TGF-β2-induced NEAT1 regulates lens epithelial cell proliferation, migration and EMT by the miR-26a-5p/FANCE axis

AIM: To explore the regulatory mechanism of nuclear paraspeckle assembly transcript 1 (NEAT1) in the pathogenesis of posterior capsule opacification (PCO). METHODS: Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was executed to analyze NEAT1 and microRNA (miR)-26a-5p expression in transforming growth factor-beta 2 (TGF-β2)-disposed lens epithelial cells (LECs). The proliferation, cell cycle progression, apoptosis, and migration of TGF-β2-disposed LECs were evaluated. The relationship between NEAT1 or fanconi anemia (FA) complementation group E (FANCE) and miR-26a-5p was verified by dual-luciferase reporter assay. RESULTS: TGF-β2 induced NEAT1 expression in LECs. NEAT1 inhibition accelerated apoptosis, cell cycle arrest, decreased proliferation, epithelial-mesenchymal transition (EMT), and migration of TGF-β2-disposed LECs. NEAT1 sponged miR-26a-5p to further regulate FANCE expression. Rescue experiments presented that miR-26a-5p downregulation overturned NEAT1 silencing-mediated impacts on TGF-β2-disposed LEC biological behaviors. Additionally, FANCE overexpression reversed miR-26a-5p mimic-mediated impacts on TGF-β2-disposed LEC biological behaviors. CONCLUSION: TGF-β2-induced NEAT1 facilitates LEC proliferation, migration, and EMT by upregulating FANCE via sequestering miR-26a-5p.

Quantitative Evaluation of Human Lens Epithelial Cell Viability and Cytolysis by Distilled Water Ex Vivo

Purpose: To investigate the role of distilled water (DW) in isolated human lens epithelial cells (LECs) viability and lysis ex vivo.Methods: After immersion in DW or balanced salt solution (BSS) for 1-, 2-, and 3-minutes, respectively, the cell viability of LECs was quantitatively evaluated. In addition, the capsule samples soaked in DW or BSS for 1-, 2-, and 3-minutes were combined with rinse for 1 minute to analyze the difference of LECs shedding percentage in each subgroup. The histopathological changes of the samples after treating were observed.Results: The percentage of LECs shed in DW immersion combined with rinse was significantly higher than in DW immersion alone (p all <0.001). In the subgroup soaked in DW for 3 minutes, the death number, mortality, and the percentage of cell shedding of LECs was the most (p all <0.001). The histopathological changes showed that the cell destruction in the DW subgroup for 1-, 2-, and 3-minutes, and the transmission electron microscope results showed that the cells were partially detached from the capsule in the DW 3 minutes subgroup.Conclusions: Soaking in the DW can cause LECs death, and DW immersion combined with rinse was an effective method to remove LECs. The histopathology changes of treated DW suggested cellular necrosis was one type of LECs death mechanism.

miRNA-26b suppresses the TGF-β2-induced progression of HLE-B3 cells via the PI3K/Akt pathway

AIM: To study the effect of miR-26b on lens epithelial cells induced by transforming growth factor beta (TGF-β) 2 and the underlying signaling pathways. METHODS: Human lens epithelial cell line B-3 (HLE-B3) was incubated with TGF-β2 (5 ng/mL) and then transfected with miR-26b mimics. The expression of miR-26b was determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), while 5’-bromodeoxyuridine (BrdU) and wound-healing assays were used to measure the growth and migration of HLE-B3 cells, respectively. The expression of epithelial-mesenchymal transition (EMT) markers and the activity of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway were measured by Western blotting assay and immunofluorescence staining. Electron microscopy was also used to observe cellular morphology. RESULTS: The expression levels of miR-26b were significantly reduced in human posterior capsular opacification-attached lens tissue and TGF-β2-stimulated HLE-B3 cells. In the presence of TGF-β2, the growth, migration, and EMT of HLE-B3 cells were distinctly enhanced; these effects were attenuated by the administration of miR-26b mimics. Furthermore, the overexpression of miR-26b significantly reduced upregulation of the PI3K/Akt pathway when stimulated by TGF-β2 in HLE-B3 cells. Moreover, the addition of an activator (740 Y-P) led to the upregulation of the PI3K/Akt pathway and abolished the protective effect of miR-26b on the HLE-B3 cells that was mediated by TGF-β2. CONCLUSION: The miR-26b suppresses TGF-β2-induced growth, migration, and EMT in HLE-B3 cells by regulating the PI3K/Akt signaling pathway.

Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells

The present study aims to understand the mechanism of the lens epithelial cell’s strong anti-apoptotic capacity and survival in the mature human lens that, on the one hand, maintains lens transparency over several decades, while on the other hand, increases the risk of posterior capsule opacification (PCO). Here we compared FHL124 cells and HeLa cells, spontaneously immortalized epithelial cell lines derived from the human lens and cervical cancer cells, respectively, of their resistance to TNFα-mediated cell death. TNFα plus cycloheximide (CHX) triggered almost all of HeLa cell death. FHL124 cells, however, were unaffected and able to block caspase-8 activation as well as prevent caspase-3 and PARP-1 cleavage. Interestingly, despite spontaneous NFκB and AP-1 activation and upregulation of multiple cell survival/anti-apoptotic genes in both cell types, only FHL124 cells were able to survive the TNFα challenge. After screening and comparing the cell survival genes, cFLIP was found to be highly expressed in FHL124 cells and substantially upregulated by TNFα stimulation. FHL124 cells with a mild cFLIP knockdown manifested a profound apoptotic response to TNFα stimulus similar to HeLa cells. Most importantly, we confirmed these findings in an ex vivo lens capsular bag culture system. In conclusion, our results show that cFLIP is a critical gene that is regulating lens epithelial cell survival.