The inclusion criteria for the patients with glaucoma included in the present study were as follows: i) Patients diagnosed with glaucoma according to the latest diagnostic criteria for glaucoma developed by the Cooperative Group on Fundus Diseases of the People's Republic of China (6); and ii) patients who have undergone glaucoma surgery. Patients were excluded based on the following criteria: i) Patients with other diseases and currently receiving treatment; ii) pregnant or breast-feeding women; iii) patients who are allergic to probiotics or who have recently used/are using antibiotics; iv) patients with alcoholism (individuals who drink ≥5 bottles of beer at a time or have a blood alcohol level of ≥0.08 g/100 ml); and v) smokers, according to a previous reference (6).

Matched HS and normal (control/healthy) tissues were collected from patients (n=5) with POAG who underwent glaucoma filtration surgery at Fuyang People's Hospital (Hangzhou, China) between November 2019 and May 2020 according to previous reports (16,17). It has been reported that HS tissue has more melanocytes, and that there are fewer melanocytes in normal tissue (18). The basic clinicopathological characteristics of the patients are listed in Table I. Subsequently, to isolate HTFs from HS tissues and corneal epithelial cells (control cells) from healthy tissues. Briefly, 5x5-mm sections of Tenon's capsule were collected, minced and placed in a 35-mm culture dish containing Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc.), 50 U/ml penicillin and 50 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were allowed to migrate from the explanted tissue and were then incubated at 37˚C in 5% CO₂. Cells between the third and fifth passages were used according to a previous study (19). Next, HTFs were maintained in DMEM supplemented with 10% FBS, 1% penicillin and 1% streptomycin (all from Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO₂, as previously described (20,21). The isolated cells were observed using fluorescent staining under a fluorescence microscope (Olympus Corporation). The present study was approved by the Ethics Committee of Fuyang People's Hospital (Hangzhou, China; approval no. FPH20191011), and written informed consent was provided by all patients prior to the study onset.

3-Methyladenine (3-MA) was obtained from MedChemExpress (cat. no. HY-19312). Cells were treated with 1 mmol/l 3-MA for 24 h according to a previous study (22). In addition, TGF-β was provided by Millipore Sigma (cat. no. SAB4502954). Cells were treated with 10 ng/ml TGF-β for 24 h according to a previous study (23).

Total RNA was extracted from HS tissues, healthy tissues and HTFs (5x10⁴/ml) using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the EntiLink™ 1st Strand cDNA Synthesis kit [ELK (Wuhan) Biotechnology Co., Ltd.] according to the manufacturer's instructions. qPCR was subsequently performed using the StepOne™ Real-Time PCR System (Thermo Fisher Scientific, Inc.). EnTurbo™ SYBR Green PCR SuperMix kit (ELK Biotechnology Co., Ltd.) was used for qPCR. The thermocycling conditions were used as follows: 3 min at 95˚C, followed by 40 cycles of 10 sec at 95˚C, 30 sec at 58˚C and 30 sec at 72˚C. mRNA expression levels were normalized to β-actin levels. Relative expression levels were calculated using the 2^-ΔΔCq method (24). The following primer sequences were used for qPCR: β-actin forward, 5'-GTCCACCGCAAATGCTTCTA-3' and reverse, 5'-TGCTGTCACCTTCACCGTTC-3'; and LINC01605 forward, 5'-CAACTCATTCCCGTTACAAACA-3' and reverse, 5'-CATCTCAACTGCCTCTGTCTCC-3'.

Droplets of suspended corneal epithelial cells and HTFs (3x10⁵ cells/well) were cultured in a 24-well plate at 37˚C with 5% CO₂ and subsequently fixed with 4% paraformaldehyde for 30 min at room temperature. The following primary antibodies were diluted with 5% BSA (Beyotime Biotechnology): Anti-vimentin (1:1,000; Abcam; cat. no. ab92547), anti-keratin (1:1,000; Abcam; cat. no. ab8068) and anti-LC3 (1:1,000; Abcam; cat. no. ab192890) at 4˚C. Following primary antibody incubation overnight at 4˚C, cells were incubated with horseradish peroxidase IgG secondary antibody (1:5,000; Abcam; cat. no. ab6728) for 1 h at room temperature. Cells were washed three times with PBS. Cell nuclei were stained with 50-100 µl DAPI at room temperature for 5 min. Finally, cells were observed under a fluorescence microscope (Olympus Corporation).

Three siRNAs against LINC01605 and an siRNA-negative control (NC) were synthesized by Guangzhou RiboBio Co., Ltd. The following sequences were used: LINC01605 siRNA1, 5'-TCTTGAAGAATAAGAAGCCACAGCT-3'; LINC01605 siRNA2, 5'-GAGTCTTGAAGAATAAGAAGCCACA-3'; LINC01605 siRNA3, 5'-TAAGAAGCCACAGCTTGTCAGGGAA-3'; and siRNA-NC, 5'-GAGGTTGAATAAGAAGAACCTCACA-3'. The siRNAs (10 nM) were transfected into HTFs (3x10⁵ cells/well) using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.) for 48 h at 37˚C. After 48 h of incubation, cells were used for subsequent experimentation. The blank group was comprised of non-transfected cells.

The Cell Counting Kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology) was performed to assess cell viability. HTFs were seeded into 96-well plates at a density of 5x10³ cells/well. Next, siRNA-NC, LINC01605 siRNA1 and LINC01605 siRNA2 were transfected into HTFs using Lipofectamine^® 2000 reagent for 6 h at 37˚C. The culture medium was then changed and HTFs continued to be cultured in DMEM for 0, 24, 48 and 72 h. Next, 10 µl CCK-8 reagent was added into each well and cells were incubated for 2 h at 37˚C. Absorbance was measured at a wavelength of 450 nm using a microplate reader (Varioskan™ LUX; Thermo Fisher Scientific, Inc.).

Apoptosis was analyzed by flow cytometry. HTFs were seeded into six-well plates at a density of 5x10⁴ cells/ml. Following transfection with siRNAs, cells were stained with Annexin V-FITC and PI (Tianjin Sungene Biotech, Co., Ltd.) for 15 min in the dark at room temperature. Subsequently, the apoptotic rate (the sum of early and late apoptosis) was analyzed using a FACSCanto II flow cytometer (BD Biosciences). The data was analyzed using FlowJo software (version 10.6.2; FlowJo LLC).

Protein was extracted from HTFs using RIPA buffer (Aspen Biotechnology) and concentration was determined using a BCA kit (Aspen Biotechnology, Co., Ltd.). Proteins (30 µg/lane) were separated by 10% SDS-PAGE, transferred onto PVDF membranes and blocked with 5% non-fat milk diluted in TBS with 0.1% Tween-20 for 1 h at room temperature. The membranes were incubated overnight at 4˚C with primary antibodies against Bax (1:1,000; Abcam; cat. no. ab32503), Bcl-2 (1:1,000; Abcam; cat. no. ab32124), Pro-caspase-3 (1:1,000; Abcam; cat. no. ab32150), cleaved caspase-3 (1:1,000; Abcam; cat. no. ab2302), phosphorylated (p)-Smad2 (1:1,000; Abcam; cat. no. ab280888), Smad2 (1:1,000; Abcam; cat. no. ab40855), α-SMA (1:1,000; Abcam; cat. no. ab5694), MMP9 (1:1,000; Abcam; cat. no. ab76003), autophagy-related (ATG)7 (1:1,000; Abcam; cat. no. ab52472), p62 (1:1,000; Abcam; cat. no. ab109012), beclin 1 (1:1,000; Abcam; cat. no. ab207612), p-AMP-activated protein kinase (AMPK) (1:1,000; Abcam; cat. no. ab133448), AMPK (1:1,000; Abcam; cat. no. ab32047) and β-actin (1:1,000; Abcam; cat. no. ab8227). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (1:5,000; cat. no. ab7090; Abcam) at room temperature for 1 h. Protein bands were visualized using the Enhanced Chemiluminescence Reagent (Thermo Fisher Scientific, Inc.). β-actin was used as a loading control. Finally, the density of the blots was analyzed using AlphaEaseFC software (version 4.0; Alpha Innotech Corporation).

The Transwell migration assay was performed using 24-well Transwell chambers (Corning, Inc.). Firstly, HTFs during the logarithmic growth phase were incubated overnight at 37˚C. Next, HTFs were transfected with siRNA-NC, LINC01605 siRNA1 and LINC01605 siRNA2 using Lipofectamine 2000 reagent for 24 h at 37˚C. After that, HTFs (200 µl) were plated on the upper chamber suspended with 100 µl serum-free DMEM at 37˚C, and 600 µl complete DMEM supplemented with 10% FBS was added to the lower chamber; cells were incubated for 24 h at 37˚C. Following incubation, the migrated cells on the lower chamber were stained with 0.2% crystal violet at room temperature for 30 min and observed under a light microscope (Leica Microsystems Ltd.; cat. no. DMLB2).

Statistical analysis was performed using GraphPad Prism software (v7.0; GraphPad Software, Inc.). All experiments were performed in triplicate. The comparison between two matched samples was analyzed with paired Student's t-test. One-way ANOVA followed by Tukey's post hoc test was used to compare differences between multiple groups. All experimental data are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

Matching HS and healthy tissues were collected from patients with POAG who underwent glaucoma filtration surgery at Fuyang People's Hospital, and RT-qPCR analysis was performed to detect LINC01605 expression levels. The results demonstrated that LINC01605 expression was higher in HS tissues compared with that in normal (control/healthy) tissues (Fig. 1A). Subsequently, HTFs and corneal epithelial cells were isolated from the aforementioned tissues. In addition, it has been reported that vimentin is a fibrocyte marker (25). Vimentin is one of the main components of medium fiber and plays an important role in cytoskeleton and motility (26,27). Furthermore, keratin is the main component of cytoskeletal proteins (28). Immunofluorescence assays indicated that vimentin was highly expressed in HTFs and in corneal epithelial cells, whereas keratin was expressed at a low level in HTFs (Fig. 1B).

LINC01605 expression significantly decreased in HTFs following transfection with LINC01605 siRNAs (Fig. 1C). The results indicated that LINC01605 siRNA1 and LINC01605 siRNA2 had a stronger knockdown effect compared with LINC0165 siRNA3, thus these two siRNAs were selected for subsequent experiments (Fig. 1C). LINC01605 siRNA1 and LINC01605 siRNA2 significantly inhibited the viability of HTFs compared with siRNA-NC (Fig. 1D). These results suggested that HTFs were successfully isolated and LINC01605 siRNA significantly inhibited the viability of HTFs.

To investigate the function of LINC01605 in HTFs, flow cytometry was performed. As presented in Fig. 2A, LINC01605 knockdown significantly induced apoptosis in HTFs compared with the siRNA-NC or blank group. In addition, transfection with LINC01605 siRNAs notably increased the expression levels of apoptosis-related proteins Bax and cleaved caspase-3, and decreased Bcl-2 expression in HTFs, compared with the siRNA-NC or blank group (Fig. 2B). Collectively, these results suggested that LINC01605 knockdown significantly induced apoptosis in HTFs.

Transwell assays were performed to investigate the effect of LINC01605 on the migratory ability of HTFs. The results demonstrated that LINC01605 knockdown significantly inhibited the migratory ability of HTFs compared with the siRNA-NC or blank group (Fig. 3A). In addition, p-Smad2, α-SMA and MMP9 are important proteins in the process of fibrosis (29-31). Western blot analysis demonstrated that transfection with LINC01605 siRNAs significantly downregulated the ratio of p-Smad2/total Smad2, and the expression levels of α-SMA and MMP9 in HTFs, compared with the siRNA-NC or blank group (Fig. 3B-E). Taken together, these results suggested that LINC01605 knockdown inhibited the migratory ability and the fibrosis process of HTFs.

To determine the association between LINC01605 and autophagy, immunofluorescence staining of LC3 was performed. The results demonstrated that transfection with LINC01605 siRNA significantly decreased LC3 expression in HTFs compared with the siRNA-NC or blank group (Fig. 4A). In addition, the expression levels of autophagy-related proteins were examined. Transfection with LINC01605 siRNAs notably decreased ATG7 and beclin 1 expression, and increased p62 expression in HTFs, compared with the siRNA-NC or blank group (Fig. 4B-E). Since AMPK/mTOR has been reported to serve a crucial role in autophagy, the function of LINC01605 in this signaling pathway was investigated (32,33). Western blotting results demonstrated that transfection with LINC01605 siRNA markedly decreased p-AMPK expression in HTFs compared with the siRNA-NC or Blank group (Fig. 4B and F), indicating that LINC01605 knockdown markedly inhibited autophagy in HTFs. Moreover, it has been reported that 3-methyladenine (3-MA), which is an autophagy inhibitor, could inhibit the formation of autophagosomes (34,35). Given that HTFs were sensitive to LINC01605 siRNA2 (Fig. 1C), it was selected for use in subsequent experiments. LINC01605 siRNA2 significantly inhibited the viability of HTFs compared with the siRNA-NC or blank group, and 3-MA exacerbated this phenomenon (Fig. 4G). These results indicated that LINC01605 knockdown inhibited autophagy in HTFs.

It has been reported that TGF-β is closely associated with autophagy and fibrosis (36). To further investigate the mechanism by which LINC01605 regulates the formation of HS, rescue experiments were performed. As presented in Fig. 5A and B, LINC01605 knockdown notably inhibited the viability of HTFs by inducing apoptosis, compared with the siRNA-NC or blank group, the effects of which were reversed following treatment with TGF-β. In addition, LINC01605 knockdown notably inhibited the migration of HTFs, compared with siRNA-NC or Blank group, and the effects of which were reversed following treatment with TGF-β (Fig. 5C). Taken together, these results suggested that LINC01605 siRNA2-induced apoptosis of HTFs was reversed by TGF-β.