Scar fibroblasts were isolated from a patient (aged 63 years, female) who underwent glaucoma surgery in The Affiliated Hospital of Beihua University in August 2019, as previously described (26). The patient with glaucoma was diagnosed as previously described (27). The clinical and pathological data of patient were collected following their written informed consents. The present study was approved by the Ethics Committee of The Affiliated Hospital of Beihua University.

The scar fibroblasts were cultured in 90% RPMI-1640 medium supplemented with 10% FBS, 1% penicillin and 1% streptomycin (all from Thermo Fisher Scientific, Inc.) in an incubator with 5% CO₂ at 37°C.

HS tissues (n=5) and normal tissues (n=5) were obtained from The Affiliated Hospital of Beihua University between August 2019 and April 2020. HS and normal tissues were obtained from patients who underwent glaucoma surgery. Patients with glaucoma were diagnosed as previously described (27). Moreover, the patients who donated the HS tissues and the normal tissues were the same. The distributions of age and sex among the patients are listed in Table I. The present study was approved by the Institutional Ethical Committee of The Affiliated Hospital of Beihua University. Written informed consent was obtained from all participants.

The patient exclusion criteria were as follows: i) Individuals who suffered from other diseases and are currently under treatment; ii) pregnant and lactating women; iii) patients allergic to probiotics or have used/are using antibiotics recently; and iv) alcoholics (individuals who drank ≥5 bottles of beer at a time, or for whom the alcohol content in the blood was at ≥0.08) and smokers.

The patient inclusion criteria were as follows: Individuals diagnosed with glaucoma, according to the latest glaucoma diagnostic criteria formulated by the Ocular Fundus Disease Cooperative Group of the People's Republic of China (28), and patients who received glaucoma surgery.

Total RNA was extracted from scar fibroblasts or tissues using TRIzol^® reagent (Takara Bio, Inc.) according to the manufacturer's protocol. Subsequently, cDNA was synthesized using PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer's instructions. The temperature and duration of RT were: 37°C for 60 min and 85°C for 5 min. RT-qPCR was performed using a SYBR^® Premix Ex Taq™ II kit (Takara Bio, Inc.) on a 7900HT system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in triplicate under the following conditions: Initial denaturation for 10 min at 95°C; 40 cycles of 95°C for 15 sec and 60°C for 30 sec; and final extension for 1 min at 60°C. The primer sequences (Shanghai GenePharma Co., Ltd.) used were as follows: FAM225B forward, 5′-CCCTTGGATGCTTGGAGTGA-3′ and reverse, 5′-GCGACGGTGCTGAATCTTGT-3′; p62 forward, 5′-GAACAGATGGAGTCGGATAACTG-3′ and reverse, 5′-CTGGGAGAGGGACTCAATCAG-3′; Beclin 1 forward, 5′-GGCACAATCAATAACTTCAGGC-3′ and reverse, 5′-GGCAGCTCCTTAGATTTGTCTG-3′; for microtubule associated protein 1 light chain 3 α (LC3) forward, 5′-ACCGCTGTAAGGAGGTACAGC-3′ and reverse, 5′-GAAGCCGTCCTCGTCTTTCT-3′; autophagy related (ATG)7 forward, 5′-TTCCTCCTCTTGACATTTGCAG-3′ and reverse, 5′-TATCTTCGTCCTTTGACCTTGG-3′; and β-actin forward, 5′-GTCCACCGCAAATGCTTCTA-3′ and reverse, 5′-TGCTGTCACCTTCACCGTTC-3′. The relative expression was quantified using the 2^−ΔΔCq method (29). The internal reference gene β-actin was utilized for normalization.

The small interfering RNAs (siRNAs) against FAM225B (FAM225B siRNA1, FAM225B siRNA2 and FAM225B siRNA3; 10 nM) and negative control siRNA (siRNA-NC) were purchased from Shanghai GenePharma Co., Ltd., and were transfected into scar fibroblasts using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.) at 37°C for 48 h according to the manufacturer's instructions. Cells were incubated at 37°C for 6 h and the transfection efficiency was determined using RT-qPCR. The siRNA sequences were as follows: NC siRNA, 5′-GTCGCAGTACGCATACCTT-3′; FAM225B siRNA1, 5′-GCATTGGCCTTGACCACAT-3′; FAM225B siRNA2, 5′-GCGTATGCAGACGTTGCTT-3′; and FAM225B siRNA3, 5′-GCTGCTAAGTGGCAGGTAA-3′.

For FAM225B overexpression, scar fibroblasts were transfected with pcDNA3.1 vector (NC, 10 nM) or pcDNA3.1-FAM225B overexpression plasmid (FAM225B-OE, 10 nM) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h. The pcDNA3.1 vector and FAM225B-OE plasmid were purchased from Shanghai GenePharma Co., Ltd.

Total proteins were isolated from scar fibroblasts using a RIPA buffer (Shanghai GenePharma Co., Ltd.) and were quantified with a BCA protein assay kit (Beyotime Institute of Biotechnology). Total proteins (30 µg) were separated by 10% SDS-PAGE, and then transferred onto PVDF (Bio-Rad Laboratories, Inc.) membranes. Following blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Subsequently, membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. ab7090; 1:5,000; Abcam) at room temperature for 1 h. Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) was used to visualize the protein bands. The membranes were scanned with an Odyssey Imaging system and analyzed using the Odyssey v2.0 software (Li-Cor Biosciences). The primary antibodies used in the current study were as follows: Anti-p62 (cat. no. ab109012; 1:1,000), anti-ATG7 (cat. no. ab52472; 1:1,000), anti-Beclin 1 (cat. no. ab207612; 1:1,000), anti-Bax (cat. no. ab32503; 1:1,000), anti-Bcl-2 (cat. no. ab182858; 1:1,000), anti-cleaved caspase 3 (cat. no. ab49822; 1:1,000) and anti-β-actin (cat. no. ab8226; 1:1,000; all from Abcam). β-actin served as an internal control.

Cell viability was tested by CCK-8 kit according to the manufacturer's instructions. Briefly, scar fibroblasts were transfected with NC siRNA (10 nM), FAM225B siRNA1/siRNA2/siRNA3 (10 nM), FAM225B-OE (10 nM) or FAM225B-OE (10 nM) + 3-methyladenine (3-MA; MedChemExpress) for 0, 24, 48 and 72 h at 37°C. Then, cells were treated with 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) and incubated for an additional 2 h at 37°C. The absorbance was measured at 450 nm using a microplate reader.

Following transfected with siRNA-NC (10 nM), FAM225B siRNA2 (10 nM), FAM225B siRNA3 (10 nM), pcDNA3.1-NC (10 nM), FAM225B-OE (10 nM) or FAM225B-OE (10 nM) + 3-MA at 37°C for 72 h, scar fibroblasts were fixed with 4% paraformaldehyde at room temperature for 20 min, washed with PBS and blocked with 2% BSA (Beyotime Institute of Biotechnology) at room temperature for 30 min. Subsequently, cells were incubated with an EdU Assay/EdU Staining Proliferation kit (iFluor 647; cat. no. ab222421; Abcam) or anti-LC3 antibody (cat. no. ab63817; Abcam; 1:1,000) at 4°C overnight. Subsequently, cells were incubated with a secondary antibody [cat. no. ab6728; IgG (horseradish peroxidase); Abcam; 1:5,000] at room temperature for 1 h. The nuclei were stained with DAPI (Beyotime Institute of Biotechnology) at room temperature for 5 min. Finally, cells were observed under a fluorescent microscope at a magnification of ×200.

For migration assays, the transfected cells (density, 3×10⁴ cells/well) were resuspended in serum-free DMEM (100 µl; Thermo Fisher Scientific, Inc.) and were then plated onto the upper chamber of the Transwell 24-well plates. Moreover, the lower chamber of each well was supplemented with 600 µl DMEM containing 10% FBS. Following incubation for 24 h at 37°C, the non-migrated cells on the upper surface of the membrane were removed. Subsequently, the migrated cells on the lower surface were fixed with 4% paraformaldehyde at room temperature for 15 min, followed by staining with 0.5% crystal violet at room temperature for 30 min. The number of migrated cells was counted under a light microscope at a magnification of ×400.

The early and late apoptosis (Annexin-V⁺ PI⁻ plus Annexin-V⁺ PI⁺) of scar fibroblasts were measured via flow cytometry. Scar fibroblasts were seeded into 6-well plates at a density of 1×10⁶ cells/well. The residue was resuspended in 100 µl binding buffer following centrifugation (4°C) at 500 × g for 5 min. Subsequently, 5 µl Annexin V-FITC (BD Biosciences) and 5 µl PI (BD Biosciences) at room temperature were added into the cell suspension for 15 min. The cell apoptosis rate was measured using a flow cytometer (BD Biosciences) and the results were analyzed using FACS (BD Biosciences) with FlowJo (v10.6.2; FlowJo LLC).

ROS detection was performed using a ROS detection kit (green fluorescence; Beyotime Institute of Biotechnology). Cell suspensions were collected and stained with the ROS probe 2,7-dichlorofluorescein diacetate (Beyotime Institute of Biotechnology) at room temperature for 15 min as previously described (30). After 20 min of incubation, cells were centrifuged at 300 × g (4°C for 10 min), washed with PBS and resuspended. Finally, the relative ROS level was measured via flow cytometry (FACSAria II; BD Biosciences), and the data were analyzed using FlowJo 10.0 (BD Biosciences).

The levels of superoxide dismutase (SOD) and glutathione (GSH) in the supernatants of scar fibroblasts (collected by centrifugation at 4°C for 10 min at 300 × g) were detected using SOD ELISA kit [cat. no. CSB-EL022399; Multisciences (Lianke) Biotech Co., Ltd.] and GSH ELISA kit [cat. no. xy-E10818; Multisciences (Lianke) Biotech Co., Ltd.] according to the manufacturer's instructions.

Data are presented as the mean ± SD. The CCK-8 assay was performed in quintuplicate. Cell transfections, RT-qPCR, immunofluorescence staining, flow cytometry, western blot analysis and the Transwell migration assay were repeated three times. GraphPad Prism software (version 7.0; GraphPad Software, Inc.) was used to analyze the data. One-way ANOVA and Tukey's tests were conducted to evaluate the differences for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The morphology of scar fibroblasts isolated from a patient was observed under a microscope, and fibrotic scarring was observed (Fig. 1A). This morphology was consistent with the characteristics of scar fibroblasts (31). Moreover, the expression of FAM225B was significantly upregulated in HS tissues, compared with normal tissues (Fig. 1B). It was also found that the expression of FAM225B in scar fibroblasts was significantly downregulated following transfection with FAM225B siRNAs (Fig. 1C). In addition, FAM225B was more efficiently downregulated when scar fibroblasts were treated with FAM225B siRNA2 or siRNA3 compared with FAM225B siRNA1. Therefore, FAM225B siRNA2 and siRNA3 were selected to knockdown FAM225B expression in the subsequent experiments. The viability of scar fibroblasts was significantly decreased following transfection with FAM225B siRNA2 or siRNA3 (Fig. 1D). Consistently, knockdown of FAM225B significantly attenuated the proliferation of scar fibroblasts (Fig. 1E). Overall, these findings indicated that FAM225B knockdown significantly attenuated the proliferation of scar fibroblasts.

To evaluate the effect of FAM225B on the levels of ROS in scar fibroblasts, flow cytometric analysis was conducted. As expected, knockdown of FAM225B significantly increased ROS levels in scar fibroblasts (Fig. 2A). In addition, the levels of SOD and GSH in the supernatants of scar fibroblasts were significantly decreased by FAM225B siRNA (Fig. 2B and C). Since the levels of ROS, SOD and GSH are closely associated with cell injury (32), the aforementioned data suggested that knockdown of FAM225B could notably induce oxidative stress in scar fibroblasts.

In order to evaluate the association between FAM225B and cell autophagy, immunofluorescence staining against LC3 (autophagy indicator) was performed. As presented in Fig. 3A, the expression of LC3 in scar fibroblasts was significantly decreased following FAM225B knockdown. In addition, knockdown of FAM225B significantly upregulated p62 expression (Fig. 3B and C). By contrast, the protein expression levels of ATG7 and Beclin-1 in scar fibroblasts were significantly downregulated in cells transfected with FAM225B siRNA (Fig. 3B and C). However, knockdown of FAM225B had a very limited effect on the mRNA expression of LC3 (Fig. 3C). Since LC3, p62, ATG7 and Beclin-1 are involved in the process of cell autophagy (33–35), these factors can be considered as autophagy indicators. Thus, the results indicated that FAM225B knockdown attenuated the progression of HS in vitro by inhibiting autophagy.

To evaluate cell apoptosis, flow cytometric analysis (Annexin V and PI staining) was performed. As presented in Fig. 4A, knockdown of FAM225B significantly induced scar fibroblast apoptosis. Furthermore, FAM225B siRNA significantly increased the expression levels of Bax and cleaved caspase 3, and downregulated those of Bcl-2 in scar fibroblasts (Fig. 4B). FAM225B knockdown also significantly inhibited the migratory ability of scar fibroblasts (Fig. 4C). Therefore, knockdown of FAM225B markedly induced apoptosis and attenuated the migration of scar fibroblasts.

In order to further confirm the association between FAM225B and cell autophagy in scar fibroblasts, cells were overexpressed with FAM225B, and then the efficiency of overexpression was detected. As expected, pcDNA3.1-FAM225B significantly increased the expression levels of FAM225B in scar fibroblasts (Fig. 5A). In addition, the viability of scar fibroblasts was significantly enhanced by FAM225B overexpression, which was partially reversed following treatment of fibroblasts with the autophagy inhibitor 3-MA (Fig. 5B). Consistently, the FAM225B overexpression-mediated increase of scar fibroblast proliferation was significantly inhibited by 3-MA (Fig. 5C). Furthermore, overexpression of FAM225B significantly enhanced the migratory ability of scar fibroblasts, which was notably reversed by 3-MA (Fig. 5D). Collectively, these results suggested that overexpression of FAM225B significantly promoted the proliferation of scar fibroblasts by inhibiting autophagy.