INTRODUCTION:Diabetic cataract (DC) is a common ocular complication of diabetes. Mitofusin 2 (MFN2), a mitochondrial fusion protein, is involved in the pathogenesis of cataract and diabetic complications. However, its role and molecular mechanisms in DC remain unclear. MATERIALS AND METHODS:DC models in rats were induced by intraperitoneal injection of streptozocin (STZ) for 12 weeks. We measured the body weight of rats, blood glucose concentrations, sorbitol dehydrogenase (SDH) activity and advanced glycation end products (AGE) content in the lenses of rats. MFN2 mRNA and protein expression levels in the lenses were detected by RT-qPCR and western blot assays. In vitro, human lens epithelial (HLE) B3 cells were treated for 48 h with 25 mM glucose (high glucose, HG) to induce cell damage. To determine the role of MFN2 in HG-induced cell damage, HLE-B3 cells were transfected with lentivirus loaded with MFN2 overexpression plasmid or short hairpin RNA (shRNA) to overexpress or knock down MFN2 expression, followed by HG exposure. Cell viability was assessed by CCK-8 assay. Flow cytometry was used to detect cell apoptosis and reactive oxygen species (ROS) level. JC-1 staining showed the changes in mitochondrial membrane potential (Δψm). The mediators related to apoptosis, mitochondrial damage, and autophagy were determined. RESULTS:STZ-administrated rats showed reduced body weight, increased blood glucose levels, elevated SDH activity and AGE content, suggesting successful establishment of the DC rat model. Interestingly, MFN2 expression was significantly downregulated in DC rat lens and HG-induced HLE-B3 cells. Further analysis showed that under HG conditions, MFN2 overexpression enhanced cell viability and inhibited apoptosis accompanied by decreased Bax, cleaved caspase-9 and increased Bcl-2 expression in HLE-B3 cells. MFN2 overexpression also suppressed the mitochondrial damage elicited by HG as manifested by reduced ROS production, recovered Δψm and increased mitochondrial cytochrome c (Cyto c) level. Moreover, MFN2 overexpression increased LC3BⅡ/LC3BⅠ ratio and Beclin-1 expression, but decreased p62 level, and blocked the phosphorylation of mTOR in HG-treated HLE-B3 cells. In contrast, MFN2 silencing exerted opposite effects. CONCLUSIONS:Presented findings indicate that MFN2 expression may be essential for preventing lens epithelial cell apoptosis during development of diabetic cataract.

Purpose:The purpose of this study was to investigate the role of lncRNA H19 in epithelial-mesenchymal transition (EMT) and its molecular mechanism in fibrotic cataracts.Methods:TGF-β2-induced EMT was induced in human lens epithelial cell line (HLECs) and rat lens explants to mimic posterior capsular opacification (PCO) in vitro and in vivo. Anterior subcapsular cataract (ASC) was induced in C57BL/6J mice. The long noncoding RNA (lncRNA) H19 (H19) expression was detected by RT-qPCR. Whole-mount staining of lens anterior capsule was used to detect α-SMA and vimentin. Lentiviruses carrying shRNA or H19 vector were transfected in HLECs to knockdown or overexpress H19. Cell migration and proliferation were characterized by EdU, Transwell, and scratch assay. EMT level was detected by Western blotting and immunofluorescence. The rAAV2 carrying mouse H19 shRNA was injected into ASC model mouse anterior chambers as a gene therapy to determine its therapeutic potential.Results:PCO and ASC models were built successfully. We found H19 upregulation in PCO and ASC models in vivo and in vitro. Overexpression of H19 by lentivirus transfection increased cell migration, proliferation, and EMT. In addition, H19 knockdown by lentivirus suppressed cell migration, proliferation, and EMT levels in HLECs. Moreover, transfection of rAAV2 H19 shRNA alleviated fibrotic area in ASC mouse lens anterior capsules.Conclusions:Excessive H19 participates in lens fibrosis. Overexpression of H19 increases, whereas knockdown of H19 ameliorates HLECs migration, proliferation, and EMT. These results demonstrate H19 might be a potential target for fibrotic cataracts.

Objective:To establish an in vitro capsular bag model and compare the inhibitory effects of different 360° square-edge intraocular lens (IOL) on lens epithelial cells (LECs) migration. Methods:In vitro capsular bag model with posterior capsule opacification (PCO) was established using Transwell compartment, cell climbing slices, human collagen type Ⅳ, and IOL.The models were divided into Plate-loop HydroSmart group, C-loop HydroSmart group, and C-compensation-loop Hydrophobic group according to the different square-edge IOL implanted.A blank control group was set using the Transwell compartment without IOL.The early PCO pathological manifestations in lens epithelial cell line SRA01/04 cultured in the Transwell compartment were observed with an inverted microscope.The cell morphology in different groups was observed by hematoxylin and eosin staining.The cell counting and cell migration inhibition rate of anterior capsule and posterior capsule were calculated by Transwell assay and cell-exclusion zone assay, respectively. Results:The early pathological characteristics of PCO, such as early Soemmering ring and small Elschnig pearl, could be found in cells in the in vitro capsular bag model after 48-hour culture.The migrating cells in model groups were fibrous.No changes mentioned above were found in blank control group.The number of migrating cells in the anterior capsule of Plate-loop HydroSmart group, C-loop HydroSmart group, C-compensation-loop Hydrophobic group was 18.80±5.53, 24.67±9.80, and 34.47±10.80, respectively, and the number of migrating cells in the optical area of the posterior capsule of the three groups was 56.43±9.00, 162.20±16.38, and 121.30±12.01, respectively.The cell migration inhibition rate in the anterior capsule of Plate-loop HydroSmart group, C-loop HydroSmart group, C-compensation-loop Hydrophobic group was (92.02±1.94)%, (89.76±3.10)%, (86.27±4.54)%, respectively, and the cell migration inhibition rate in optical area of the posterior capsule of the three groups was (91.60±3.65)%, (70.14±5.35)%, (78.43±3.48)%, respectively.The number of migrating cells in the anterior capsule was lower and the cell migration rate inhibition was higher in Plate-loop HydroSmart group than C-compensation-loop Hydrophobic group, with significant differences (both at P<0.05). The number of migrating cells in the optical area of the posterior capsule and the cell migration inhibition rate was greater than those of C-loop HydroSmart group and C-compensation-loop Hydrophobic group, showing statistically significant differences (all at P<0.001). Conclusions:The in vitro capsular bag model can be used in PCO research.Compared with C-loop HydroSmart IOL and C-compensation-loop Hydrophobic IOL, Plate-loop HydroSmart IOL can more effectively inhibit the migration of LECs to the optical area of the posterior capsule.

Mitofusin 2 (Mfn2) mediates the mitochondrial fusion in dynamic balance between mitochondrial fission and fusion. This study aimed to investigate the role of Mfn2 in mice corneal dysplasia with conditional knockout (CKO) technique. The Mfn2 CKO mice model was established with the Cre-loxP system. Each offspring of Le-Cre +/-; Mfn2 fl/fl (Mfn2 CKO) mice and Mfn2fl/fl (Mfn2 WT) mice was identified by polymerase chain reaction (PCR). Macroscopic observation, immunohistostaining and HE staining were used to evaluate the corneal morphologic development in Mfn2 CKO mice and Mfn2 WT mice. The cells proliferation and apoptosis were detected by BrdU labeling and TUNEL assay. Real-time PCR was used to detect mRNA expression of corneal markers (K12, Col1α1, Pax6, keratocan and NSE). Results showed that Mfn2 CKO mice showed increased corneal thickness, small eyeball from E15.5 to P60 and small eye crack after birth. The corneal stromal thickness significantly increased in Mfn2 CKO mice, and the random arrangement fibers of the corneal stroma increased in Mfn2 CKO mice. The proliferative cells in the cornea of Mfn2 CKO mice were less than in Mfn2 WT mice while the apoptotic cells in the cornea of Mfn2 CKO mice were increased. K12 and Pax6 expression decreased in the cornea and the Col1α1 expression increased in Mfn2 CKO mice as compared to Mfn2 WT mice. The expression of corneal stromal marker Col1α1 in the Mfn2 CKO mice was significantly higher than that in the Mfn2 WT mice. Corneal thickness was mainly caused by corneal stroma collagen proliferation. In conclusion, Mfn2 deletion affects corneal development, especially because of collagen hyperplasia in the corneal stroma.

The odd‐skipped related 1 ( OSR 1 ) gene encodes a zinc‐finger transcription factor. The expression and significance of OSR 1 in human tumors remains unclear. We found that OSR 1 was downregulated in lung cancers, and its expression was correlated with poor differentiation. Overexpression of OSR 1 by OSR 1 gene transfection into H1299 cells (H1299‐ OSR 1 ) inhibited the proliferation and invasion of lung cancer cells. Knockdown of OSR 1 with small interfering (si) RNA against OSR 1 in A549 cells (A549‐si OSR 1 ) enhanced the proliferation and invasion of lung cancer cells. Western blot analysis showed that the expression level of GSK 3β increased, while that of p‐ GSK 3β, nuclear β‐catenin, cyclin D1, c‐Myc and matrix metallopeptidase 7 significantly decreased in the H1299‐ OSR 1 cells, and this pattern was reversed in the A549‐si OSR 1 cells compared to that in the control cells. Furthermore, upregulation of sex‐determining region Y‐box 9 ( SOX 9) by SOX 9 gene transfection increased the expression of β‐catenin, which was inhibited by OSR 1. The mRNA and protein expression levels of SOX 9 and β‐catenin were reduced in H1299‐ OSR 1 cells and increased in A549‐si OSR 1 cells. In conclusion, the expression of OSR 1 was more reduced in lung cancer tissues than in normal lung tissues, and was correlated with poor differentiation. OSR 1 downregulated the activity of the Wnt signaling pathway by suppressing the expression of SOX 9 and β‐catenin.

Background: Nemo-like kinase (NLK) is an evolutionarily conserved MAP kinaserelated kinase involved in the pathogenesis of several human cancers. Objective: The aim of this study was to investigate the expression and role of NLK in lung cancers, and its underlying mechanisms. Methods: We examined the expression of NLK in lung cancer tissues through western blot analysis. We enhanced or knocked down NLK expression by gene transfection or RNA interference, respectively, in lung cancer cells, and examined expression alterations of key proteins in the Wnt signaling pathway and in epithelial-mesenchymal transition (EMT). We also examined the roles of NLK in the proliferation and invasiveness of lung cancer cells by cell proliferation, colony formation, and Matrigel invasion assays. Results: NLK expression was found to be significantly higher in lung cancer tissue samples than in corresponding healthy lung tissue samples. Overexpression of NLK correlated with poor prognosis of patients with lung cancer. Overexpression of NLK upregulated β-catenin, TCF4, and Wnt target genes such as cyclin D1, c-Myc, and MMP7. N-cadherin and TWIST, the key proteins in EMT, were upregulated, while E-cadherin expression was reduced. Additionally, proliferation, colony formation, and invasion turned out to be enhanced in NLK-overexpressing cells. After NLK knockdown in lung cancer cells, we obtained the opposite results. Conclusion: NLK is overexpressed in lung cancers and indicates poor prognosis. Overexpression of NLK activates the Wnt signaling pathway and EMT and promotes the proliferation and invasiveness of lung cancer cells.