Background
After extracapsular cataract extraction or after ocular trauma crystal rupture, the remaining cortex and capsular membrane become cloudy are called after cataract, also known as secondary cataract [
1]. Modern secondary cataract mainly refers to posterior capsular opacification (PCO) [
2,
3]. Current studies suggest that the proliferation, migration and epithelial-mesenchymal transformation (EMT) of lens epithelial cells (LECs) are the key factors for PCO formation [
4,
5]. The EMT process of LECs plays a central role in the formation of PCO, which can lead to cell adhesion and loss of the apical-basal polarity of the mesenchymal phenotype, leading to the production of fibroblasts [
6,
7]. Although the cause of PCO has been clarified, the molecular targets that influence its occurrence still need to be further explored. At present, transforming growth factor-β2 (TGF-β2)-induced LECs are considered to be an effective way to construct an in vitro model of PCO [
8,
9], which provides a convenient experimental model for us to carry out relevant research on PCO.
Long non-coding RNA (lncRNA) is a non-coding protein that participates in the regulation of various processes in cells [
10]. Research has confirmed that lncRNA can regulate cell growth, differentiation and apoptosis, and is related to many diseases progression [
11,
12]. More importantly, many lncRNAs have been shown to regulate LECs proliferation, migration and EMT, such as FEZF1-AS1, HOTAIR and MIAT [
13‐
15]. Nuclear enriched abundant transcript 1 (NEAT1) is a nuclear-restricted lncRNA that is abnormally expressed in many diseases and is thought to be associated with disease progression. Xiong et al. report that NEAT1 promotes proliferation, migration and invasion of cells to enhance the progression of breast cancer [
16]. And Wang et al. show that NEAT1 regulates the EMT process of diabetic nephropathy [
17]. Therefore, NEAT1 may play a vital function in cell proliferation, migration and EMT process. In PCO, Dong et al. conducts microarray analysis on the LECs of PCO patients and normal humans and reveals that the expression of NEAT1 in PCO is significantly increased [
18]. However, it is unclear whether NEAT1 participates in the regulation of PCO progress.
Studies on the functions of microRNAs (miRNAs) have been largely confirmed by many researchers. In the regulatory network of lncRNA-miRNA-messenger RNA (mRNA), the role of miRNA as a bridge between lncRNA and target genes has also become the key to elucidate the molecular mechanism of lncRNA [
19,
20]. MiR-486-5p is involved in the mediation of proliferation, metastasis and EMT of many diseases, including breast cancer and hepatocellular carcinoma [
21,
22]. The study confirms that miR-486-5p shows a low expression trend in TGF-β2-induced LECs and can participate in the proliferation, invasion and EMT of TGF-β2-induced LECs [
23].
Drosophila mothers against decapentaplegic 4 (SMAD4) belongs to the SMAD family and is activated by transmembrane serine/threonine receptor kinases, such as TGF-β receptors [
24]. SMAD family proteins play an essential role in the transduction of TGF-β signaling from cell surface receptors to the nucleus [
25]. The effects of SMAD4 on LECs proliferation and EMT have been reported in many studies [
18,
26]. Therefore, the study of SMAD4 and TGF-β/SMAD signaling pathway will help us better understand the factors affecting the progression of PCO. Our study is aimed to explore the role of NEAT1 in PCO progression and the potential mechanism, hoping to provide a novel molecular target for the exploration of the prevention and control of PCO.
Materials and methods
Sample tissues collection
This study was approved by the ethics committee of The First Affiliated Hospital of Zhengzhou University and was performed in accordance with the Declaration of Helsinki. Posterior capsular tissues were obtained from 30 PCO patients (30 eyes, age range was 50–75, free of other ocular diseases) in The First Affiliated Hospital of Zhengzhou University. All patients were diagnosed with PCO and were graded: 6 cases of grade I, 21 cases of grade II, and 3 cases of grade III. Similarly, we also obtained normal posterior capsular tissues from 30 organ donors (age range was 45–72). Written informed consent was signed from each patient and donor.
Cell culture, TGF-β2 treatment and cell transfection
Human LECs (SRA01/04) were bought from Biovector (Beijing, China). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 12,100-046, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; 10437028, Invitrogen) and 1% penicillin/streptomycin (15140148, Invitrogen). SRA01/04 cells were incubated at 37 °C with 5% CO2 incubator. When cells reached 60% confluence, SRA01/04 cells were treated with different concentrations of TGF-β2 (0, 1, 5, and 10 ng/mL) for 48 h, and the expression of NEAT1, miR-486-5p and SMAD4 could be detected. Similarly, when cells reached 50% confluence, transfection could be performed, followed by treatment with 10 ng/mL TGF-β2. All vectors and oligonucleotides were purchased from RiboBio (Guangzhou, China), including NEAT1 small interfering RNA and pcDNA overexpression vector (si-NEAT1: 5′-GAGCAATGACCCCGGTGACG-3′and NEAT1: F 5′-TTGGGACAGTFFACGTGTGG-3′, R 5′-TCAAGTCCAGCAGAGCA-3′) or their negative controls (si-NC: 5′-TAGATACCCCCAGGCCTACC-3′; and pcDNA: 5′-TAGAAGGCACAGTCGAGG-3′), miR-486-5p mimic and inhibitor (miR-486-5p: 5′-UCCUGUACUGAGCUGCCCCGAG-3′; and anti-miR-486-5p: 5′-CUCGGGGCAGCUCAGUACAGGA-3′) or their negative controls (miR-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′; and anti-miR-NC: 5′-CAGUACUUUUGUGUAGUACAA-3′), SMAD4 overexpression vector (SMAD4: F 5′-CGGACATTACTGGCCTGTTC-3′, R 5′-TAGGGCAGCTTGAAGGAAACC-3′) and its negative control (pcDNA: 5′-TAGAAGGCACAGTCGAGG-3′). Lipofectamine 3000 (L3000015, Invitrogen) was employed to transfect all vectors and oligonucleotides into SRA01/04 cells.
Quantitative real-time polymerase chain reaction (qRT-PCR)
TRIzol reagent (15596-026, Invitrogen) was used to extract total RNA, and BeyoRT II cDNA Synthesis Kit (D7170S, Beyotime, Shanghai, China) was employed to synthesize cDNA. The NEAT1 and SMAD4 expression was measured using SYBR Green (11762500, Invitrogen) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was designated as an internal control. The miR-486-5p expression was determined by TaqMan MicroRNA Assay (4440887, Applied Biosystems, Foster City, CA, USA) and U6 was employed as an internal control. All primers were as follows: NEAT1: F 5′-TTGGGACAGTFFACGTGTGG-3′, R 5′-TCAAGTCCAGCAGAGCA-3′; SMAD4: F 5′-CGGACATTACTGGCCTGTTC-3′, R 5′-TAGGGCAGCTTGAAGGAAACC-3′; miR-486-5p: F 5′-CGCGTCCTGTACTGAGCTGCC-3′, R 5′-ATCCAGTGCAGGGTCCGAGG-3′; U6: F 5′-CTCGCTTCGGCAGCACA-3′, R 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH: F 5′-ACAGTCAGCCGCATCTTCT-3′, R 5′-GACAAGCTTCCCGTTCTCAG-3′. Data were analyzed using the 2−ΔΔCt method.
Cell proliferation assay
This assay was employed using the 3-(4, 5-dimethyl-2 thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) Assay Kit (C0009, Beyotime). SRA01/04 cells were collected at 24 h after transfection and inoculated on 96-well plates. After cells were attached to the wall, 10 μL MTT solution was added to each well and incubated in the incubator for 4 h. After removed supernatant, Formazan solvent was added to each well and shock dissolved for 10 min. Cell absorbance was measured at 490 nm and cell viability was calculated. Non-treated and non-transfected cells were used as Control.
Transwell assay
This assay was performed using transwell chambers (3422, Corning Inc., Corning, NY, USA), which were pre-coated with Matrigel (354234, Corning Inc.) to measure the number of invaded cells and non-coated to determine the number of migrated cells. After treatment or transfection for 24 h, SRA01/04 cells were seeded in the upper chambers, which were filled with serum-free DMEM. In the lower chambers, DMEM contained 10% FBS was added. After 24 h, the cells were fixed with 4% methanol, stained using 0.1% crystal violet. The cells were photographed (100×) and counted using a microscope (DM500, Leica, Wetzlar, Germany). Non-treated and non-transfected cells were used as Control.
Western blot (WB) analysis
SRA01/04 cells were lysed using RIPA reagent (P0013K, Beyotime) containing protease inhibitor cocktail (Beyotime). After that, total protein was quantified using BCA Kit (Beyotime). 30 μg protein samples were subjected to 10% separating gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Next, the membranes were blocked with 5% nonfat milk, incubated with primary antibodies and probed with secondary antibody (bs-0295G, 1:20,000). Chemistar ECL Western Blotting Substrate (180-501, Tanon, Shanghai, China) was used to visualize the protein bands. All antibodies were obtained from Bioss (Beijing, China), and the primary antibodies containing E-cadherin (bs-1519R, 1:1000), Vimentin (bs-23063R, 1:1000), α-SMA (bsm-52392R, 1:5000), SMAD4 (bs-23966R, 1:1000), SMAD2 (bs-0718R, 1:1000), p-SMAD2 (bs-20341R, 1:300), SMAD3 (bsm-52224R, 1:1000), p-SMAD3 (bs-5235R, 1:1500), and GAPDH (bs-10900R, 1:2000). All antibodies were diluted with Primary Antibody Dilution Buffer (P0023A, Beyotime). Non-treated and non-transfected cells were used as Control.
Dual-luciferase reporter assay
The fragments of NEAT1 and SMAD4 3′UTR containing the predicted miR-486-5p binding sites or mutant binding sites were amplified and cloned into pGL3 reporter vector (Promega, Madison, WI, USA), recorded as wild-type and mutant-type NEAT1 or SMAD4 3′UTR reporter vectors (NEAT1-WT/MUT or SMAD4 3′UTR-WT/MUT). SRA01/04 cells were co-transfected with the reporter vectors and miR-486-5p mimic or miR-NC, and the luciferase activities were evaluated using the Dual-Lucy Assay Kit (D0010, Solarbio, Beijing, China).
RNA immunoprecipitation (RIP) assay
Magna RIP Kit (17-700) was bought from Millipore. Magnetic beads were pre-coated with antibodies against immunoglobulin G (IgG) or argonaute2 (Ago2) overnight at 4 °C. SRA01/04 cells were lysed and then incubated with magnetic beads. After washed with RIP buffer, total RNA was isolated and the abundances of NEAT1, miR-486-5p and SMAD4 were measured by qRT-PCR.
Biotin-labeled RNA pull-down assay
The biotinylated miR-486-5p (bio-miR-486-5p) probe and negative control (bio-miR-NC) probe were synthesized by Sangon (Shanghai, China) and transfected into SRA01/04 cells. After incubated for 48 h, the cells were harvested and then incubated with Dynabeads M-280 Streptavidin (11205D, Invitrogen). The enrichment of NEAT1 and SMAD4 was determined by qRT-PCR.
Statistical analysis
Each experiment was carried out at least three times. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad, La Jolla, CA, USA). Data were represented as mean ± standard deviation. Student’s t-test or one-way analysis of variance was used for statistical analysis. The correlation between miR-486-5p and NEAT1 or SMAD4 was determined using Pearson correlation analysis. P < 0.05 indicated statistical significance.
Discussion
PCO often leads to progressive loss of vision in patients and can cause vision loss in severe cases, so it brings a lot of inconvenience to the life of patients [
27]. At present, Nd-YAG laser capsulotomy is often used for the treatment of PCO, but postoperative complications are still possible [
28]. Therefore, a better understanding of the factors affecting the pathogenesis of PCO is conducive to the development of new strategies to prevent and alleviate the development of PCO. NEAT1 is often considered an oncogene in cancer because of its role in promoting proliferation, metastasis, and EMT [
29,
30]. Dong et al. found that NEAT1 and MALAT1 were highly expressed in PCO, and confirmed that MALAT1 could promote EMT of TGF-β2-induced LECs by regulating the miR-26a/SMAD4 axis [
18]. However, the role of NEAT1 in TGF-β2-induced LECs is unclear. Similarly with the previous study, our study found that NEAT1 was highly expressed in posterior capsular tissues of PCO patients and TGF-β2-stimulated LECs. But not only the EMT of cells, our research also explored the effect of lncRNA on cell proliferation and metastasis. The inhibitory effect of NEAT1 on the proliferation, metastasis and EMT of TGF-β2-stimulated LECs indicated that NEAT1 was a key factor for LECs to maintain normal biological function. Consistent with previous findings [
18], our results provide new evidence for NEAT1 as a target for the treatment of PCO.
The involvement of miR-486-5p in disease progression in the form of low expression had been well documented. For example, miR-486-5p was believed to interact with lncRNA DLGAP1-AS1 to participate in the regulation of hepatocellular carcinoma cell proliferation by DLGAP1-AS1 [
31]. Also, miR-486-5p had been reported to modulate the EMT process in papillary thyroid cancer by regulating KIAA1199 expression [
32]. Therefore, the negative influence of miR-486-5p on cell proliferation, metastasis and EMT has been widely confirmed. In our study, we found that the expression trend of miR-486-5p in posterior capsular tissues of PCO patients and TGF-β2-stimulated LECs was opposite to NEAT1, and verified the interaction between the two through bioinformatics. At the same time, the reversing effect of anti-miR-486-5p on the function of si-NEAT1 also confirmed the negative regulatory effect of miR-486-5p on the proliferation, migration, invasion and EMT of TGF-β2-stimulated LECs. The anti-proliferation, anti-metastasis and anti-EMT effects of miR-486-5p on LECs also were verified by our data, which was consistent with the results of Liu et al. [
23]. Hence, miR-486-5p might also be an effective molecular target to prevent PCO progression.
SMAD has been a focus of research because it is a key protein in the TGF-β/SMAD signaling pathway that mediates cell growth and differentiation [
25]. Some studies have shown that the low expression of SMAD4 is believed to inhibit cell proliferation, metastasis and EMT, such as in colon cancer and esophageal squamous cell carcinoma [
33,
34]. Herein, we suggested that SMAD4 had an increased expression in posterior capsular tissues of PCO patients and TGF-β2-stimulated LECs. The inverting effect of SMAD4 on the function of miR-486-5p mimic confirmed that SMAD4 was the target of miR-486-5p. More importantly, the pro-proliferation, pro-metastasis and pro-EMT effects of SMAD4 on LECs also was demonstrated by this study, which was consistent with previous research [
18,
26]. In addition, we also indicated the regulatory effect of the NEAT1/miR-486-5p/SMAD4 axis on p-SMAD2 and p-SMAD3 expression, which confirmed that TGF-β/SMAD signaling pathway was the downstream pathway of the NEAT1/miR-486-5p/SMAD4 axis. These results provided a perfect molecular mechanism for NEAT1 to regulate the progress of PCO.
Of course, there are still some deficiencies in our current research. In the rescue experiment, we found that the reversal effect of miR-486-5p inhibitor on NEAT1 silencing function is partial, so this indicates that there may be other miRNAs involved in the regulation of NEAT1 on the biological functions of LECs. Similarly, the reversal effect of SMAD4 on miR-486-5p function is also partial, which indicates that there may be other targets involved in the regulation of miR-486-5p on the biological functions of LECs. In future research, we will focus on exploring more mechanisms by which NEAT1 regulates the biological functions of LECs, in order to provide new ideas for the treatment of PCO.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.