SNHG3 cooperates with ELAVL2 to modulate cell apoptosis and extracellular matrix accumulation by stabilizing SNAI2 in human trabecular meshwork cells under oxidative stress

Glaucoma is the main reason for irreversible blindness, and pathological increased intraocular pressure is the leading risk factor for glaucoma. It is reported that trabecular meshwork cell injury is closely associated with the elevated intraocular pressure. The current study aimed to investigate the role of small nucleolar RNA host gene 3 (SNHG3) in human trabecular meshwork (HTM) cells under oxidative stress. A series of experiments including real‐time quantitative polymerase chain reaction, subcellular fractionation assay, western blot analysis, cell counting kit‐8 assay, RNA pull down, flow cytometry analysis, and RNA immunoprecipitation assay were used to explore the biological function and regulatory mechanism of SNHG3 in HTM cells under oxidative stress. First, we observed that H2O2 induced SNHG3 upregulation in HTM cells. Then, we found that SNHG3 silencing alleviated H2O2‐induced oxidative damage in HTM cells. Moreover, snail family transcriptional repressor 2 (SNAI2) knockdown alleviated the oxidative damage induced by H2O2 in HTM cells. Mechanistically, SNHG3 bound with ELAV like RNA binding protein 2 (ELAVL2) to stabilize SNAI2. Finally, SNAI2 overexpression counteracted the effect of SNHG3 silencing on H2O2‐treated HTM cells. In conclusion, our results demonstrated that SNHG3 cooperated with ELAVL2 to modulate cell apoptosis and extracellular matrix accumulation by stabilizing SNAI2 in HTM cells under oxidative stress.


| INTRODUCTION
Glaucoma is characterized by atrophy and depression of optic papilla, visual field defect and visual acuity decline. 1,2 Pathological elevation of intraocular pressure and inadequate blood supply of optic nerve are the main reasons for glaucoma. 3,4 Previous research has showed that trabecular meshwork (TM) cells exert crucial effect on aqueous humor circulation, and TM cell injury is closely related to the elevated intraocular pressure. 5,6 Therefore, finding novel biomarkers that In primary open angle glaucoma, increased intraocular pressure results in deformation at the optic nerve head; especially, intraocular pressure elevation leads to the deformation at the lamina cribrosa region where extracellular matrix (ECM) molecules, such as fibronectin and collagen, tend to accumulate. [7][8][9] Changes in the levels of regulators of ECM homeostasis such as matrix metalloproteinases (MMPs) occur in the primary open angle glaucoma lamina cribrosa. 10 MMPs are zinc-dependent endopeptidases, which degrade ECM components such as fibronectin and collagen. 11,12 MMPs are crucial regulators of aqueous humor outflow for they can remodel TM ECM and keep a stable outflow resistance and ensuing intraocular pressure. [13][14][15] Therefore, MMPs are promising therapeutic targets for glaucoma treatment due to their capability to regulate aqueous humor outflow. 13,14 Long noncoding RNAs (lncRNAs) are a category of noncoding RNAs (ncRNAs), with over 200 nucleotides in size. 16,17 LncRNAs are related to multiple cellular functions, and most of the cellular functions involve the binding of lncRNAs with one or more RNA-binding proteins (RBPs) to increase the stability of target messenger RNAs (mRNAs). [18][19][20][21] LncRNAs are active regulators in a variety of diseases, including glaucoma. [22][23][24] For example, lncRNA GAS5 knockdown relieves glaucoma in rat models via decreasing the apoptosis of retinal ganglion cells. 25 LncRNA MALAT1 regulates the apoptosis of retinal ganglion cells via the PI3K/Akt pathway in glaucomatous rats. 26 LncRNA MEG3 participates in glaucoma progression by facilitating the autophagy of retinal ganglion cells. 27 LncRNA small nucleolar RNA host gene 3 (SNHG3) has been found to participate in regulating the development of several diseases. [28][29][30] Importantly, SNHG3 has been found to be related to neurodegeneration, 31 and it has been reported to be upregulated in H 2 O 2 -stimulated HTM cells. 32 Accordingly, we predicted that SNHG3 may exert certain effect on glaucoma.
DNA damage is related to various neurodegenerative diseases, including glaucoma. 32,33 In comparison with healthy controls, oxidative DNA damage is distinctly increased in the TM of patients with glaucoma. 32  containing 10% fetal bovine serum (Invitrogen, USA), 100 U/mL penicillin (Sigma-Aldrich, USA) and 100 μg/ml streptomycin (Sigma-Aldrich, USA), and then stored in the humidified atmosphere with 5% CO 2 at 37 C.

| Cell treatment
The cultured HTM cells were treated with disparate concentrations of H 2 O 2 (0-300 μM; Beyotime Ins. Of Bio., Shanghai, China) in a serumfree medium for 2 h to establish an in vitro oxidative cellular damage model. Non-disposed basal cells acted as a control.

| Cell counting kit-8 assay
The viability of HTM cells were detected by using cell counting kit

| Western blot
Transfected cells were lysed applying radioimmunoprecipitation assay buffer (Thermo, USA), and cultured for 15 min at 4 C. Then, the lysate was centrifuged, and the concentrations of the proteins were measured using a BCA Protein Assay Kit (Thermo, USA). The proteins were isolated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and moved onto PVDF membranes (Thermo, USA). Then, 5% skim milk was used to block the membranes for 1 h. Next, the membranes were incubated with primary antibodies overnight at 4 C. The primary antibodies were obtained from Abcam company (Shanghai, China): SNAI2 (ab51772), ELAVL2 (ab72603), collagen I (ab34710), fibronectin (ab2413), collagen III (ab7778), MMP3 (ab52915), MMP9 (ab76003) and GAPDH (ab8245). Subsequently, secondary antibody was used to be incubated with the membranes for 2 h at room temperature. Finally, an enhanced chemiluminescence kit (GE Healthcare, Chicago, IL) was adopted to observe the signals. Each experiment was repeated three times.

| Reverse transcription quantitative polymerase chain reaction
TRIzol reagent (ThermoFisher Scientific Invitrogen, USA) was used to extract total RNA. Then, total RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kits

| Subcellular fractionation assay
The nuclear and cytoplasmic fractions were separated from HTM cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Total RNA was isolated from the nuclear and cytoplasmic fractions using TRIzol (Invitrogen). RNAs isolated from the nucleus or cytoplasm were performed analyzed by RT-qPCR analysis. The levels of U6 (nucleus control), GAPDH (cytoplasm control) and SNHG3 were respectively measured. Each experiment was repeated three times.

| Data statistics
Data analysis was conducted using SPSS 23.0 (IBM SPSS, Chicago, IL, USA). All data are shown as the means ± SD. Statistical significance among more than two groups was calculated using one-way analysis of variance and Tukey's post-hoc test. Difference between two groups was evaluated using Student's t test. Each experiment was repeated three times. p < .05 was regarded to be statistically significant.  (Figure 1(B)). Additionally, the levels of extracellular matrix (ECM) proteins collagen I, collagen III, fibronectin, MMP3, and MMP9 were increased by H 2 O 2 in HTM cells (Figure 1(C)). Moreover, SNHG3 expression was upregulated in HTM cells (Figure 1(D)). Additionally, SNHG3 expression in sh-SNHG3#1 group was lower than in sh-SNHG3#2 group, suggesting that the knockdown efficacy of sh-SNHG3#1 was better than that of sh-SNHG3#2. Thus, sh-SNHG3#1 was used in following experiments. Then, CCK-8 assay demonstrated SNHG3 knockdown promoted the viability of H 2 O 2stimulated HTM cells (Figure 2(B)). Flow cytometry analysis showed that SNHG3 silencing suppressed the apoptosis of H 2 O 2 -treated HTM cells (Figure 2(C)). Moreover, the levels of ECM proteins collagen III, collagen I, fibronectin, MMP3, and MMP9 were reduced by SNHG3 silencing in H 2 O 2 -stimulated HTM cells (Figure 2(D)).

| SNAI2 knockdown alleviated H 2 O 2 -induced oxidative damage in HTM cells
SNAI2 has been widely reported to regulate ECM proteins, 34

| SNHG3 bound with ELAVL2 to stabilize SNAI2
Subsequently, we explored the molecular mechanism of SNHG3 in HTM cells. First, we found that SNHG3 mainly existed in the cytoplasm of cells by using online tool (http://www.csbio.sjtu.edu.cn/ bioinf/lncLocator/) (Figure 4(A)). Then, we further found that SNHG3 mainly existed in the cytoplasm of HTM cells (Figure 4(B)).
In conclusion, SNHG3 cooperated with ELAVL2 protein to increase SNA12 mRNA stability, thereby suppressing viability and promoting apoptosis and ECM accumulation in H 2 O 2 -treated HTM cells (Figure (6)).  39,40 and glaucoma. 23,24 Notably, SNHG3 has been found to be related to neurodegeneration, 31  SNAI2 is also known as SLUG, SLUGH, SLUGH1, SNAIL2, or WS2D; it has been reported to regulate a wide range of human diseases [41][42][43] . Importantly, previous research has proved that SNAI2 is involved in the regulation of neurodegenerative diseases, 44 and it may participate in the modulation of glaucoma. 45  LncRNAs can cooperate with specific proteins to increase mRNA stability or induce mRNA decay in cytoplasm. 46 For example, LncRNA LINC00707 interacts with mRNA stabilizing protein HuR to increase the stability of VAV3/F11R in gastric cancer. 47 LncRNA LAST interacts with CNBP to promote CCND1 mRNA stability in human cells. 48 Previously, the regulatory mechanism of SNHG3 has been explored in some human diseases. 29,30,49 Additionally, ELAVL2, a member of drosophila ELAV family, is widely expressed in peripheral and central nervous system and protects hippocampal CA3 pyramidal neurons against seizure. 50 In this study, we observed that SNHG3 bound with ELAVL2 to stabilize SNAI2 in H 2 O 2 -treated HTM cells. Rescue assays confirmed that SNAI2 overexpression counteracted the effects of SNHG3 silencing on viability, apoptosis and ECM protein (collagen I, collagen III, fibronectin, MMP3, and MMP9) levels in H 2 O 2 -stimulated HTM cells.

| DISCUSSION
In conclusion, our results illustrated that SNHG3 interacted with ELAVL2 to regulate cell proliferation, apoptosis and ECM accumula-

CONFLICT OF INTEREST
The authors declare that there are no competing interests in this study.