Ethics declaration

The experimental animals used in this medical research were handled in conformity to the approval granted by the Animal Committee of the Fifth Affiliated Hospital of Southern Medical University, following thorough discussion and review.

Bioinformatics analysis

Targets of lncRNA MIAT and miR-130a-3p were analyzed via the online site Starbase (https://starbase.sysu.edu.cn/agoClipRNA.php?source=lncRNA) (This database, based on CLIP-seq experimental data, possesses high biological credibility).

miRNA downstream regulatory genes were obtained via the online analysis websites mirwalk (http://mirwalk.umm.uni-heidelberg.de/), miRmap (https://mirmap.ezlab.org/app/) and microT_CDS (http://dianalab.e-ce.uth. gr/html/dianauniverse/index.php?r = microT_CDS) (These tools all perform analyses based on sequence complementarity and evolutionary conservation), and the Venn diagrams of target genes were drawn by utilizing the online tool sangerbox (http://vip.sangerbox.com/home.html) to acquire the intersected genes. PPI of those candidate targets were further obtained via the database STRING (https://string-db.org). The top 10 targets of the PPI network were ranked in accordance with the number of adjacent nodes and the R software “count” package.

RT-qPCR

The isolation of RNA extracted from tissues and cells was performed by Trizol reagent (15596026, Invitrogen). For miRNA expression, cDNAs for miRNAs were synthesized through the Mir-X™ miRNA First-Strand Synthesis Kit, which was sourced from Takara; SYBR Premix Ex Taq II (Takara) was employed for RT-qPCR reactions. Regarding mRNA and lncRNA assessment: RNA was converted into cDNA through reverse transcription by utilizing PrimeScript RT reagent Kit (Takara), and RT-qPCR was implemented on the synthesized cDNA through SYBR Premix Ex Taq II (Takara). Data analysis was counted by implementing 2-ΔΔCt values and U6 or GAPDH normalization, and the primer sequences were displayed in Table 1.

Table 1 Primer sequence for RT-qPCRLuciferase report assay

Fragments of MIAT-WT, MIAT-MUT, Pdgfra-WT, and Pdgfra-MUT including miR-130a-3p binding site and mutant binding site underwent insertion into luciferase reporter vectors (pMIR-reporter), sourced from Thermo Fisher Scientific, to construct MIAT-WT, MIAT-MUT, Pdgfra-WT, and Pdgfra-MUT reporter vectors. Mouse retinal microglia (PXMC-C271, Suzhou Starfish Biotechnology Co., Ltd., Jiangsu, China) were cotransfected with miR-130a-3p mimic or mimic-NC and these mentioned reporter vectors by implementing Lipofectamine 3000 (Invitrogen). Mouse retinal microglia supernatants were collected after 24 h (Wang et al. 2019; Wei et al. 2020).

RIP

RIP was implemented by EZ-Magna RIP Kit (Millipore). Anti-Ago2 antibody and normal Anti-IgG were cultivated at 4 °C for 1 h with magnetic beads (Millipore). Next, some cell lysate was utilized as a blank NC (Input), and the other part of the cell lysate was cultivated with magnetic beads overnight. Afterwards, RT-qPCR was executed to measure the enrichment of Pdgfra, MIAT and miR-130a-3p (Wei et al. 2020).

RNA pull down assay

Following transfection with biotinylated miR-130a-3p (MUT and WT 50 nM each), cells were gathered, vortexed, followed by grown on a specific cell lysis solution (Ambion). Next, the sample cell lysate (50 mL) was distributed, and the left lysate was cultivated with M-280 streptavidin magnetic beads (Sigma) at 4℃ for 3 h. Afterwards, RNA was isolated and RT-qPCR was carried out (Lal et al. 2011; Wang et al. 2017).

Establishment and grouping of BCCAO mouse models

Male WT C57BL/6 J mice (10–12 weeks) were selected and maintained in a pathogen-free environment with ad libitum access to water and food. As previously reported (Manouchehrian et al. 2015), CRHI mouse models were established by BCCAO. Briefly, mice were sedated through intraperitoneal administratio of 0.3% sodium pentobarbital. For low perfusion and sham surgery, the mice were subjected to 5% isoflurane anesthesia and 2% isoflurane oxygen anesthesia maintaining. A minor cut was made on the neck to expose the common carotid artery. For low perfusion, metal coils were set as follows: pitch: 0.50 mm; wire diameter: 0.08 mm; ID: 0.18 mm; total length: 2.5 mm; surface: gold-plated (Invitrotech Co, LTD, shimogas-cho Kusatsu) and wrapped around the common carotid artery to reduce blood flow by approximately 70%. Afterward, the anesthesia was discontinued, and the incision was closed using local anesthesia Marcain (1.25 mg/kg, Bupivacaine, Apoteket, ume < s:1 > , Sweden). The sham-operated mice received the same treatment but without the implanted coils. At the 17th week postoperatively, the mice were euthanized with 5% isoflurane and their eyes were extracted. Eyes were immediately fixed with 4% PFA after enucleation. In sh-MIAT- or oe-Pdgfra-treated mice, adeno-associated virus (AAV) solutions (1 μL, 1012 v.g/mL) loaded specific targets were delivered into the vitreous 4 w prior to modeling to maximize transfection efficiency. The miR-130a-3p antagomir-containing solution (1 μL, 20 μM) was injected intravitreally prior to modeling.

ELISA

Orbital blood samples were centrifuged to obtain the supernatants. An ELISA kit (Abcam) was applied to test the contents of IL-1β and IL-18 in mouse serum. The absorbance values were assessed with a microplate reader (BS-1101, Nanjing DeTie) at 492 nm, with a blank control well zeroing, and the contents of the detected indicators were referred to the standard curve according to the A-value of each sample (Fu et al. 2023).

Histological test

After fixation in a FAS eye fixator (Servicebio, Wuhan, China) overnight, the eyes were subjected to paraffin-sectioned into slices of 15 μm and HE staining. Servicebio was applied for sectioning and staining. Retinal thickness was tested from the GCL to the edge of the outer nuclear layer by implementing ImageJ software (Lou et al. 2022).

TUNEL staining

Retinal cell apoptosis was tested by implementing a modified TUNEL (fluorescence) method. Specifically, cell apoptosis of slices was measured by a TUNEL-based red light in situ cell death detection kit (from Roche). Afterward, paraffin-embedded sections were pretreated with graded xylene, ethanol and proteinase K, and these slices were cultivated in permeabilization solution. Afterwards, each section was combined with 50 μL of TUNEL reaction solution and cultured. The slides underwent three rinsing procedures, each time for 5 min, stained by DAPI for 3 min and flat-mounted. Nine fields of view were randomly selected from each retina with Zeiss confocal fluorescence microscope (Leica Microsystems, Bensheim, Germany). To evaluate apoptosis, the proportion of TUNEL-positive cells among the total cell count was calculated (Ding et al. 2023).

Immunofluorescence assay

After cold anesthesia, the mice were transcardially perfused by saline and 4% PFA sequentially, and their right eye was enucleated and fixed in PFA (4%). Next, the isolation of the entire retina was performed, and it was placed in Triton X-100 cold solution (0.3%) and blocked with donkey serum for 1 h (10%; Jackson immunresearch Laboratories), with Tween-20 (0.05%; Sigma-Aldrich) and bovine serum albumin (1%) at ambient temperature. Rabbit anti-iba1 (ab245230, 1:100, Abcam) antibodies were utilized for microglia and focal death was assessed by GSDMD (ab245230, 1:100, Abcam) antibody overnight. To visualize immunoreactive proteins, incubation was conducted adopting goat anti-rabbit secondary antibodies sourced from Thermo Fisher Scientific (1:10,000). Images were photoed by laser scanning confocal microscope (Carl Zeiss, LSM700) (Ding et al. 2023).

RNA in situ hybridization (RNA-ISH)

The retinal slices were fixed in 4% PFA PBS and sectioned at 14–20 μm. Digoxin-labeled probes were prepared according to the Diagnostics protocol. Briefly, the MIAT DNA fragment was amplified from retinal cDNA by the subsequent primers (from 5' to 3'), and sub-cloned into the pBluescript vectors, from which the MIAT probe was transcribed. RNAscope was executed as per the guidelines outlined in the RNAscope®Multiplex Fluorescent v2. MIAT probes were provided by Advanced Cell Diagnostics (Lou et al. 2022).

Statistics

SPSS 21.0 (IBM SPSS Statistics) was implemented to process statistical data. Measurement data were depicted as mean ± SD, and an independent sample t-test was implemented for two-group comparisons, whereas one-way ANOVA was conducted for multi-group comparisons, with Tukey’s for post hoc tests. P < 0.05 was defined as a statistically significant difference.