Glaucoma is an irreversible disease that causes blindness. Formation of a hypertrophic scar (HS) is the main cause of failure of glaucoma surgery. The long non-coding RNA LINC01605 is closely associated with the formation of HS; however, the function of LINC01605 in the formation and development of HS remains unclear. For this study, firstly, human Tenon's capsule fibroblasts (HTFs) and corneal epithelial cells (control cells) were collected from patients (n=5) with POAG who underwent glaucoma filtration surgery at Fuyang People's Hospital. Immunofluorescence analysis was performed to detect the expression levels of vimentin (one of the main components of medium fiber and plays an important role in the cytoskeleton and motility), keratin (the main component of cytoskeletal proteins) and LC3 (an autophagy marker). In addition, reverse transcription-quantitative PCR analysis was performed to detect LINC01605 expression. Besides, the Cell Counting Kit-8 assay was performed to assess the viability of human Tenon's capsule fibroblasts (HTFs). Next, flow cytometry was performed to detect HTF apoptosis. Furthermore, western blot analysis was performed for Bax, Bcl-2, Pro-caspase-3, cleaved caspase-3, phosphorylated (p-)Smad2, Smad2, α-SMA, MMP9, ATG7, p62, beclin 1, p-AMPK and AMPK in HTFs to determine the mechanism by which LINC01605 regulates the formation and development of HS. Moreover, a Transwell assay was performed to detect the migratory ability of HTFs. The results demonstrated that LINC01605 was significantly upregulated in HS tissues compared with that in normal (control/healthy) tissues. In addition, vimentin was highly expressed in HTFs, whereas keratin was expressed at a low level. Also, in HTFs, LINC01605 knockdown inhibited cell viability by inducing apoptosis, decreasing Smad2 activation and inhibiting autophagy. Furthermore, LINC01605 knockdown significantly inhibited the migratory ability of HTFs. Transfection with LINC01605 small interference RNAs significantly downregulated the expression levels of p-Smad2, α-SMA and MMP9 in HTFs. Furthermore, LINC01605 knockdown notably inhibited the viability and migration, and induced the apoptosis of HTFs, the effects of which were reversed following treatment with TGF-β. Taken together, the results of the present study suggested that LINC01605 knockdown may inhibit the viability of HTFs by inducing the apoptotic pathway. These findings may provide novel directions for the treatment of HS.
Glaucoma is a common eye disease caused by optic nerve atrophy and visual field defect (
Long non-coding RNAs (lncRNAs) are a class of non-coding RNA molecules of >200 nucleotides in length that are located in the nucleus or in the cytoplasm and have no or little protein coding function (
Therefore, present study aimed to investigate the role of LINC01605 in HS to identify novel potential treatment strategies. To explore the effects of LINC01605 on the formation of HS, human Tenon's capsule fibroblasts (HTFs) and corneal epithelial cells (control cells) were collected and cultured
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 (
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 (
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 (
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 (
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 (
LINC01605 expression significantly decreased in HTFs following transfection with LINC01605 siRNAs (
To investigate the function of LINC01605 in HTFs, flow cytometry was performed. As presented in
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 (
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 (
It has been reported that TGF-β is closely associated with autophagy and fibrosis (
It has been reported that LINC01605 serves an important role in the formation of HS (
A previous study reported that the formation of HS was attributed to the inhibition of apoptosis in p53-deficient mice (
LC3 is widely used for the detection of autophagy levels (
TGF-β is closely associated with autophagy and fibrosis (
The present study has certain limitations. For example, the mechanism through which LINC01605 regulates the Smad pathway remains unclear. In addition, the association between LINC01605 and cell autophagy has not been extensively investigated. RNA
In conclusion, results from the present study demonstrated that LINC01605 knockdown inhibited the viability of HTFs by inducing apoptosis and suggested that LINC01605 knockdown may provide novel directions for the treatment of HS.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
QS made major contributions to the conception and design of the study, as well as drafting and revising the manuscript. YY and HL were responsible for data acquisition, including conducting the experiments, data analysis, data interpretation and manuscript revision. QS, YY and HL confirm the authenticity of all the raw data. All authors agreed to be accountable for all aspects of the work. All authors have read and approved the final manuscript.
The present study was approved by the Ethics Committee of Fuyang People's Hospital (Hangzhou, China), and written informed consent was provided by all patients prior to the study start.
Not applicable.
The authors declare that they have no competing interests.
Successful isolation of HTFs and LINC01605 knockdown significantly inhibits the viability of HTFs from patients with POAG. (A) RT-qPCR was performed to detect the expression of LINC01605 in HS or normal (control/healthy) corneal tissues. (B) Immunofluorescence staining was performed to detect the expression of vimentin and keratin in corneal epithelial cells and HTF. Scale bar, 25 µm. (C) HTFs were transfected with siRNA-NC, LINC01605 siRNA1, LINC01605 siRNA2 or LINC01605 siRNA3, and RT-qPCR was performed to detect the expression levels of LINC01605. (D) Cell Counting Kit-8 assay was used to detect the viability of HTFs. n=3; **P<0.01 vs. siRNA-NC or healthy/blank group. HS, hypertrophic scar; HTFs, human Tenon's capsule fibroblasts; NC, negative control; OD, optical density; POAG, primary open-angle glaucoma; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.
Knockdown of LINC01605 induces HTF apoptosis. (A) HTFs were transfected with siRNA-NC, LINC01605 siRNA1 or LINC01605 siRNA2. Flow cytometry was performed to detect HTF apoptosis. (B) Western blot assays were performed to determine the protein expression levels of Bax, Bcl-2, Pro-caspase-3 and cleaved caspase-3 in HTFs. n=3; **P<0.01 vs. siRNA-NC or blank group. HTFs, human Tenon's capsule fibroblasts; NC, negative control; siRNA, small interfering RNA.
Knockdown of LINC01605 inhibits HTF migration and the marker of fibrotic phenotype (p-Smad2, α-SMA and MMP9). (A) Transwell assay was performed to detect the migratory ability of HTFs. Scale bar, 50 µm. (B) Western blot assays were performed to detect the (C) ratio of p-Smad2/total Smad2, and the expression of (D) α-SMA and (E) MMP9 in HTFs. n=3; **P<0.01 vs. siRNA-NC or blank group. α-SMA, α-smooth muscle actin HTFs, human Tenon's capsule fibroblasts; NC, negative control; p, phosphorylated; siRNA, small interfering RNA.
Knockdown of LINC01605 inhibits HTF autophagy. (A) Immunofluorescence staining was performed to detect the expression of LC3 in HTFs. Scale bar, 10 µm. (B) Western blot assays were performed to detect and semi-quantify the expression levels of (C) ATG7, (D) p62, (E) beclin 1, (F) p-AMPK and AMPK in HTFs. (G) Cell Counting Kit-8 assay was used to detect the viability of HTFs. n=3; **P<0.01 vs. siRNA-NC or Blank group; ##P<0.01 vs. LINC01605 siRNA2 group. AMPK, AMP-activated protein kinase; ATG7, autophagy-related 7; HTFs, human Tenon's capsule fibroblasts; NC, negative control; p, phosphorylated; siRNA, small interfering RNA.
LINC01605 siRNA2-induced apoptosis of HTFs is reversed by TGF-β. HTFs were transfected with siRNA-NC, LINC01605 siRNA2 or LINC01605 siRNA2 + TGF-β. (A) Cell Counting Kit-8 assay was used to detect the viability of transfected HTFs. (B) Flow cytometry was used to determine apoptotic rates. (C) Transwell assay was performed to examine the migratory ability of the transfected HTFs. Scale bar, 50 µm. n=3; **P<0.01 vs. siRNA-NC or blank group; ##P<0.01 vs. LINC01605 siRNA2 group. HTFs, human Tenon's capsule fibroblasts; NC, negative control; OD, optical density; siRNA, small interfering RNA.
Basic clinicopathological characteristics of the patients.
Patient | Age, years | Sex | Date of hospital admission, month/year |
---|---|---|---|
1 | 67 | Female | 11/2019 |
2 | 72 | Male | 11/2019 |
3 | 54 | Female | 03/2020 |
4 | 62 | Female | 04/2020 |
5 | 71 | Male | 05/2020 |