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Introduction

Cerebral aneurysm (CA) is a cystic dilation of cerebral vascular wall caused by congenital or postnatal factors (1,2). CA is characterized by the destruction of the integrity of arterial wall, endothelial dysfunction and extracellular matrix disorder (3). There are reports that 20-50% of patients with CA will experience the rupture of CA, leading to subarachnoid hemorrhage with high disability rate, high mortality rate and poor prognosis (4-7). Unfortunately, most patients are painless and asymptomatic when the aneurysm is not ruptured, which is a challenge for early treatment (8). At present, the occurrence and rupture of CA is closely associated with inflammation (9), and macrophages are the key effector cells (10). Monocyte chemoattractant protein-1 (MCP-1) induces macrophages to infiltrate CA walls and stimulates macrophages to differentiate into inflammatory phenotypes, thus affecting the formation and rupture of CA (11). In addition, the low wall shear stress of the aneurysm wall induces the continuous expression of MCP-1 in vascular smooth muscle cells (12,13). However, the pathogenesis of aneurysm remains to be explored. Therefore, it is urgent to identify useful biomarkers for the diagnosis and prognosis of CA.

MicroRNAs (miRNAs) are short strands of noncoding RNAs composed of 20-25 nucleotides (14,15). miRNAs can inhibit the expression of target genes by binding with the 3'-untranslated region (UTR) region of target mRNA to inhibit the translation of mRNA or promote its degradation, thus regulating most biological processes such as cellular proliferation, migration, invasion and apoptosis (16-19). The dysregulation of miRNAs is frequently found in the pathogenesis of inflammatory diseases (20). For example, Kawano and Nakamachi (21) found that miR-124 can regulate the process of rheumatoid arthritis, and researchers also confirmed that the inhibition of miR-124 can activate the inflammatory response of retina and central neurons (22,23). The formation and rupture of CA are attributed to the inflammatory dysfunction of endothelial cells, and some researchers have confirmed that miRNAs are associated with the formation of CA (24,25). Therefore, it is speculated that miRNAs are involved in the inflammatory process of CA. It has been found that miR-124-5p can regulate macrophage phagocytosis and inhibit inflammatory pathways (26,27). In addition, miR-124-5p has been shown to regulate angiogenesis (27). However, the role and molecular mechanism of miR-124-5p in CA has not been confirmed. The present study aims to explore the effect and the molecular mechanism of miR-124-5p on the occurrence and development of CA.

Materials and methods

Clinical samples

Peripheral blood samples from 30 patients with CA and 30 healthy individuals were collected from the Handan Central Hospital between August 2018 and January 2020. Before surgery, all patients did not receive treatment. Written informed consent was obtained from all participants and their families. The institutional ethics committee of Handan Central Hospital has approved this study on August 20, 2018.

In vitro CA model construction

Human umbilical vein endothelial cells (HUVECs) were chosen as the model cells in the present study, since it is a common cell in vascular system in vitro research, and are easier to obtain compared with other endothelial cells (28). In addition, the cellular function of HUVECs inhibited by inflammatory smooth muscle cells that are associated with CA (28). HUVECs purchased from Thermo Fisher Scientific, Inc. (cat. no. C0155C), and the cells were cultured in 37˚C with 5% CO2 for 2 days in DMEM complete medium (Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.). HUVECs were plated in 24-well plates (3x105 cells/well) and treated with 10 ng/ml IL-1b (Invitrogen; Thermo Fisher Scientific, Inc.) for 1 day at 37˚C to establish the inflammatory model of CA.

Reverse transcription-quantitative (RT-q)PCR

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract the total RNA according to the manufacturer's instructions. The cDNA was synthesized using the First Strand cDNA Synthesis kit at 55˚C for 30 min (Thermo Fisher Scientific, Inc.). Then, the mixture system including SYBR Green qPCR Master mix, cDNA templates and primers were applied for qPCR according to standard methods (95˚C for 3 min to carry on pre-denaturation; 95˚C for 30 sec to carry out denaturation; 55˚C for 30 sec to subject to annealing; and 72˚C for 60 sec to subject to elongation) to detect the expression of miR-124-5p or FoxO1. Finally, 1.5% agarose gel was used to electrophorese and visualize the products of PCR. The relative expression was calculated by the 2-ΔΔCq method (29). U6 small nuclear RNA and GAPDH were used as the internal controls of miR-124-5p and FoxO1, respectively. The primer sequences were: miR-124-5p forward, 5'-CGCGTGTTCACAGCGGAC-3'; miR-124-5p reverse, 5'-AGTGCAGGGTCCGAGGTATT-3'; U6 forward, 5'-GCTTCGGCAGCACATATACTAAAAT-3'; U6 reverse, 5'-CGCTTCACGAATTTGCGTGTCAT-3'; FoxO1 forward, 5'-GGCTGAGGGTTAGTGAGCAG-3'; FoxO1 reverse, 5'-AAAGGGAGTTGGTGAAAGACA-3' (30); GAPDH forward, 5'-TCGACAGTCAGCCGCATCTTCTTT-3'; and GAPDH reverse, 5'-GCCCAATACGACCAAATCCGTTGA-3' (31).

Cell transfection

miR-124-5p-inhibitor, miR-124-5p-mimics, FoxO1-small interfering (si)RNA (si-FoxO1) and negative controls (inhibitor-NC, mimic-NC and si-NC) were designed and construct by Shanghai Gene Pharmaceutical Technology Comsenz Inc. In addition, the Lipofectamine™ 3000 Transfection kit was purchased from Thermo Fisher Scientific, Inc. Three different siRNAs were used to knockdown FoxO1 and si-FoxO1-1 was screened out as the most specific for subsequent experiments (Fig. S1). The cells were cultured with trypsin for 1 h, and then the cells were transfected according to the instructions of the Lipofectamine™ 3000 Transfection kit. Subsequently, the cells were cultured in an incubator with 5% CO2 at 37˚C for 48 h. Meanwhile, the transfection efficiency of miR-124-5p-mimic/inhibitor was also detected by PCR (Fig. S2).

Enzyme-linked immunosorbent assay (ELISA)

Human IL-6 ELISA kit (cat. no. ab178013; Abcam), Human TNF alpha ELISA kit (cat. no. ab181421; Abcam) and Human IL-8 ELISA kit (cat. no. ab214030; Abcam) were used to detect the levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α). According to the instructions of the kits, 50 µl samples or standards were added to the appropriate wells of 96-well plates that were incubated with antibodies cocktail for 1 h at 37˚C. Then, each well was washed by 350 µl 1X Wash Buffer PT for 3 times. Next, 100 µl tetramethylbenzidine development solution were added into each well and incubated for 10 min in the dark. Finally, the results were detected by a Microplate Reader at 450 nm after the color reaction stopped. Each sample was assayed using three replicates.

Western blotting

The expression of nuclear factor (NF)-κB, intercellular adhesion molecule (ICAM)-1 and FoxO1 were detected by western blotting. The HUVECs were lysed by RIPA total protein lysate (150 mM NaCl, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 7.4, 1% NP-40) to obtain protein samples. The BCA protein quantitative detection kit was used to determine the protein concentration, according to the instructions. Firstly, the protein standard solution was prepared and added to the 96-well plates according to the gradient concentration, and the appropriate concentration of sample protein was added. Next, BCA solution (A:B=50:1) was added to the protein solution and incubated at 37˚C for 30 min. Finally, the absorbance was measured and the standard curve was drawn to determine the concentration of sample protein. Next, 40 µg protein of each group was loaded into 10% SDS-PAGE gel, and GAPDH was used as the internal control to carry out concentrated gel electrophoresis and separation gel electrophoresis. Then, the transmembrane electrophoresis was implemented by using the polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% bovine serum albumin (BSA) for 1 h at 37˚C. The membrane was then incubated with primary antibodies purchased from Abcam, including anti-NF-NF-κB (cat. no. ab16502; 0.5 µg/ml), anti-ICAM-1 (cat. no. ab109361; 1:10,000), anti-FoxO1 (cat. no. ab70382; 1:10,000) and anti-GADPH (cat. no. ab9485; 1/12,500) antibodies, for 14 h at 4˚C. Subsequently, the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (cat. no. ab205718; Abcam; 1:50,000) was added to the PVDF membranes and incubated for 90 min at 37˚C. Eventually, the PVDF membrane was put into the gel imager with ECL luminescent reagent for exposure for 30 sec. The proteins were quantified using ImageJ software (v1.8.0; National Institutes of Health).

Dual luciferase reporter assay

The binding site of FoxO1 in miR-124-5p was predicted using the TargetScan website (http://www.targetscan.org/mmu_72/), and the double luciferase reporter gene analysis was applied to identify the targeting association between miR-124-5p and FoxO1. The FoxO1 3'-UTR containing the miR-124-5p binding sites was synthesized and inserted into the pGL3 control vector between the BamH1 site and Xhal site to construct the FoxO1 wild-type (FoxO1-WT) reporter vector. The FoxO1-WT reporter vector (100 ng) and miR-124-5p mimics (50 nM) or mimic-NC (100 nM) were co-transfected into the cells at 37˚C for 8 h using Lipofectamine™ 3000 Transfection kit. The aforementioned process was also applied to the corresponding FoxO1 mutant (FoxO1 MUT) reporter vector. The activity of luciferase was determined by the Dual-Luciferase Reporter Assay System kit (cat. no. E1910; Shanghai Haoran Biotechnology Co., Ltd.) after transfection for 48 h.

Transwell migration/invasion assay

The migration and invasion abilities of the HUVECs were detected by using Transwell chambers. After digestion and washing, the cells at the logarithmic growth stage were resuspended in serum-free medium at a cell concentration of 2x105 cells/ml. Next, 650 µl medium containing 10% FBS was added to the lower chamber, 150 µl cell suspension was added to the upper chamber, and the chamber was incubated for 24 h at 37˚C. Then, the cells on the upper surface of the membrane were removed slowly using a cotton swab. Subsequently, the cells on the lower surface of the membrane were fixed with 4% methanol for 30 min at room temperature after the membrane was dried, and the cells were stained by Giemsa for 25 min at room temperature. Ultimately images were captured with a Leica DC 300F light microscope (magnification, x400) and the cells were counted using the ImageJ software (v1.8.0; National Institutes of Health) to assess the ability of cell migration. The invasion assay was performed as aforementioned for the migration assay, except that Matrigel gel diluted by serum-free DMEM medium was added to the upper chamber.

Statistical analysis

All experiments were repeated three times, and all data are presented as means ± SD. The data were evaluated using statistical approach, including one-way ANOVA followed by Sidak's multiple comparisons test or Student's t-test, using the GraphPad Prism 7.0 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

Downregulation of miR-124-5p in CA in vivo and in vitro

The difference in the expression of miR-124-5p in the peripheral blood of 30 patients with CA and 30 healthy individuals was analyzed by using RT-qPCR. The results showed that the expression of miR-124-5p was lower in patients with CA compared with that of healthy individuals (Fig. 1A). In addition, according to previous studies, It is known that IL-1β can activate vascular endothelial cells and induce their secretion of adhesion factors such as ICAM-1, in order to recruit inflammatory cells and trigger the inflammatory reaction of arterial walls, thus forming aneurysm (32). In the present study, HUVECs were used as an in vitro model system, and the IL-1β were used to stimulate HUVECs to simulate the inflammatory state of CA. Then, the expression of miR-124-5p was detected in HUVECs that were simulated by IL-1β and the non-stimulated HUVECs using RT-qPCR. The results showed that the expression of miR-124-5p in the IL-1β group was significantly lower compared with that of the control group (Fig. 1B).

Figure 1 Downregulation of miR-124-5p in cerebral aneurysm in vivo and in vitro. (A) The expression of miR-124-5p in the peripheral blood of 30 patients with CA and 30 normal individuals was detected by RT-qPCR. (B) The expression of miR-124-5p in HUVECs stimulated by IL-1β and normal HUVECs was detected by RT-qPCR. **P<0.01; ****P<0.0001 vs. control. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; IL-1β, interleukin-1β.

Inhibition of miR-124-5p can enhance the cell function of HUVECs and promote the release of inflammatory factors

The changes in the cell function of HUVECs and the associated inflammatory factors were detected following the downregulation of miR-124-5p by the miR-124-5p-inhibitor. The results of RT-qPCR showed that IL-1β could inhibit the expression of miR-124-5p, and there was no significant difference between the IL-1β group and IL-1β + miR-124-5p-inhibitor-negative control (NC) group (Fig. 2A). Furthermore, the application of miR-124-5p-inhibitor could enhance the inhibitory effect of miR-124-5p compared with the IL-1β + miR-124-5p-inhibitor-NC group (Fig. 2A). The results of the cell invasion assay indicated that the IL-1β group had higher invasion ability compared with the control group, and the invasion ability was further enhanced by miR-124-5p-inhibitor compared with the miR-124-5p-inhibitor-NC (Fig. 2B). Compared with the control group, the migration ability of HUVECs was strengthened by IL-1β, and miR-124-5p-inhibitor further promoted the cell migration compared with the IL-1β + miR-124-5p-inhibitor-NC group (Fig. 2C). Meanwhile, western blotting and ELISA were used to detect the expression of inflammatory factors. Western blotting showed that the expression of NF-κB and ICAM-1 was increased in the IL-1β group compared with the control group, and the transfection of miR-124-5p-inhibitor enhanced the expression of inflammatory factors compared with the IL-1β + miR-124-5p-inhibitor-NC group (Fig. 2D). The results of ELISA indicated that IL-1β upregulated the expression levels of IL-6, IL-8 and TNF-α compared with the control group, and the miR-124-5p-inhibitor further promoted the increase in the expression of inflammatory factors (Fig. 2E-G). The aforementioned experimental results demonstrate that the inhibition of miR-124-5p could enhance the cellular function of HUVECs and promote the release of inflammatory factors.

Figure 2 Inhibition of miR-124-5p can enhance the cell function of HUVECs and promote the release of inflammatory factors. In this group of experiment, HUVECs were treated differently and divided into four groups: Control group; IL-1β group; IL-1β + miR-124-5p-inhibitor-NC; IL-1β + miR-124-5p-inhibitor. The transfection of miR-124-5p-inhibitor decreased the expression of miR-124-5p, and miR-124-5p-inhibitor-NC was the negative control. (A) The changes in miR-124-5p expression in HUVECs were detected by reverse transcription-quantitative PCR. The (B) invasion and (C) migration abilities of HUVECs were detected by Transwell assay. Scale bar, 50 µm. (D) The changes in the expression of ICAM-1 and NF-κB in HUVECs were detected by western blotting. GAPDH was used as the internal reference. The expression of (E) IL-6, (F) IL-8 and (G) TNF-α in HUVECs was detected by enzyme-linked immunosorbent assay. **P<0.01, ***P<0.001, ****P<0.0001 vs. control; #P<0.05, ###P<0.001, ####P<0.0001, vs. IL-1β + miR-124-5p-inhibitor-NC. miR, microRNA; NC, negative control; IL-1β/6/8, interleukin-1β/6/8; TNF-α, tumor necrosis factor-α; ICAM1, intercellular adhesion molecule; NF-κB, nuclear factor-κB.

FoxO1 is the target gene of miR-124-5p

It was predicted that FoxO1 is the target gene of miR-124-5p by using the TargetScan website (Fig. 3A); this was verified using dual luciferase reporter and western blotting assays. The results of the dual luciferase assay showed decreased luciferase activity in the FoxO1 3'-UTR wild-type group, following transfection with miR-124-5p-mimics, compared with the miR-124-5p-mimics-NC group, but there was no significant change in the FoxO1 3'-UTR mutant group (Fig. 3B). Results of western blotting displayed that the miR-124-5p-inhibitor increased the expression of FoxO1, while miR-124-5p-mimics decreased the expression of FoxO1 (Fig. 3C). The results of RT-qPCR also showed that the miR-124-5p-mimics group had lower expression of FoxO1 compared with the control group, and the miR-124-5p-inhibitor group had the opposite results (Fig. 3D). All the experimental results indicated that FoxO1 was the target gene of miR-124-5p, and miR-124-5p could negatively regulate FoxO1.

Figure 3 FoxO1 is the target gene of miR-124-5p. (A) The binding sites of miR-124-5p and FoxO1 obtained from the TargetScan website. (B) Dual luciferase reporter assay was used to detect whether wild-type-FoxO1 is the target gene of miR-124-5p, and the mutant-FoxO1 was the control. miR-124-5p-mimics were used to simulate the high-level expression of endogenous mature miR-124-5p in cells, and the miR-124-5p-mimics-NC was the negative control. (C) Western blotting was applied to detect whether the expression of FoxO1 in HUVECs changed with the expression of miR-124-5p, and the quantitative analysis of protein bands was also implemented by using Image J. GAPDH was used as the internal reference. (D) The changes in relative mRNA level of FoxO1 was detected by reverse transcription-quantitative PCR. *P<0.05, **P<0.01 vs. miR-124-5p-mimics-NC; #P<0.05, ##P<0.01 vs. miR-124-5p-inhibitor-NC. miR, microRNA; NC, negative control; UTR, untranslated region.

miR-124-5p regulates the cell migration and invasion, as well as the release of inflammatory factors, through mediating FoxO1 expression

A series of experiments were conducted to verify the interaction of FoxO1 with miR-124-5p in HUVECs. The results of western blotting showed that the upregulated expression of FoxO1 induced by miR-124-5p-inhibitor was weakened by the si-FoxO1 (Fig. 4A). The results of cell invasion and migration experiments indicated that inhibition of miR-124-5p promoted the invasion and migration of HUVECs, which was weakened after the cells were co-transfected with miR-124-5p-inhibitor and si-FoxO1 (Fig. 4B and C). At the same time, the increase in NF-κB, ICAM-1, IL-6, IL-8 and TNF-α that was induced by miR-124-5p-inhibitor were downregulated by FoxO1 knockdown (Fig. 4A, D-F). All the experimental results showed that inhibition of miR-124-5p promoted the cell function and the release of inflammatory factors of HUVECs, which could be reversed by FoxO1 knockdown. Hence, miR-124-5p regulated the cell function and the release of inflammatory factors of HUVECs through mediating FoxO1 expression.

Figure 4 miR-124-5p regulates cell migration and invasion, as well as the release of inflammatory factors, through mediating FoxO1 expression. (A) The changes in the expression levels of FoxO1, ICAM-1 and NF-κB in HUVECs after transfecting with siRNA-FoxO1 was detected by western blotting. GAPDH was used as the internal reference. The changes in (B) invasion and (C) migration abilities of HUVECs were detected by using Transwell assays after FoxO1 knockdown. Scale bar, 50 µm. The changes in (D) IL-6, (E) IL-8 and (F) TNF-α of HUVECs were detected by enzyme-linked immunosorbent assay after FoxO1 knockdown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. control; ##P<0.01, ###P<0.001, ####P<0.0001 vs. IL-β; $$P<0.01, $$$P<0.001, $$$$P<0.0001 vs. IL-1β + miR-124-5p-Inhibitor-si-NC. miR, microRNA; NC, negative control; TNF-α, tumor necrosis factor-α; ICAM1, intercellular adhesion molecule; NF-κB, nuclear factor-κB; siRNA/si-, small interfering RNA; IL-6/8, interleukin-6/8.

Discussion

The present study demonstrated that the expression of miR-124-5p in patients with CA and in HUVECs that were stimulated by IL-1β was lower compared with that in healthy individuals or cells. The lower expression of miR-124-5p could promote the ability of cell invasion, migration and the inflammatory response of HUVECs. Then, FoxO1 was predicted as a target gene of miR-124-5p. The inhibition of miR-124-5p promoted the cell migration, cell invasion and the release of inflammatory factors, which could be reversed by FoxO1 knockdown. Hence, it was speculated that miR-124-5p could delay the progression of CA by inhibiting the expression of FoxO1.

The environmental and genetic factors of the formation of CA have been reported, but the pathological factors of its occurrence and development have not been fully acquainted (33). Furthermore, the most recognized factors that promote the development of CA are inflammation and hemodynamics (34). NF-κB is one of the key components to regulate the expression of proinflammatory genes and it induces the expression of proinflammatory cytokines, chemokines and adhesion molecules (35). It has been reported that the expression of NF-κB in CA is significantly higher compare with normal tissues and the activation of NF-κB is a risk factor in the occurrence and development of CA (36-38). In inflammatory reaction, endotoxin and some chemokines can promote the expression of ICAM-1 in endothelial cells and leukocytes in the wall of the porch (39). Furthermore, the activated leukocytes adhere to the vascular wall by ICAM-1, and then pass through the vessel wall and enter the focus of infection (39). TNF-α plays a role in vascular inflammation when the vascular inflammation is closely associate with the progression of aneurysm (40-42). Some studies have confirmed that hemodynamic stress increases the expression of TNF-α, and TNF-α inhibitors can inhibit the formation of aneurysm and delay the disease process of aneurysm (42,43). Studies have shown that cytokines such as IL-6 and IL-8 can downregulate procollagen biosynthesis at the transcription level, while the decrease in arterial wall collagen is a significant pathological feature of CA (44). Since the aforementioned inflammatory factors are closely associated with the occurrence and process of CA, the present study examined the changes in these inflammatory factors under various experimental treatments.

Previous studies have shown that miRNAs can affect multiple signaling pathways to regulate the cell function, such as the ability of migration and invasion of cancer cells (45-48). However, according to the research of miR-124-5p in diseases, the effect of miR-124-5p on the ability of migration and invasion for glioblastoma multiforme cells is not obvious (27). Because the influence of miR-124-5p on the cell function of CA cells has not been studied, the present study attempted to study about CA by using HUVECs. The results indicated that the expression level of miR-124-5p in patients with CA or in cells was lower compared with that in normal individuals or cells. It was also found that the miR-124-5p inhibitor increased the ability of cell migration and invasion of HUVECs stimulated by IL-1β. It has been reported that miRNAs can regulate the expression of a variety of inflammatory factors; for example, Wu et al (49) found that miR-124-5p can relieve the cerebral injury that is caused by inflammation by downregulating the expression of IL-6 and TNF-α. The present study further indicated that the decrease in miR-124-5p could increase the release of inflammatory factors such as IL-6, IL-8, TNF-α, ICAM-1 and NF-κB.

In order to further explore the molecular mechanism of miR-124-5p in CA, target gene FoxO1 of miR-124-5p was identified by using the TargetScan website. Previous studies have shown that FoxO1 knockdown can inhibit inflammatory response; for example, Malik et al (50) found that FoxO1 knockdown can decrease the expression of IL-1β, thus, relieving the symptoms of diabetic retinopathy, and Wu et al (51) found that the allergic inflammation in asthmatic patients can be lowered by the decrease in IL-9 that is regulated by knocking down FoxO1. The present results indicated that inhibition of miR-124-5p enhanced the release of inflammatory factors (IL-6, IL-8, TNF-α, NF-κB and ICAM-1), which was reversed by FoxO1 knockdown. Thus, the present study showed the inhibition on the inflammation caused by FoxO1 knockdown is in agreement with the previous reports. Furthermore, the promotion of migration and invasion of HUVECs induced by miR-124-5p inhibitors was weakened by the introduction of FoxO1 siRNA. Therefore, miR-124-5p regulated the migration and invasion abilities and the release of inflammatory factors of HUVECs through mediating FoxO1 expression.

The present study focused on the regulation of miR-124-5p and FoxO1 on the cellular function and the release of inflammatory factors by HUVECs in vitro, however, whether miR-124-5p/FoxO1 pathway is also associated with inflammatory process or cellular function in CA animal models is the focus of the follow-up study. In addition, whether factors (such as matrix metalloproteinases) associated with CA in vivo or in vitro are regulated by miR-124-5p/FoxO1 pathway will also be studied in the future.

In conclusion, miR-124-5p was able to inhibit the cellular function and the release of inflammatory factors of HUVECs by downregulating the expression of FoxO1, which might prevent the occurrence of CA or delay the development of CA. Therefore, the present results provide the potential biomarkers and signaling pathway for the treatment of CA, and supply another way to explore the molecular mechanism of the occurrence and development of CA.

Supplementary Material

Screening of specific siRNA and detection of transfection efficiency. (A) Western blotting and (B) reverse transcription-quantitative PCR assays were used to detect the specific siRNA for FoxO1, and (A) the quantification of proteins was performed by GraphPad Prism. **P<0.01, ***P<0.001, ****P<0.0001 vs. control; ##P<0.01, ###P<0.001, ####P<0.0001 vs. si-NC. siRNA/si-, small interfering RNA; NC, negative control.

miR-124-5p-mimics/inhibitors were successfully transfected into the cells. Reverse transcription-quantitative assay was used to detect the transfection efficiency of (A) miR-124-5p-mimics and (B) miR-124-5p-inhibitors. **P<0.01, ****P<0.0001 vs. control; ##P<0.01, ####P<0.0001 vs. mimic-NC/inhibitor-NC. miR, microRNA; NC, negative control.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

RKW and GYY designed experiments; YYS, GYL and HTY collected data; XJL, KFL and XZ analyzed experimental results. RKW wrote the manuscript; GYY approved the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The institutional ethics committee of Handan Central Hospital has approved this study. Written informed consent was obtained from all participants and their families.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References