Role of LINC01592 in TGF-β1-induced epithelial-mesenchymal transition of retinal pigment epithelial cells

Regulation of long-chain non-coding RNA01592 (LINC01592) in the process of transforming retinal pigment epithelial (RPE) cells into mesenchymal cells following induction by transforming growth factor beat 1 (TGF-β1) was investigated by interfering with LINC01592 expression in human RPE (hRPE) cells. LINC01592 expression in hRPE cells was significantly increased following treatment with 10 ng/mL TGF-β1 for 48 h. Expression of E-cadherin and Snail were decreased in hRPE cells following induction with TGF-β1 compared with the control group (P < 0.05). Following induction by TGF-β1, expression of E-cadherin, alpha-smooth muscle actin (α-SMA), and Snail were significantly lower in the LINC01592-knockdown group compared with the negative control group (P < 0.05). LINC01592 overexpression significantly enhanced the viability, proliferation, and migration of hRPE cells induced by TGF-β1 (P < 0.05). Following induction by TGF-β1, E-cadherin expression was significantly decreased and α-SMA and Snail expression were significantly increased in the LINC01592-overexpression group compared with the negative control group (P < 0.05). RPE cells induced by TGF-β1 exhibited epithelial-mesenchymal transition (EMT). Inhibiting LINC01592 expression could significantly reduce TGF-β1-induced EMT of hRPE cells. The regulatory effect of LINC01592 on EMT in hRPE cells induced by TGF-β1 provides a novel treatment for proliferative vitreoretinopathy.


INTRODUCTION
Proliferative vitreoretinopathy (PVR), a complication associated with retinal detachment surgery and trauma [1,2], occurs when simulation and migration of retinal pigment epithelial (RPE) cells is induced by cytokines and oxidative stress, which eventually leads to visual impairment and blindness [3,4]. Epithelial-mesenchymal transformation (EMT) plays an indispensable role in oxidative stress, stem cell differentiation, growth, and wound healing, but also promotes the occurrence and development of cell fibrosis and cancer pathologies [5,6]. Previous studies confirmed that EMT of RPE cells mediated by transforming growth factor beta 1 (TGF-β1) is the main reason for pathological changes associated with PVR [7,8]. RPE cells convert from an epithelial to mesenchymal phenotype and participate in EMT [9,10].
The transformation of cell differentiation is mediated by key transcription factors such as Snail, a zinc-finger box binding protein and basic helix transcription factor [11].
In addition, long noncoding RNA (lncRNA) such as lnc-ATB, linc-RoR, and HOTAIR have been shown to induce EMT in tumor epithelial cells [12,13]. Moreover, studies have shown that lncRNA plays an important role in triggering EMT in epithelial cells during tumor metastasis [14,15]. MALAT1 can promote the proliferation, migration, and epiretinal membrane formation of RPE cells in PVR [16,17]. In addition, it was confirmed that downregulation of MALAT1 could inhibit the induction of EMT by TGF-β1 in ARPE-19 cells, and significantly reduced the upregulation of EMT-related transcription factors Snail, SLUG, and ZEB1 in RPE cells [18,19].
Results of our previous lncRNA microarray analysis showed that LINC01592 expression was significantly increased in hRPE cells following induction by TGF-β1 compared with the control group, indicating an important role for LINC01592 in regulation of hRPE cell proliferation. In the present study, we investigated the role of LINC01592 in the process of TGF-β1induced EMT in hRPE cells.

TGF-β1 induced EMT in hRPE cells
After 48-h intervention with 10 ng/mL TGF-β1, hRPE cells were transformed into loosely arranged spindleshaped cells, indicating their transition from an epithelial to mesenchymal phenotype ( Figure 1).
Expression of E-cadherin, alpha-smooth muscle actin (α-SMA), and Snail (an EMT-related transcription factor) was decreased in hRPE cells of the experimental group compared with the control group following induction with TGF-β1 for 48 h (P < 0.05, Figure 2).

TGF-β1 induced LINC01592 expression in hRPE cells
Our results show that LINC01952 expression in hRPE cells was significantly increased after 48-h intervention with TGF-β1 compared with the control group (P < 0.05, Figure 3).

LINC01592 knockdown inhibited TGF-β 1-induced EMT in hRPE cells
Our results indicate significantly decreased E-cadherin expression and significantly increased α-SMA expression in hRPE cells of the TGF-β1 group (P < 0.05). In addition, LINC01592 expression was increased in the TGF-β1 group (P < 0.05). Expression of E-cadherin in the LINC01592-knockdown plus TGF-β1 induction (LINC01592-KD + TGF-β1) group was significantly lower (P < 0.05) than in the negative control plus TGF-β1-induction (LINC01592-KD-NC + TGF-β1) group, which also exhibited significantly increased α-SMA expression (P < 0.05). These results indicate that downregulation of LINC01592 inhibited the AGING decrease of E-cadherin and increase of α-SMA in hRPE cells induced by TGF-β1, which suggests that TGF-β1induced EMT of hRPE cells can be inhibited ( Figure 4).

LINC01592 knockdown reduced proliferation and migration of hRPE cells
The results of cell migration-scratch testing indicated no significant difference between LINC01592-KD and   AGING LINC01592-KD-NC groups (P > 0.05). However, the residual scratch area of both LINC01592-KD + TGF-β1 and LINC01592-KD-NC + TGF-β1 groups was significantly lower compared with LINC01592-KD and LINC01592-KD-NC groups (P < 0.05), respectively. Moreover, the residual scratch area of the LINC01592-KD-NC + TGF-β1 group was significantly lower compared with the LINC01592-KD-NC group (P < 0.05). The residual scratch area of the LINC01592-KD-NC + TGF-β1 group was significantly lower compared with the LINC01592-KD + TGF-β1 group (P < 0.05). These results suggest that following induction by TGF-β1, proliferation and migration of hRPE cells was decreased in response to reduced LINC01592 expression ( Figure 6). Cell Counting Kit 8 (CCK-8) results revealed no significant difference in viability between cells in LINC01592-KD and LINC01592-KD-NC groups (P > 0.05). Moreover, no significant differences were observed among LINC01592-KD + TGF-β1, LINC01592-KD-NC + TGF-β1, and LINC01592-KD-NC + TGF-β1 groups (P > 0.05). Compared with the LINC01592-KD-NC group, both LINC01592-KD and LINC01592 groups exhibited significantly higher cell viability (P < 0.05). Viability of the LINC01592-KD + TGF-β1 group was significantly lower than that of the LINC01592-KD-NC + TGF-β1 group (P < 0.05). These results suggest that following induction by TGF-β1, viability of hRPE cells was decreased in response to reduced LINC01592 expression ( Figure 6).

LINC01592 overexpression enhanced the proliferation and migration of hRPE cells
The results of cell migration-scratch testing indicated no significant difference in residual scratch area between LINC01592-OE and LINC01592-OE-NC groups (P > 0.05). However, the residual scratch areas of LINC01592-OE + TGF-β1 and LINC01592-OE-NC + TGF-β1 groups were significantly lower compared with LINC01592-OE and LINC01592-OE-NC groups, respectively (P < 0.05). Moreover, the residual scratch area of the LINC01592-OE + TGF-β1 group was AGING significantly lower compared with the LINC01592-OE-NC + TGF-β1 group (P < 0.05). These results suggest that LINC01592 overexpression enhanced the proliferation and migration of hRPE cells following induction by TGF-β1.
CCK-8 assay results indicated no significant difference in cell viability between LINC01592-OE and LINC01592-OE-NC groups (P > 0.05). However, compared with LINC01592-OE-NC and LINC01592-OE-NC groups, cell viability of the LINC01592-OE + TGF-β1 group was significantly increased (P < 0.05). Importantly, viability of the LINC01592-OE+TGF-β1 group was significantly higher compared with LINC01592-OE and LINC01592-OE-NC groups (P < 0.05). These results suggest that overexpression of LINC01592 increased the viability of hRPE cells following induction by TGF-β1 (Figure 9).

DISCUSSION
PVR is a type of ocular fibrous disease characterized by the formation of a contractile epiretinal membrane, the main cellular component of which is RPE cells. EMT occurs when RPE cells detach from the damaged retina and migrate into the vitreous cavity or subretinal space, whereby they are stimulated by various cytokines [20][21][22][23]. After RPE cells acquire a mesenchymal phenotype, their migration, invasiveness, and anti-apoptotic ability are enhanced, and they begin to produce extracellular matrix [24][25][26][27]. RPE cells that undergo the EMT process change from epithelial cells to fibroblast-like cells and participate in the formation of an epiretinal membrane [28][29][30]. Following retinal damage, TGF-β1 released from vitreous or serum is the main factor stimulating EMT in RPE cells [31]. Although EMT has been confirmed as the main pathogenic factor of PVR in RPE cells, the mechanism by which EMT occurs RPE cells remains unclear. In the present study, expression of E-cadherin was decreased but that of α-SMA and Snail was increased following TGF-β1 induction. These results confirmed that EMT could occur in RPE cells 48 h after TGF-β1 intervention.
LINC01592 is a 2367-bp lncRNA located in two bands of the 13 region of chromosome 8. In this study, RNA was extracted from hRPE cells treated with TGF-β1 for 48 h. RT-PCR assay results confirmed that LINC01592 expression was significantly increased in RPE cells treated with TGF-β1, suggesting the potential involvement of LINC01592 in regulation of EMT in hRPE cells during the development of PVR.
Our results suggest that inhibiting LINC01592 expression not only inhibited TGF-β-induced EMT of hRPE cells but also reduced their proliferation and migration. In addition, increased expression of the EMT-related transcription factor Snail induced by TGF-β1 was inhibited by knockdown of LINC01592 expression. Previous studies implicated Snail in some signaling pathways associated with EMT, which suggests that LINC01952 may regulate EMT in AGING RPE cells by participating in a signaling pathway involving Snail. However, the specific signaling pathways affected by LINC01592 and Snail in TGF-β1-induced EMT of hRPE cells require further study.
Following induction by TGF-β1, LINC01592 overexpression could promote the EMT of hRPE cells and enhance their proliferation and migration ability. In addition, LINC01592 overexpression enhanced expression of the EMT transcription factor Snail following induction by TGF-β1. These results indicate that LINC01592 not only participated in the process of EMT in hRPE cells induced by TGF-β1 but also regulated their proliferation and migration and promoted the EMT process.

AGING
Following EMT of RPE cells, they produce and participate in the formation of an epiretinal membrane the main pathogenic factor of PVR. In this study, TGF-β1 was used to induce EMT in hRPE cells. The effect of lncRNA on EMT, proliferation, and migration of hRPE cells was confirmed by interfering with LINC01592 expression. Reducing LINC01592 expression could inhibit the EMT process of hRPE cells following induction by TGF-β1, thus realizing the possibility of inhibiting the occurrence and development of PVR. In addition, we found that LINC01592 may regulate EMT in hRPE cells by participating in a signaling pathway involving the transcription factor Snail. TGF-β1 promoted EMT of hRPE cells; LINC01592 could regulate the process of TGF-β1-induced EMT of hRPE cells, and reduced expression of LINC01592 inhibited the EMT process. The regulatory effect of LINC01592 on TGF-β1induced epithelial interstitialization of hRPE cells may involve signaling pathways involving Snail.
Our findings confirm that LINC01592 is related to the occurrence and development of PVR. At present, no report has described the mechanism by which LINC01592 participates in the pathogenesis of EMT in hRPE cells. To provide a new target for gene therapy of PVR, the present study elucidated the role of LINC01592 in the process of TGF-β1-induced EMT in hRPE cells.

MATERIALS AND METHODS
All procedures of this experiment were approved by the First Affiliated Hospital of Harbin Medical University (Harbin, China) ethics committee and conformed with Association for Research in Vision and Ophthalmology guidelines for ophthalmic and vision studies.

EMT of hRPE cells following induction by TGF-β1
Donated eyeballs were from the eye bank of First Affiliated Hospital of Harbin Medical University. hRPE cells were carefully collected and then treated with 0.25% trypsin for 1 h. hRPE cells were inoculated in six-well plates and cultured in an incubator at 37° C and 5% CO2 for 12 h, until the cells completely adhered. hRPE cells were used for experiments after they reached confluence.

Western blot
Cell debris and lysates were centrifuged at 12000 r/min for 15 min. After collecting the supernatant, the protein concentration was determined according to the instructions of a bicinchoninic acid assay kit. A 12% gel was prepared and 30 μg of protein was loaded into each lane. Proteins were subsequently transferred to polyvinylidene fluoride membranes, which were blocked in 5% skimmed milk powder in phosphatebuffered saline containing Tween 20 (PBS-T), placed on a horizontal shaker, and sealed for 1 h. Next, membranes were incubated with mouse anti-human Ecadherin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-human α-SMA (1:500, Santa Cruz Biotechnology), rabbit anti-human Snail (1:1000, Santa Cruz Biotechnology), and/or rabbit anti-human GAPDH (1:1000, Santa Cruz Biotechnology) antibodies at room temperature for 2 h, followed by 4° C for 12 h. Subsequently, membranes were washed three times (10 min each) with PBS-T on a decolorizing shaking bed, followed by incubation with appropriate secondary antibodies in a horizontal AGING shaking bed at room temperature for 1 h. Membranes were analyzed according to instructions of an enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, USA) [32].

Real-time quantitative polymerase chain reaction (RT-qPCR)
RNA extracts were treated with RNase-Free H2O. After discarding the culture medium from six-well plates, cells were washed twice with PBS at 4° C. Next, 150 µL of RNA was added to each well along with extract Buffer R-I from the kit, and the mixture was pipetted up and down 8-10 times. The mixture containing cell debris and lysate was transferred to a 1.5-mL centrifuge tube. RNA was extracted according to the instructions of an RNA extraction kit (Invitrogen).
After thermal denaturation of RNA at 65° C for 5 min, RNA was immediately cooled on ice. The reaction liquid (4 µL of 4× DN Master Mix, 1 µg of RNA template, 11 µL of Nuclease-free Water) was stirred gently and evenly, then incubated at 37° C for 5 min. Reverse transcription was carried out and reactions were prepared on ice as follows: 4 µg/L of 4× DN Master Mix, 1 µg of RNA template, 11 µg/L Nuclease-free Water, and 4 µg/L of 5× RT Master Mix II. Reactions were carried out at the following temperatures: 37° C for 15 min, 50° C for 5 min, 98° C 5 min, and then maintained at 4° C. The DNA solution was stored at -20° C after the reaction. Subsequently, reactions containing 6.4 µL of sterilized distilled water, 6 pmol/0.6 µL of forward primer, 6 pmol/0.6 µL of reverse primer, 0.4 µL of 50× ROX reference dye, and 2 µL of DNA solution were prepared on ice. PCR was carried out under the following conditions: pre-denaturation at 95° C 60 s, denaturation at 95° C 15 s, extension at 60° C 30 s, and final extension at 60° C for 60 s (a total of 40 cycles).
Cell migration-scratch test hRPE cells (3 × 10 5 per well of six-well plate) were inoculated in DMEM/F12 medium containing 10% FBS at 37° C and 5% CO2 for 24 h. A zigzag scratch was made perpendicular to the plate orifice. Next, cells were washed twice with PBS. Wells were divided into LINC01592-KD + TGF-β1, KD-NC + TGF-β1, LINC01592-OE + TGF-β1, and OE-NC + TGF-β1 groups. The concentration of TGF-β1 was adjusted to 10 ng/mL and a constant volume of 2 mL was added in serum-free medium for LINC01592-KD, KD-NC, LINC01592-OE, and OE-NC groups. Samples were analyzed after 0, 24, and 48 h of incubation at 37° C with 5% CO2.
Cell viability was calculated using the following formula: Cell viability (%) = [A (medication) -B (blank)]/[C (0 medication) -B (blank)] × 100, whereby A is the absorption of experimental group wells with cells, CCK-8 solution, and culture medium, following transfection with plasmid and intervention with or without TGF-β1; B is the absorption of wells with CCK-8 solution and culture medium, but without cells; and C is the absorption of control group wells with cells, CCK-8 solution, and culture medium, but without plasmid transfection or TGF-β1 intervention of [34]. All experiments were repeated three times.

Statistical analysis
SPSS22.0 software was used for statistical analysis. The data were analyzed by one-way ANOVA and doubletailed t-test.

AUTHOR CONTRIBUTIONS
Y.S. and Z.T. performed the experiment. Y.S. and F.W. wrote the manuscript. F.W. supervised the study. All authors read and approved the final manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.

FUNDING
The Natural Science Grant of the Heilongjiang Province of China (H2018035, LH2020H040) and the Innovation and Development Foundation of First Affiliated Hospital of Harbin Medical University (2018L002).