Circ_0047835 Combines with miR-144-3p to Promote the Proliferation, Invasion, Migration, and Fibrosis of TGF-β1-Treated Human Tenon’s Capsule Fibroblasts by Upregulating SP1

Abstract Purpose Glaucoma is the leading cause of blindness worldwide with complex pathogenesis. Circular RNAs (circRNAs) play critical roles in various diseases, including glaucoma. The purpose of this study was to investigate the role of circ_0047835 and underlying mechanisms in the development of fibrosis after glaucoma filtration surgery. Methods Human Tenon’s capsule fibroblasts (HTFs) were stimulated using transforming growth factor-β1 (TGF-β1) to mimic a cellular model of glaucoma in vitro. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2’-deoxyuridine (EdU) assay. Cell invasion and migration were detected by transwell assay and wound healing assay, respectively. Western blot assay was used to measure protein levels. The expression levels of circ_0047835, microRNA-144-3p (miR-144-3p) and specific protein 1 (SP1) mRNA were determined by real-time quantitative polymerase chain reaction (RT-qPCR). The interaction between miR-144-3p and circ_0047835 or SP1 was confirmed by dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. Results Circ_0047835 expression was elevated in glaucoma tissues and TGF-β1-treated HTFs. Circ_0047835 or SP1 knockdown suppressed the proliferation, migration, invasion, and fibrosis of TGF-β1-treated HTFs. MiR-144-3p was a target of circ_0047835, and miR-144-3p inhibition reversed the effects of circ_0047835 knockdown in TGF-β1-treated HTFs. Moreover, SP1 was identified as a target of miR-144-3p, and miR-144-3p overexpression weakened TGF-β1-induced proliferation, migration, invasion, and fibrosis by targeting SP1 in HTFs. Furthermore, circ_0047835 combined with miR-144-3p to regulate SP1 expression. Conclusion Circ_0047835 might contribute to fibrosis progression after glaucoma surgery by regulating the miR-144-3p/SP1 axis.


Introduction
Glaucoma is one of the most common chronic eye diseases and is characterized by optic disc cupping and irreversible apoptosis of retinal ganglion cells. 1 Current therapeutic methods for glaucoma mainly include surgery and pharmaceuticals; however, scar formation due to fibrosis and excessive proliferation of human Tenon's capsule fibroblasts (HTFs) is a major factor leading to unsatisfactory therapeutic effects. 2,3 Thus, understanding the molecular mechanisms related to the pathology of glaucoma is a critical step in optimizing treatment strategies for glaucoma patients.
After glaucoma surgery, transforming growth factor-b1 (TGF-b1) can act as a central player in conjunctival scar formation, fibrosis, and systemic wound healing. 4 TGF-b1 can increase the activity of HTFs and then promotes cell proliferation, mobility and fibrosis, finally inducing the transdifferentiation of HTFs into myofibroblasts. [5][6][7] TGF-b1-treated HTF model is used to study the mechanisms of fibrosis and glaucoma surgery failure. Circular RNAs (circRNAs), an important type of noncoding RNA (ncRNAs), are generated by back-splicing of precursor mRNA and can form covalently closed loops without 5'-end cap or 3'-end poly-A tail. 8 Owing to their circular structures, circRNAs are more stable and not easily affected by RNases. 9 A growing body of evidence shows that circRNAs are pivotal regulators in cell behaviors and the progression of multiple diseases, including glaucoma. 10,11 Circ_0047835 is located on chr18:57569878-57571538 and is derived from phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) gene (http://www.circbase.org). A previous study indicated that circ_0047835 was upregulated in human trabecular meshwork cells (HTMCs) treated with H 2 O 2 (cell model of primary open-angle glaucoma). 12 However, the role and the underlying mechanism of circ_0047835 have not been explored in glaucoma.
CircRNAs can serve as competing endogenous RNAs to interact with microRNAs (miRNAs; another class of ncRNAs), thereby affecting target gene expression and further regulating cell behaviors. 13,14 Accumulating evidence shows that dysregulated expression of miRNAs is tightly related to glaucoma progression. 15 MiR-144-3p is a potential therapeutic target for glaucoma. 16 Nevertheless, the association between circ_0047835 and miR-144-3p is still unclear in glaucoma. A previous study showed that miR-29b inhibited the proliferation of TGF-b1-HTFs by targeting specific protein 1 (SP1). 17 However, whether SP1 can be targeted by miR-144-3p has not been studied.
In our work, we detected circ_0047835, miR-144-3p and SP1 expression in glaucoma tissue samples and TGF-b1exposed HTFs. Moreover, we explored the function of circ_0047835 in HTFs stimulated with TGF-b1 for the first time. Moreover, we assembled the potential network of circ_0047835/miR-144-3p/SP1 to determine the mechanism of circ_0047835 in regulating fibrosis after glaucoma filtration surgery. The aim of our work is to provide new biomarkers for the treatment of glaucoma.

Specimen collection
Glaucoma fascia tissue specimens (Tenon's capsules; n ¼ 25) were collected from glaucoma patients who underwent operation at the Affiliated Huaian No.1 People's Hospital of Nanjing Medical University. Nonglaucoma fascia tissue specimens (Tenon's capsules; n ¼ 25) were collected from patients who had undergone trauma surgery. After being collected, these tissues were quickly frozen in liquid nitrogen. This study had acquired authorization from the Ethics Committee of the Affiliated Huaian No.1 People's Hospital of Nanjing Medical University. The research was carried out in accordance with the World Medical Association Declaration of Helsinki, and all subjects provided written informed consent.

Cell culture and transfection
HTFs isolated from post-mortem non-glaucoma eye donors were bought from BeNa Culture Collection (Beijing, China). DMEM (Invitrogen, Carlsbad, CA, USA) including 10% fetal bovine serum (FBS; Invitrogen) was used to incubate HTFs. HTFs were maintained in standard conditions (37 C; 5% CO 2 ). HTFs were exposed to TGF-b1 (PeproTech, Rocky Hill, NJ, USA) to construct a glaucoma model in vitro.

Cell proliferation assays
For Cell Counting Kit-8 (CCK-8) assay, HTFs were inoculated into a 96-well plate and incubated for a specific time to detect cell viability. Next, CCK-8 reagent (10 mL; Beyotime, Jiangsu, China) was added to each well. After incubation for 3 h, we used a microplate reader (Bio-Rad) to detect the absorbance at 450 nm.
To test cell proliferation and DNA synthesis, 5-ethynyl-2'-deoxyuridine (EdU) assay was conducted using EdU Cell Proliferation Kit (Beyotime). Shortly, we seeded HTFs into 24-well plates. After treatments, HTFs were incubated with EdU (10 lM) for 2 h and fixed with 4% paraformaldehyde. Next, we used 0.5% TritonX-100 to permeabilize cells. After that, the cells were incubated with Click Additive solution for 0.5 h in the dark. The nucleic acids were stained with DAPI. Finally, fluorescence microscope (200Â; Leica, Wetzlar, Germany) was employed to determine EdU positive cells.

Invasion and migration assays
Transwell assay was used to analyze HTFs invasion. Shortly, the top surface of the chamber was coated with Matrigel (BD Biosciences, San Jose, CA, USA) at 37 C for 0.5 h. Thereafter, HTFs re-suspended in serum-free medium (200 lL) were seeded into the top chamber. Meanwhile, complete medium (0.6 mL) was placed into the lower chamber. After 24 h of incubation, HTFs in the top chamber were discarded, and HTFs invaded onto bottom chambers were fixed with paraformaldehyde (Beyotime). After being stained with crystal violet solution (Beyotime), invaded cells were photographed using a microscope (Leica) at Â100 magnification.
Wound healing assay was used to assess the migration of HTFs. Briefly, HTFs were inoculated into a 24-well plate. A sterile pipette tip (200 lL) was employed to scratch wounds when cell confluence reached about 80%. After washing the cells using PBS, images of each wounded monolayer were captured at 0 h and 24 h using a microscope (Leica).

Western blot assay
Cells or tissues were harvested and lysed with cell lysis buffer. After quantification of total protein, protein samples (about 30 lg/lane) were loaded onto SDS-PAGE and transferred onto PVDF membranes (2 h, 100 V). We used 5% skim milk to block the membranes, and then the membranes were incubated with primary antibodies at 4 Covernight. After incubation with a secondary antibody at room temperature for 2 h, enhanced chemiluminescence solution (Vazyme, Nanjing, China) was used to observe the protein bands. The antibodies were all bought from Abcam  After extracting total RNA with TRIzol Reagent (Invitrogen), reverse transcription was conducted using Primescript RT Reagent (for circRNA and mRNA; TaKaRa, Kusatsu, Japan) or miRNA First-Strand Synthesis Kit (for miRNA; Clontech, Mountain View, CA, USA). Thereafter, RT-qPCR was performed on CFX96Touch system (Bio-Rad) with SYBR TM Green PCR Master Mix (Clontech). RNA levels were analyzed by the 2 ÀDDCt method. U6 and GAPDH were utilized as the reference controls for miR-144-3p and circ_0047835/SP1, respectively. Primers used in this study were exhibited as follows: circ_0047835 (sense,

CircRNA identification
RNase R was used to degrade linear RNAs. Total RNA was incubated for half an hour at 37 C with/without RNase R (Seebio, Shanghai, China). Lastly, the enrichment of circ_0047835 and GAPDH was assessed by RT-qPCR.
Oligo (dT) 18 primers can only amplify RNA with poly (A) tail, but random primers can amplify all RNAs. Circ_0047835 and GAPDH were amplified with random or oligo (dT) 18 primers. Lastly, RT-qPCR was conducted to confirm whether circ_0047835 contained poly (A) tail.

RNA immunoprecipitation (RIP)
We used the EZ-Magna RIP Kit (Millipore, Billerica, MA, USA) for RNA immunoprecipitation. Shortly, cells were lysed using RIP lysis buffer, and the cell extracts were incubated with magnetic beads coated with antibody against either immunoglobulin G (anti-IgG; as a control) or Argonaute2 (anti-AGO2). After incubation for 6 h at 4 C, the beads were digested with Proteinase K for 0.5 h at 55 C, and immunoprecipitated RNA was subjected to RT-qPCR.

Statistical analysis
All data from at least three independent experiments were displayed as mean ± standard deviation. Difference analysis was performed using Student's t-test (two groups) or a oneway analysis of variance with Tukey test (multiple groups) on GraphPad Prism 7. Pearson's correlation coefficient was used to detect the correlation between miR-144-3p and circ_0047835 or SP1. p < 0.05 indicates statistically significant.

TGF-b1 promoted the proliferation, migration, invasion and fibrosis of HTFs
First, we explored the effects of TGF-b1 on the proliferation, migration and invasion of HTFs. CCK-8 and EdU assays showed that TGF-b1 treatment increased cell viability and DNA synthesis in HTFs (Figure 1(A,B)), indicating that TGF-b1 promoted HTFs proliferation. Moreover, transwell and wound healing assays indicated that the invasion and migration of HTFs were dose-dependently increased by TGF-b1 treatment (Figure 1(C-E)). In addition, western blot assay showed that the protein levels of PCNA (a growthpromoting protein), a-SMA and Fibronectin (fibrosis markers) were enhanced by TGF-b1 stimulation ( Figure  1(F)). These results indicated that TGF-b1-stimulated HTFs could act as a glaucoma cell model.

Knockdown of circ_0047835 suppressed TGF-b1-induced proliferation, invasion, migration, and fibrosis of HTFs
To explore whether circ_0047835 was dysregulated in glaucoma, the expression of circ_0047835 was detected by RT-qPCR. As presented in Figure 2(A), circ_0047835 expression was higher in Tenon's tissues from glaucoma patients obtained during glaucoma surgery compared to Tenon's tissues from non-glaucoma patients obtained during trauma surgery. Likewise, circ_0047835 expression was also increased by TGF-b1 in HTFs in a dose-dependent manner (Figure 2(B)). RNase R can digest linear RNA but not circular RNA. As expected, circ_0047835 expression was not affected by RNase R but GAPDH level was reduced by RNase R (Figure 2(C)), indicating the cyclic structure of circ_0047835. As displayed in Figure 2(D), circ_0047835 expression was lower in the oligo(dT) 18 primer group than in the random primer group, implying the absence of a poly (A) tail for circ_0047835. Knockdown of circ_0047835 reversed the promoting effect of TGF-b1 on circ_0047835 expression in HTFs (Figure 2(E)). We found that circ_0047835 downregulation attenuated TGF-b1-induced proliferation, invasion and migration of HTFs ( Figure  2(F-I)). In addition, circ_0047835 knockdown weakened the promoting effects of TGF-b1 on PCNA, a-SMA and Fibronectin expression in HTFs (Figure 2(J)). Taken together, these results indicated that circ_0047835 knockdown inhibited the proliferation, invasion, migration, and fibrosis of TGF-b1-treated HTFs.

MiR-144-3p was directly targeted by circ_0047835
CircRNAs serves as molecular sponges of miRNAs to regulate cell behaviors. Hence, the downstream targets of circ_0047835 were predicted by circinteractome. Several binding sites between circ_0047835 and miR-144-3p were identified (Figure 3(A)), indicating that miR-144-3p was a target of circ_0047835. Transfection of miR-144-3p markedly increased miR-144-3p expression in HTFs ( Figure  3(B)). Dual-luciferase reporter assay and RIP assay were performed to identify whether miR-144-3p could directly bind to circ_0047835. The data showed that miR-144-3p transfection reduced the luciferase activity of WT-circ_0047835 in HTFs, whereas the luciferase activity of MUT-circ_0047835 was not affected after overexpression of miR-144-3p ( Figure  3(C)). Moreover, both circ_0047835 and miR-144-3p were obviously accumulated in the anti-Ago2 group than in the anti-IgG group (Figure 3(D)). The expression of miR-144-3p was reduced in glaucoma tissues relative to Tenon's tissues from non-glaucoma patients obtained during trauma surgery and was negatively correlated with circ_0047835 expression in glaucoma tissues (Figure 3(E,F)). In addition, TGF-b1 exposure dose-dependently decreased miR-144-3p expression in HTFs (Figure 3(G)). These results confirmed that circ_0047835 combined with miR-144-3p.

SP1 was directly targeted by miR-144-3p
To investigate the regulatory mechanism of miR-144-3p in TGF-b1-treated HIFs, we screened the target genes of miR-144-3p using starBase. As displayed in Figure 5(A), miR-144-3p contained some complementary sites of SP1 3'UTR. The data of dual-luciferase reporter assay suggested that miR-144-3p upregulation remarkably inhibited the luciferase activity of WT-SP1 3'UTR but not that of MUT-SP1 3'UTR ( Figure 5(B)). RIP assay revealed that both miR-144-3p and SP1 were greatly enriched in the Ago2 group in contrast to the IgG group ( Figure 5(C)). Moreover, SP1 mRNA expression was increased in glaucoma tissues when compared with Tenon's tissues from non-glaucoma patients obtained during trauma surgery and was positively correlated with miR-144-3p expression in glaucoma tissues ( Figure 5(D,E)). In addition, SP1 protein level was also enhanced in glaucoma tissues ( Figure 5(F)). Besides, TGF-b1 treatment dose-dependently elevated SP1 protein expression in HTFs ( Figure 5(G)). To sum up, SP1 was a target of miR-144-3p.

Circ_0047835 interacted with miR-144-3p to regulate SP1 expression
We have demonstrated that circ_0047835 was a sponge of miR-144-3p, which targeted SP1. Thus, we explored whether circ_0047835 regulated SP1 expression by sponging miR-144-3p. As shown in Figure 8(A,B), SP1 mRNA and protein expression were dramatically downregulated after circ_0047835 knockdown in TGF-b1-treated HIFs, which could be recovered by inhibition of miR-144-3p, further supporting the regulatory of the circ_0047835/miR-144-3p/ SP1 axis in glaucoma in vitro.

Discussion
Glaucoma is still the main cause of blindness with complicated etiology and pathogenesis. Uncontrolled HTFs proliferation is the main reason of poor prognosis after glaucoma filtration surgery. 18 Therefore, finding effective strategies to resist the proliferation, mobility and fibrosis of HTFs is critical for the treatment of filtering glaucoma. In this work, the expression of circ_0047835 was examined in glaucoma tissue samples as well as TGF-b-treated HTFs. Moreover, the biological functions and the underlying mechanism of circ_0047835 in glaucoma were explored, which might provide a potential treatment strategy for glaucoma. CircRNAs are stable, abundant, and conserved in tissues and plasma and are usually dysregulated in human diseases. 19 Due to these characteristics, circRNAs have the potential as potential therapeutic targets or biomarkers for many diseases. 20 As for eye diseases, some circRNAs have been identified to be abnormally expressed by high-throughput sequencing and bioinformatics analysis. 21,22 Moreover, the roles of few circRNAs have been explored in glaucoma, such as circular RNA-ZRANB1 23 and circular RNA-ZNF609. 24 Nevertheless, the roles and the mechanisms of multiple circRNAs in glaucoma remain largely unclear, and further research is still needed. Shen and her colleagues explored a series of circRNAs in primary open-angle glaucoma cell model and found circ_0047835 was one of upregulated circRNAs. 12 In line with the previous research, circ_0047835 was upregulated in glaucoma tissues and TGF-b1-induced HIFs in our research. Since the biological role of circ_0047835 in glaucoma has never been investigated before, we investigated the influence of circ_0047835 on human Tenon's capsule fibroblast proliferation, invasion, migration, and fibrosis using TGF-b1-induced HIFs. We found that circ_0047835 knockdown weakened TGF-b1triggered proliferation, invasion, migration, and fibrosis of HIFs, indicating that reducing the expression of circ_0047835 might be a treatment strategy for glaucoma.
CircRNAs are reported to bind to miRNAs to release the miRNA-targeted mRNAs, which can regulate downstream gene expression at the transcriptional level. 25 Although the circRNA-miRNA-mRNA regulatory network has been reported in various diseases, more detailed analyses of this regulatory network in glaucoma have not been performed. To further clarify the mechanism by which circ_0047835 regulated proliferation, invasion, migration, and fibrosis of TGF-b1-induced HIFs, circ_0047835-assoctaed miRNA was predicted and validated. We confirmed that miR-144-3p was targeted by circ_0047835. MiR-144-3p can function as a cancer promoter or suppressor in different tumors. [26][27][28] Additionally, a recent study proved that miR-144-3p was low-expressed in serum of primary open-angle glaucoma patients, and overexpression of miR-144-3p was a potential target for glaucoma treatment. 16 Consistently, a low level of miR-144-3p was detected in TGF-b1-exposed HIFs and glaucoma tissues in our study. Moreover, miR-144-3p inhibition counteracted the suppressive impacts of circ_0047835 knockdown on the proliferation, invasion, migration, and fibrosis of TGF-b1-treated HIFs, indicating that circ_0047835 functioned by targeting miR-144-3p in TGF-b1-treated HIFs.
In conclusion, circ_0047835 was upregulated in TGF-b1stimulated HTFs and glaucoma tissues. In addition, circ_0047835 served as a sponge for miR-144-3p to upregulate SP1 expression, thus promoting the proliferation, invasion, migration, and fibrosis of TGF-b1-treated HTFs ( Figure S1). Despite the presence of non-glaucoma Tenon's tissues in the present study, patients with severe ocular trauma are not ideal controls in fibrosis research as severe trauma itself can lead to conjunctival and subconjunctival fibrosis. A better control group should be squint surgery patients with no glaucoma, no previous incisional conjunctival surgery, and no trauma. This issue should be considered when evaluating the present work. Nevertheless, our results may help to develop therapeutic targets and diagnostic biomarkers for glaucoma patients.

Ethical approval
The present study was approved by the ethical review committee of The Affiliated Huaian No.1 People's Hospital of Nanjing Medical University. Written informed consent was obtained from all enrolled patients.

Consent form
Patients agree to participate in this work.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.