ß2 treated and untreated pLECs, which is the first observation of ferroptosis related genes in primary human lens epithelial cells under TGF-ß2 stimulation.

Let-7c-3p suppresses lens epithelial-mesenchymal transition by inhibiting cadherin-11 expression in fibrotic cataract.

Fibrotic cataract, including anterior subcapsular cataract (ASC) and posterior capsule opacification, always lead to visual impairment. Epithelial-mesenchymal transition (EMT) is a well-known event that causes phenotypic alterations in lens epithelial cells (LECs) during lens fibrosis. Accumulating studies have demonstrated that microRNAs are important regulators of EMT and fibrosis. However, the evidence explaining how microRNAs modulate the behavior and alter the cellular phenotypes of the lens epithelium in fibrotic cataract is insufficient. In this study, we found that hsa-let-7c-3p is downregulated in LECs in human ASC in vivo as well as in TGFß2-induced EMT in vitro, indicating that hsa-let-7c-3p may participate in modulating the profibrotic processes in the lens. We then demonstrated that overexpression of hsa-let-7c-3p markedly suppressed human LEC proliferation and migration and attenuated TGFß2-induced EMT and injury-induced ASC in a mouse model. In addition, hsa-let-7c-3p mediated lens fibrosis by directly targeting the CDH11 gene, which encodes cadherin-11 protein, an important mediator in the EMT signaling pathway. It decreased cadherin-11 protein expression at the posttranscriptional level but not at the transcriptional level by binding to a specific site in the 3-untranslated region (3'-UTR) of CDH11 mRNA. Moreover, blockade of cadherin-11 expression with a specific short hairpin RNA reversed TGFß2-induced EMT in LECs in vitro. Collectively, these data demonstrated that hsa-let-7c-3p plays a clear role in attenuating ASC development and may be a novel candidate therapeutic for halting fibrosis and maintaining vision.

Vascular endothelial cell-derived exosomal miR-1246 facilitates posterior capsule opacification development by targeting GSK-3ß in diabetes mellitus.

Posterior capsule opacification (PCO) is a serious complication after cataract surgery. Diabetes could increase the occurrence of PCO, but the mechanism is still unclear. The purpose of this study is to investigate the role of small extracellular vesicles (sEVs) derived from diabetic aqueous humor in PCO process. Intraoperatively-derived aqueous humor sEVs from patients with diabetic related cataract (DRC) promoted the epithelial-mesenchymal transition (EMT) and metastasis of human lens epithelial cells (LECs). Via mouse PCO surgical model and DiI labeled fluorescence detection of sEVs, the sEVs derived from vascular endothelium were discovered directly contacting with LECs. Furthermore, we demonstrated that high-glucose-cultured human umbilical vein endothelial cells (HUVEC) -derived sEVs facilitated EMT process of HLE-B3 using co-culture model in vitro. miRNA-seq data and GEO datasets analysis revealed that miR-1246 was essential in EMT process with diabetes. The miR-1246 was highly expressed in diabetic aqueous humor sEVs and high-glucose-treated vascular-endothelial-cell-derived sEVs. Moreover, miR-1246 promoted the metastasis and EMT process of HLE-B3 cells by directly targeting GSK-3ß. Inhibiting miR-1246 could negatively regulated EMT. This finding might serve as a potential therapy for diabetic PCO.

Circ-GGA3 promotes the biological functions of human lens epithelial cells depending on the regulation of miR-497-5p/SMAD4 axis.

The cause of posterior capsular opacification (PCO) is the dysfunction of lens epithelial cells (LECs). Circular RNA (circRNA) was found to regulate cell biological functions, including LECs. However, the role of circ-GGA3 in PCO formation is unclear. Quantitative real-time PCR was used to measure the expression of circ-GGA3, miR-497-5p and SMAD4. Cell proliferation, invasion and migration were determined via MTT assay, EdU staining, transwell assay and wound healing assay. The protein expression of epithelial-mesenchymal transition (EMT) markers, fibrosis markers, TGF-ß/SMAD pathway markers and SMAD4 were determined by western blot assay. The interaction between miR-497-5p and circ-GGA3 or SMAD4 was confirmed using dual-luciferase reporter assay. Circ-GGA3 was highly expressed in PCO patients, and its silencing inhibited the proliferation, invasion, migration, EMT process and fibrosis of TGF-ß2-induced LECs. Circ-GGA3 could sponge miR-497-5p to regulate SMAD4. Further experiments revealed that miR-497-5p inhibitor recovered the negative regulation of circ-GGA3 knockdown on the biological functions of TGF-ß2-induced LECs, and SMAD4 overexpression also abolished the suppressive effect of miR-497-5p. In addition, circ-GGA3/miR-497-5p/SMAD4 axis could activate the TGF-ß/SMAD pathway. Our results indicated that circ-GGA3 could enhance the biological functions of LECs, suggesting that circ-GGA3 might be a potential target for PCO therapy.

Circ-POLR3A accelerates TGF-ß2-induced promotion in cell viability, migration, and invasion of lens epithelial cells via miR-31/TXNIP signaling cascade.

Posterior capsular opacification (PCO) is the major complication after cataract surgery and can result in secondary vision loss. Circular RNAs (circRNAs) are reported to play critical regulatory roles in multiple cell biological processes. The most common working mechanism of circRNAs is by acting as microRNA sponges. Here, we analyzed the role and mechanism of circRNA RNA polymerase III subunit A (POLR3A) in PCO. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell motility was assessed by transwell and wound healing assays. Dual-luciferase reporter and RNA-pull-down assays were performed to verify the interaction between microRNA-31 (miR-31) and circ-POLR3A or thioredoxin interacting protein (TXNIP). PCO cell model was established by treating SRA01/04 cells with transforming growth factor-ß2 (TGF-ß2). We found that TGF-ß2 enhanced SRA01/04 cell viability, migration, and invasion abilities. Circ-POLR3A expression was upregulated in PCO tissues and TGF-ß2-induced SRA01/04 cells. TGF-ß2 promoted the viability and motility of SRA01/04 cells largely by upregulating circ-POLR3A. Circ-POLR3A negatively regulated the miR-31 level by directly interacting with it. Circ-POLR3A absence-induced influences in TGF-ß2-induced SRA01/04 cells were partly reversed by silencing miR-31. miR-31 is directly bound to the 3'-untranslated region of TXNIP. TXNIP overexpression largely attenuated miR-31 overexpression-mediated effects in TGF-ß2-induced SRA01/04 cells. Circ-POLR3A could elevate the protein expression of TXNIP by sponging miR-31. Exosomes were involved in mediating the delivery of circ-POLR3A in SRA01/04 cells. In conclusion, circ-POLR3A contributed to TGF-ß2-induced promotion of cell viability, migration, and invasion of SRA01/04 cells by targeting miR-31/TXNIP axis.

Exosome-transmitted circ-CARD6 facilitates posterior capsule opacification development by miR-31/FGF7 axis.

BACKGROUND: Posterior capsular opacification (PCO) is the major vision-disrupting complication arising after cataract surgery. Circular RNAs (circRNAs) are biological active RNAs which were involved in various physiological functions. So far, the role of circRNA caspase recruitment domain family member 6 (circ-CARD6) in PCO is still unclear. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of circ-CARD6, microRNA 31 (miR-31) and fibroblast growth factor 7 (FGF7) message RNA (mRNA). Western blot was used to analyze the protein expression. Transmission electron microscopy (TEM) was employed to capture the exosome image. The proliferation and metastasis were analyzed by cell counting kit-8 (CCK8), transwell and wound healing assays. The potential binding sequences between miR-31 and circ-CARD6 or FGF7 were respectively predicted by Circinteractome and Targetscan online tool, and verified by dual-luciferase reporter and RNA binding protein immunoprecipitation (RIP) assays. RESULTS: Exosome-transmitted circ-CARD6 was highly expressed in PCO tissues and TGF-ß2-treated SRA01/04 cells. Circ-CARD6 deletion repressed the proliferation, metastasis, EMT process and MAPK pathway, which was reversed by anti-miR-31 in TGF-ß2-treated SRA01/04 cells. Meanwhile, circ-CARD6 sponged miR-31 which directly targeted FGF7 in TGF-ß2-treated SRA01/04 cells. FGF7 overexpression allayed miR-31 overexpression-induced suppression in proliferation, metastasis, EMT process and MAPK pathway. Besides, circ-CARD6 regulated FGF7 expression by sponging miR-31. CONCLUSION: Circ-CARD6 promoted PCO development via miR-31/FGF7 axis. This finding might contribute to the development of the targeted therapy for PCO.

The effect of sex on the mouse lens transcriptome.

The transcriptome of mammalian tissues differs between males and females, and these differences can change across the lifespan, likely regulating known sexual dimorphisms in disease prevalence and severity. Cataract, the most prevalent disease of the ocular lens, occurs at similar rates in young individuals, but its incidence is elevated in older women compared to men of the same age. However, the influence of sex on the lens transcriptome was unknown. RNAseq based transcriptomic profiling of young adult C57BL/6J mouse lens epithelial and fiber cells revealed that few genes are differentially expressed between the sexes. In contrast, lens cells from aged (24 month old) male and female C57BL/6J mice differentially expressed many genes, including several whose expression is lens preferred. Like cataracts, posterior capsular opacification (PCO), a major sequela of cataract surgery, may also be more prevalent in women. Lens epithelial cells isolated from mouse eyes 24 h after lens fiber cell removal exhibited numerous transcriptomic differences between the sexes, including genes implicated in complement cascades and extracellular matrix regulation, and these differences are much more pronounced in aged mice than in young mice. These results provide an unbiased basis for future studies on how sex affects the lens response to aging, cataract development, and cataract surgery.

miR-30a reverses TGF-ß2-induced migration and EMT in posterior capsular opacification by targeting Smad2.

Posterior capsular opacification (PCO) leads to secondary vision loss following cataract surgery. TGF-ß2 and miRNA play important roles in PCO. The aim of this study was to investigate the reciprocal crosstalk between miR-30a and TGF-ß2/Smad2 during PCO progression. The expressions of and relationship between miR-30a and Smad2 were detected by RT-qPCR. Migration and epithelial-mesenchymal transition (EMT) were used to evaluate the functions of miR-30a and TGF-ß2/Smad2. We found that miR-30a was downregulated by TGF-ß2 and that it suppressed migration and EMT induced by TGF-ß2. Moreover, we identified Smad2 as a direct target of miR-30a, suggesting that miR-30a may function partly through regulating Smad2. Altogether, we verified the function of and crosstalk between miR-30a and TGF-ß2. We also provide evidence that miR-30a may serve as a potential candidate for PCO treatment.

The role of HIF-1α in the TGF-ß2-mediated epithelial-to-mesenchymal transition of human lens epithelial cells.

Human lens epithelial cells (HLE) undergo mesenchymal transition and become fibrotic during posterior capsule opacification (PCO), which is a frequent complication after cataract surgery. TGF-ß2 has been implicated in this fibrosis. Previous studies have focused on the role of hypoxia-inducible factor-1α (HIF-1α) in fibrotic diseases, but the role of HIF-1α in the TGF-ß2-mediated fibrosis in HLE is not known. TGF-ß2 treatment (10 ng/mL, 48 h) increased the HIF-1α levels along with the EMT markers in cultured human lens epithelial cells (FHL124 cells). The increase in HIF-1α corresponded to an increase in VEGF-A in the culture medium. However, exogenous addition of VEGF-A (up to 10 ng/mL) did not alter the EMT marker levels in HLE. Addition of a prolyl hydroxylase inhibitor, dimethyloxalylglycine (DMOG, up to 10 µM), enhanced the levels of HIF-1α, and secreted VEGF-A but did not alter the EMT marker levels. However, treatment of cells with a HIF-1α translational inhibitor, KC7F2, significantly reduced the TGF-ß2-mediated EMT response. This was accompanied by a reduction in the ERK phosphorylation and nuclear translocation of Snail and Slug. Together, these data suggest that HIF-1α is important for the TGF-ß2-mediated EMT of human lens epithelial cells.

Notch1 signaling induces epithelial-mesenchymal transition in lens epithelium cells during hypoxia.

BACKGROUND: Posterior Capsular Opacification (PCO) is one of the most common complications of cataract surgery which can result in severe visual damage. Epithelial-Mesenchymal Transition (EMT) of lens epithelium cells (LEC) is the pathological basis of PCO. Recent research showed that hypoxia acted as an inducer of EMT through a Notch1/Snail1/E-cadherin pathway. However, it remains unclear whether the Notch1/Snail1/E-cadherin pathway is involved in PCO under hypoxia. METHODS: The morphology of SRA01/04 cells treating with Cobalt Chloride (CoCl2) was observed and the markers of EMT and Notch1/Snail1/E-cadherin pathway were analyzed by Western blot and Immunocytochemistry assay. Transwell invasion assay and Wound healing assay were used to detected the effect of p3 × FLAG-CMV-7-NICD1 transfection on the SRA01/04 cells. RESULTS: The SRA01/04 cells lost cell polarity and cell junction culturing with CoCl2. The expression of Keratin, Hypoxia-inducible factor-1 alpha (HIF-1α), Notch1, Snail1were upregulated, on the other side, Fibronectin and E-cadherin were downregulated in hypoxia. Furthermore, the overexpression of Notch1 induced the expression of E-cadherin and increased the invasion and migration ability of SRA01/04 cells. CONCLUSIONS: These results suggest that Notch1/Snail1/E-cadherin pathway facilitates the EMT through HIF-1α in SRA01/04 cells during hypoxia and promotes LEC motility.

Long noncoding RNA expression profile in HLE B-3 cells during TGF-ß2-induced epithelial-mesenchymal transition.

BACKGROUND: Recent evidence has shown that long noncoding RNAs (lncRNAs) are involved in the process of epithelial-mesenchymal transition (EMT). However, little research has focused on the expression profile of lncRNAs during EMT in human lens epithelial cells (LECs) and their functions have not yet been described. METHODS: Dysregulated lncRNAs and mRNAs in normal human lens epithelial B-3(HLE B-3) cells and during transforming growth factor ß2(TGF-ß2)-induced EMT were analyzed via lncRNA microarray. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analyses of differentially expressed mRNAs were performed to identify their functions and pathologic pathways. Six candidate lncRNAs were validated via quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR) to confirm the microarray data. RESULTS: A total of 775 lncRNAs (325 up-regulated and 450 down-regulated) and 935 mRNAs (329 up-regulated and 606 down-regulated) were differentially expressed in HLE B-3 cells during TGF-ß2-induced EMT compared to normal HLE B-3 cells. GO and KEGG Pathway analyses indicated the functions of differentially expressed mRNAs in the TGF-ß2-induced EMT in HLE B-3 cells. qRT-PCR confirmed the trends indicated in microarray analysis for all 6 candidate lncRNAs. CONCLUSION: Our study lays the foundation for future research in lncRNAs related to EMT in HLE B-3 cells and could provide new avenues for the prevention and treatment of posterior capsule opacification (PCO).

Bit1-a potential positive regulator of epithelial-mesenchymal transition in lens epithelial cells.

PURPOSE: Posterior capsule opacification (PCO) is a common postoperative complication of cataract surgery. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is an important initial step of PCO pathogenesis. We have previously shown that Bit1 expresses in rat LECs. In this study, we aim to investigate the role of Bit1 in the EMT of human LECs. METHODS: The expression of Bit1 was firstly detected in human PCO-attached LECs and human lens cell line SRA01/04 by real-time PCR and immunofluorescence staining. The proliferation and migration of Bit1 knockdown SRA01/04 cells were evaluated by cell counting, wound-healing assay, and transwell migration assay. The EMT of LECs was triggered by TGF-ß2, and then the effect of Bit1 on EMT with a key biomarker of α-smooth muscle actin (α-SMA) was analyzed by siRNA knockdown assay, and the reversal of EMT was validated by real-time PCR and western blot. RESULTS: Bit1 was obviously augmented in LECs derived from PCO tissues and Bit1 expressed with high levels in the cytoplasm of cultured SRA01/04 cells. Cell proliferation, invasion, and migration, as well as α-SMA expression, were significantly decreased in Bit1 knockdown SRA01/04 cells. While TGF-ß2 elevated Bit1 and α-SMA expression levels in a dose-dependent manner, with peak levels at 10 ng/ml of TGF-ß2 treatment, suppression of Bit1 in SRA01/04 cells reversed the EMT process. TGF-ß2 induced elevation of α-SMA expression level, as well as migration, and invasion abilities were all suppressed by Bit1 deficiency. CONCLUSIONS: These findings reveal that Bit1 promotes TGF-ß2 induced α-SMA expression and acts as an positive regulator of EMT. Suppressing Bit1 inhibits the proliferation, migration, and EMT of LECs. Bit1 may be a potential novel therapeutic target for the prevention and treatment of PCO.

[TAK1 promotes epithelial-mesenchymal transition of lens epithelial cells].

OBJECTIVE: Transforming growth factor-ß-activated kinase-1 (TAK1) is thought to play a key role in the initiation of Smad-independent TGF-ß signaling. This study investigated the role of TAK1 in the epithelial-mesenchymal transition (EMT) lens epithelial cells. METHODS: TAK1 was overexpressed in the HLE B-3 cell line by transfecting TAK1-pcDNA3 and TAK1-binding protein 1 (TAB1)-pcDNA3 plasmids. The expression levels of TAK1, phospho-TAK1, E-cadherin, and fibronectin were detected by Western blot analysis and immunocytofluorescence to analyze the effects of overexpression. The levels of α-SMA and type I collagen were analyzed by real-time PCR. Quantitative data were analyzed by Student's t test or one-way analysis of variance (ANOVA) (multiple comparisons using LSD test). RESULTS: Western blot analysis showed in the TAK1-pcDNA3 plasmids group, expression of TAK1 proteins (1.00±0.03) with a maximum upregulation of approximately 80% at 24 h than it was in the control group (0.19±0.09)(t=8.02, P< 0.01); Western blot analysis showed in the TAB1-pcDNA3 plasmids group, expression of TAB1 proteins (1.00±0.02) with a maximum upregulation of approximately 78% at 24 h than it was in the control group (0.22±0.08)(t=7.63, P<0.01). The levels of E-cadherin/Beta-actin had significant differences among control, overexpression of TAK1 together with TAB1, overexpression of TAK1, and overexpression of TAB1 (1.00±0.02, 0.12±0.03, 0.98±0.09, 0.92±0.08;F=31.03, P<0.01). The levels of fibronectin/Beta-actin had significant differences among control, overexpression of TAK1 together with TAB1, overexpression of TAK1, and overexpression of TAB1 (0.11±0.03, 1.00±0.05, 0.16±0.04, 0.21±0.05;F=35.12, P<0.01). Overexpression of TAK1 with TAB1 resulted in upregulated expression of fibronectin, and downregulated expression of E-cadherin. The expression of E-cadherin was increased and the expression of fibronectin was decreased by TAK1 siRNA and TAK1 chemical inhibitors in the presence of TGF-ß2. CONCLUSION: These data reveal that TAK1 can induce the EMT of HLE cells, and the inhibition of TAK1 phosphorylation may be a potential novel therapeutic target for the prevention and treatment of posterior capsule opacification. (Chin J Ophthalmol, 2016, 52: 278-284).

αA-crystallin gene CpG islands hypermethylation in nuclear cataract after pars plana vitrectomy.

PURPOSE: To investigate the DNA methylation status of αA-crystallin gene in cataract secondary to pars plana vitrectomy. METHODS: Anterior capsular membranes of 40 eyes of 40 patients with cataract secondary to vitrectomy were collected. Another 20 eyes of 20 patients who received pars plana vitrectomy and phacoemulsification in the primary procedure, were recruited as control. Methylation status of the CpG islands of αA-crystallin gene was analyzed by pyrosequencing. Expression of αA-crystallin was evaluated by real-time polymerase chain reaction and western blot. RESULTS: In the post vitrectomy group, five patients with posterior subcapsular opacity and four patients with cortical opacity were excluded from further analysis. The remaining 31 patients with nuclear cataract were assigned into two groups according to tamponade types: 19 of octafluoropropane (C3F8) and 12 of silicone oil (SiO). The average nuclear color grading was elevated both in C3F8 and SiO groups after vitrectomy. Compared to the control group, hypermethylation of the CpG islands in the αA-crystallin gene promoter was found in both post vitrectomy groups, accompanied by significantly reduced αA-crystallin expression. No statistically significant differences were found between the C3F8 and SiO groups either for DNA methylation status or αA-crystallin expression. CONCLUSIONS: CpG islands hypermethylation of αA-crystallin gene may be involved in nuclear cataract formation after pars plana vitrectomy.

Massive formation of square array junctions dramatically alters cell shape but does not cause lens opacity in the cav1-KO mice.

The wavy square array junctions are composed of truncated aquaporin-0 (AQP0) proteins typically distributed in the deep cortical and nuclear fibers in wild-type lenses. These junctions may help maintain the narrowed extracellular spaces between fiber cells to minimize light scattering. Herein, we investigate the impact of the cell shape changes, due to abnormal formation of extensive square array junctions, on the lens opacification in the caveolin-1 knockout mice. The cav1-KO and wild-type mice at age 1-22 months were used. By light microscopy examinations, cav1-KO lenses at age 1-18 months were transparent in both cortical and nuclear regions, whereas some lenses older than 18 months old exhibited nuclear cataracts. Scanning EM consistently observed the massive formation of ridge-and-valley membrane surfaces in young fibers at approximately 150 µm deep in all cav1-KO lenses studied. In contrast, the typical ridge-and-valleys were only seen in mature fibers deeper than 400 µm in wild-type lenses. The resulting extensive ridge-and-valleys dramatically altered the overall cell shape in cav1-KO lenses. Remarkably, despite dramatic shape changes, these deformed fiber cells remained intact and made close contact with their neighboring cells. By freeze-fracture TEM, ridge-and-valleys exhibited the typical orthogonal arrangement of 6.6 nm square array intramembrane particles and displayed the narrowed extracellular spaces. Immunofluorescence analysis showed that AQP0 C-terminus labeling was significantly decreased in outer cortical fibers in cav1-KO lenses. However, freeze-fracture immunogold labeling showed that the AQP0 C-terminus antibody was sparsely distributed on the wavy square array junctions, suggesting that the cleavage of AQP0 C-termini might not yet be complete. The cav1-KO lenses with nuclear cataracts showed complete cellular breakdown and large globule formation in the lens nucleus. This study suggests that despite dramatic cell shape changes, the massive formation of wavy square array junctions in intact fibers may provide additional adhesive support for maintaining the narrowed extracellular spaces that are crucial for the transparency of cav1-KO lenses.

MiRNA-26b inhibits the proliferation, migration, and epithelial-mesenchymal transition of lens epithelial cells.

MicroRNAs (miRNAs) are a class of small endogenous gene regulators that play important roles in various developmental and pathological processes. However, little is known about the precise identity and functions of miR-26b in posterior capsule opacification (PCO). In this study, we report that the expression of miR-26b is decreased in human PCO-attached lens epithelial cells (LECs) and SRA01/04 cells in the presence of TGF-ß2. Overexpression of miR-26b inhibited the proliferation of LECs based on MTT assays and BrdU incorporation assays. In addition, the overexpression of miR-26b inhibited the migration ability of LECs, as shown by wound-healing and transwell migration assays. The overexpression of miR-26b increased the level of the lens epithelial marker E-cadherin and reduced the levels of mesenchymal-related proteins, such as fibronectin, a-SMA, and type I collagen, in SRA01/04 cells in the presence of TGF-ß2. Furthermore, the upregulation of E-cadherin and downregulation of mesenchymal-related proteins were induced in human PCO-attached LECs transfected with miR-26b mimics. We further demonstrated that Smad4 and COX-2 are targets of miR-26b in LECs using luciferase reporter assays. These data reveal that miR-26b can inhibit the proliferation, migration, and EMT of lens epithelial cells, and restoration of miRNA-26b may be a potential, novel therapeutic target for the prevention and treatment of posterior capsule opacification.

Knockout of TGF-ß receptor II by CRISPR/Cas9 delays mesenchymal transition of Lens epithelium and posterior capsule opacification.

Posterior capsule opacification (PCO) is the most common postoperative complication of cataract surgery. Transforming growth factor-ß (TGF-ß) is related to epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) that is proven to induce PCO formation in clinical and experimental studies. In this study, CRISPR sequences targeting exon of TGF-ßRII were knocked out with lentiviral transfection in LECs. Rabbits' PCO model was established and recombinant adeno-associated virus (AAV) for transferring the gRNA of TGF ßRII were intravitreally injected. SgRNA inhibited TGF-ßRII expression and human LECs proliferation. In TGF-ßRII knockout group, LECs motility and migration were suppressed, N-cadherin and vimentin expressions were significantly decreased, whereas E-cadherin was increased. The animal model showed that TGF-ßRII knockout in vivo was effective in suppressing PCO. The current study suggested that the CRISPR/Cas9 endonuclease system could suppress TGF-ßRII secretion, which participates in the EMT procedure of LECs in vitro and PCO in vivo. These findings might provide a new gene-editing approach and insight into a novel therapeutic strategy for PCO.

Circ_0000099/miR-223-3p/CTGF Regulates the Growth, Metastasis, and EMT Processes in TGF-ß2-Stimulated Human Lens Epithelial Cells.

PURPOSE: Posterior capsule opacification (PCO) is the major complication of visual impairment after cataract surgery. Circular RNAs (circRNAs) are involved in the development of many diseases. The purpose of this study was to explore the role and molecular mechanism of circ_0000099 in PCO. METHODS: SRA01/04 cells were treated with TGF-ß2 to establish a PCO cell model. The expression of circ_0000099, miR-223-3p, and connective tissue growth factor (CTGF) mRNA was determined by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot assay was used to analyze the protein expression. Cell proliferation, migration, and invasion were analyzed by (4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-ethynyl-2 '-Deoxyuridine (EdU), transwell, and wound healing tests. The circ_0000099/miR-223-3p/CTGF relationship was verified by dual luciferase reporter gene and RNA binding protein immunoprecipitation (RIP) assays. RESULTS: TGF-ß2 treatment promoted SRA01/04 cell proliferation invasion, migration, and EMT. Circ_0000099 expression was increased in POC patients and TGF-ß2-treated SRA01/04 cells.Knockdown of circ_0000099 suppressed TGF-ß2-induced proliferation, invasion, migration, and EMT in SRA01/04 cells. miR-223-3p was identified as the target of circ_0000099, and miR-223-3p inhibitor might partly abolish the repression of circ_0000099 silencing on TGF-ß2-triggered SRA01/04 cell disorders. MiR-223-3p directly targeted CTGF. Knockdown of CTGF suppressed TGF-ß2-induced SRA01/04 cell injury. Circ_0000099 can regulate CTGF expression by targeting miR-223-3p. CONCLUSIONS: Circ_0000099 silencing might relieve TGF-2-induced SRA01/04 cell injury by the miR-223-3p/CTGF axis, providing new avenues for the prevention and treatment of PCO.

Elevated tropomyosin expression is associated with epithelial-mesenchymal transition of lens epithelial cells.

Injury to lens epithelial cells (LECs) leads to epithelial-mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2ß suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α-smooth muscle actin (α-SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2ß following EMT/PCO formation. Expression of Tpm1α/2ß was up-regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non-cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2ß mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1α/2ß, as evidenced by increased expression of α-SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm-based inhibitors for postponing PCO and cataractogenesis.

Inhibition of Posterior Capsule Opacification by Adenovirus-Mediated Delivery of Short Hairpin RNAs Targeting TERT in a Rabbit Model.

PURPOSE: Posterior capsule opacification (PCO) is the most common postoperative complication after cataract surgery and cannot yet be eliminated. Here, we investigated the inhibitory effects of telomerase reverse transcriptase (TERT) gene silencing on PCO in a rabbit model. METHODS: After rabbit lens epithelial cells (LECs) were treated with adenovirus containing short hairpin RNAs (shRNA) targeting TERT (shTERT group), adenovirus containing scramble nonsense control shRNA (shNC group) or PBS (control group), quantitative real-time polymerase chain reaction and Western blotting were used to measure the expression levels of TERT, and a scratch assay was performed to assess the LEC migration. New Zealand white rabbits underwent sham cataract surgery followed by an injection of adenovirus carrying shTERT into their capsule bag. The intraocular pressure and anterior segment inflammation were evaluated on certain days, and EMT markers (α-SMA and E-cadherin) were evaluated by Western blotting and immunofluorescence. The telomerase activity of the capsule bag was detected by ELISA. At 28 d postoperatively, hematoxylin and eosin staining of the cornea and iris and electron microscopy of the posterior capsule were performed. RESULTS: Application of shTERT to LECs downregulated the expression levels of TERT mRNA and protein. The scratch assay results showed a decrease in the migration of LECs in the shTERT group. In vivo, shTERT decreased PCO formation after cataract surgery in rabbits and downregulated the expression of EMT markers, as determined by Western blotting and immunofluorescence. In addition, telomerase activity was suppressed in the capsule bag. Despite slight inflammation in the iris, histologic results revealed no toxic effects in the cornea and iris. CONCLUSION: TERT silencing effectively reduces the migration and proliferation of LECs and the formation of PCO. Our findings suggest that TERT silencing may be a potential preventive strategy for PCO.