Clinical experiments and grouping
A total of 30 paired peripheral blood samples were collected from patients with stroke and healthy volunteers at the Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University. Patients were excluded if any other central nervous system disease was identified. Venous blood samples (5 ml) were drawn from all participants. The serum was isolated by centrifuging at 1,000 × g at 4°C for 5 min and then stored in liquid nitrogen. The study received approval from the the Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University (YX-2021-071-01).
Cell lines and culture
Human PC12 cells cells lines (rat adrenal pheochromocytoma cell line) and 293T cells were purchased from ScienCell company (San Diego, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibico, USA) in a humidified atmosphere of 5% CO2 at 37 °C.
To model OGD/R injury, the medium was discarded and cells were washed with phosphate buffer saline (PBS) three times and incubated in Serum/glucose-free DMEM. The cells were subsequantly subjected to a hypoxic chamber at 37°C (95% N2 and 5% CO2). Cells in the Control group were cultured under normoxic conditions in the meantime. After OGD exposure, all cells were re-perfused for 12 h under normal culture conditions at 37°C (95% O2 and 5% CO2).
To assess the levels of miR-3144-5p, FOXD2-AS1 and KCTD15 in ischemic stroke patients, peripheral blood samples were drawn from these stroke patients within 3 hours of stroke onset, as well as the healthy individuals. All blood samples underwent RT-qPCR to measure miR-3144-5p, FOXD2-AS1 and KCTD15 levels in serum by using TaqMan miRNA assays (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol.
Total RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and further cDNA synthesis was performed with a reverse transcription kit (Promega Corporation, Madison, WI, USA). The relative expression levels of RNA were calculated using 2-△△Ct method.
Transfection and grouping
Before OGD/R, PC12 cells were transfected with miR-3144-5p mimic, miR-3144-5p inhibitor, shR-FOXD2-AS1 or FOXD2-AS1 plasmid by Lipo 3000 Transfection Reagent (Thermo Fisher Scientific, Inc.) as previously described. After 6 hours, the medium was replaced by fresh DMEM.
Dual luciferase reporter assay
Luciferase reporter plasmids including the wild-type and mutant sequences of FOXD2-AS1 and KCTD15 were constructed (Genewiz, China). Cells were each seeded with a proper density in 12-well plates. Then, all plasmids were transfected into the prepared cells for 24 h. After post-transfection, cells were harvested and the firefly luciferase activity was measured using a dual-luciferase reporter assay system (Promega, USA). The relative luciferase activity was normalized to Renilla luciferase activity. Transfection was repeated in triplicate.
Cell proliferation assay
Cell viability was measured by using the Cell Counting Kit-8 (CCK-8) assay (Beyotime, China). All treated cells were seeded into 96-well plates and were starved in a serum-free medium for another 24 h. Following cells were incubated with CCK-8 reagent, the absorbance at 450 nm was detected via a microplate reader.
Western blot analysis
Briefly,cells were lysed with RIPA buffer (Beyotime, China). After centrifuge, the BCA Protein Assay Reagent (Beyotime, China) was applied to quantify the protein concentration of lysates. Western blot was performed in accordance with the standard protocol anywhere. BAX, BCL2 and tubulin antibodies were obtained from Cell Signal Technology (CST, USA). Then, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies. Finally, the blots were visualized with a commercially available enhanced chemiluminescence (ECL) analysis kit (Merck Millipore, USA). The bands were analyzed with Image J® software.
Neuronal apoptosis was detected by using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Briefly, the cells fixed with ice-cold 4% paraformaldehyde followed by permeabilization with 0.1% (v/v) Triton X-100 (Sigma) for 5 min. Then, cells were incubated with TUNEL Detection Kit (Beyotime, China) in a wet and dark environment. The nuclei were then stained with 4,6-diphenylamine-2′-phenylhydrazine hydrochloride (DAPI). Afterwards, the number of apoptotic cells were determined. Positive TUNEL staining was visualized on the confocal microscope and were quantified using ImageJ. The apoptotic index is the percentage of TUNEL-positive cells (positive cells/100% total cells).
All statistical analyses were carried out by using SPSS v20.0. Data are represented as the mean ± standard deviation (SD). Statistical significance between groups was determined using Student’s t-test or the one-way ANOVA method. All experiments were repeated at least three times. Differences with a P-value less than 0.05 were defined as being statistically significant.
FOXD2-AS1 Was Down-Regulated in stroke patients blood samples and OGD/R-Induced PC12 Cells, and It Mitigated OGD/R-Induced Neuronal Injury in PC12 Cells
In order to figure out the function of FOXD2-AS1 in IS, we firstly determined the relative expression of FOXD2-AS1 in stroke patients blood samples and in brain tissues of MCAO mice by RT-qPCR. As shown in Figure 1A, a significant reduction of FOXD2-AS1 was observed in stroke patients blood samples compared with healthy controls . In addition, similar trend can be obtained when the level of FOXD2-AS1 in the brain tissues of mice with or without MCAO treatment were detected. (Figure 1B). Equivalent to previous results, the expression of FOXD2-AS1 in PC12 cells show a decrease with the increase of OGD/R treatment time, at 4 h, 8 h, 12 h and 24h respectively ly (Figure 1C). Cell viability and apoptosis were detected by CCK-8 assay and tunnel staining, respectively. The results showed that OGD/R treatment inhibited the cell viability and increased the apoptosis in PC12 cells (Figure 1D-E).
FOXD2-AS1 promoted cell viability and reduced apoptosis in OGD/R-Induced PC12 Cells
Next, we investigated the role of FOXD2-AS1 in OGD/R-Induced PC12 Cells by gain or loss functions of FOXD2-AS1. As shown in Figure 2A, FOXD2-AS1 was up-regulated by transfection of pcDNA-FOXD2-AS1 and down-regulated by transfection of sh- FOXD2-AS1 in PC12 cells. CCK-8 analysis revealed that overexpression of FOXD2-AS1 notably promoted the proliferation of PC12 cells no matter they exposed to OGD/R or not. (Figure 2B). Moreover, the cell viability was furthermore reduced by downregulated FOXD2 AS1 under OGD/R treatment (Figure 2C). As for TUNEL Assay, there was a significant reduction of TUNEL-positive cells in the FOXD2 AS1 overexpression group under OGD/R stimulation. (Figure 2D). However, when PC12 cells was transfected with the sh-FOXD2-AS1 , a reverse phenomenon that apoptotic cells markedly increased under OGD/R environment could be observed (Figure 2E). On top of that,we detected the expression of BCL2 and Bax, which were considered as apoptosis marker. The results suggested that FOXD2-AS1 overexpression offered PC12 cells protection against OGD/R stimulation while FOXD2-AS1 knockdown showed opposite results that cell apoptosis significantly elevated. Taken together, it is indicated that the augmented cell viability from FOXD2-AS1 overexpression under OGD/R stress was attributable to the reduced apoptosis.
FOXD2-AS1 Could Serve as a Competing Endogenous RNA (ceRNA) for miR-3144-5p
To further explore the function of FOXD2-AS1 in IS, miRDB and LncBase Predicted v.2. were used to investigate the potential target genes of FOXD2-AS1. There are 74 microRNAs in the two databases in total, and miR-3144-5p gained the highest score in both databases (Figure 3A-C). As shown in the Figure 3D, FOXD2-AS1 contained potential binding sites for miR-3144-5p (Figure 3D). Then the hypothesis about interaction between FOXD2-AS1 and miR-3144-5p was confirmed by DLR assay. Dual-luciferase report assay revealed that miR-3144-5p mimics reduced the luciferase activity of FOXD2-AS1 WT, but not of FOXD2-AS1 mut vector. (Figure 3D).
In addition, in order to verify whether FOXD2-AS1 takes part in regulating expression of miR-3144-5p, we overexpressed or knocked down FOXD2-AS1 in PC12 cells. The RT-qPCR showed that the level of miR-3144-5p was increased significantly after FOXD2-AS1 knockdown, and decreased when overexpressing FOXD2-AS1 in PC12 cells (Figure 3E). Meanwhile, we detected the level of miR-3144-5p in between stroke patients and healthy people blood samples. Consistent with the results mentioned before, blood samples from stroke patients had higher level of miR-3144-5p (Figure 3F). Furthermore, Pearson's correlation analysis was performed to evaluate the relationship between expression of miR-3144-5p and FOXD2-AS1. The analysis showed that the level of miR-3144-5p was negative correlated with FOXD2-AS1 (Figure 3G).
Inhibition of miR-3144-5p Alleviated OGD/ R-Induced Neuronal Injury in PC12 Cells
The cell viability of PC12 cells in the miR-3144-5p overexpression group was decreased in comparison with the NC group under OGD/R treatment, whereas the viability of PC12 cells was significantly increased by FOXD2-AS1 overexpression, although transfected miR-3144-5p (Figure 3H). Importantly, we also detected the expression of BCL2 and Bax, it indicated that the cell apoptosis was increased by miR-3144-5p, while inhibited by FOXD2-AS1 overexpression (Figure 3I). Meanwhile, the TUNEL staining showed in accordance with the previous results (Figure 3J).
KCTD15 Was Targeted by miR-3144-5p
Subsequently, we predicted the target genes of miR-3144-5p with miRDB. It indicated that KCTD15 gained the highest score in the list (Figure 4A). It was observed that KCTD15 had a conserved binding region of miR-3144-5p (Figure 4B). The result of DLR revealed that relative luciferase activity of PC12 cells was reduced by co-transfection of KCTD15 WT vector and miR-3144-5p mimics (Figure 4B), whereas relative luciferase activity of PC12 cells showed no significant change after co-transfection of KCTD15 mut vector and miR-3144-5p mimics (Figure 4B). The result of RT-qPCR showed that KCTD15 was down-regulated when cells were transfected with miR-3144-5p mimics (Figure 4C). Furthermore, KCTD15 was decreased gradually under OGD/R treatment at 4 h, 8 h, 12 h and 24 h in PC12 cells, respectively (Figure 4D). There was a positive correlation between KCTD15 and FOXD2-AS1 in stroke patients and healthy people blood samples (Figure 4E).