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Introduction

Sepsis is a systemic inflammatory disease caused by severe trauma, burn, infection and major surgery (1–3). Sepsis is often accompanied by multiple organ failure (4). Sepsis-induced excessive inflammation, immunosuppression or excessive tissue damage may increase susceptibility to secondary infection (5). Therefore, the molecules and mechanisms associated with sepsis-induced inflammatory response are important to explore. Septic shock is associated with half of patients with septic myocarditis (6); inflammatory cytokines play an important role in this process. Among them, tumor necrosis factor (TNF-α) inhibits myocardial contractility, which results in cardiac dysfunction (7).

Long non-coding RNA (lncRNA) plays an important regulatory role in the occurrence and development of inflammatory response, rheumatoid arthritis, vascular aging and cancer (8,9). LncRNA IL-1β7R is involved in the inflammatory response induced by bacterial endotoxin lipopolysaccharide (LPS) (7). LncRNA HOTAIR promotes TNF-α production in mice with LPS-induced sepsis (4). FGD5-AS1 has low expression level in periodontitis, and FGD5-AS1 overexpression could inhibit the development of periodontitis (10). However, reports on the mechanism of action of FGD5-AS1 and its role in sepsis are few.

MicroRNAs (miRNAs/miRs) are single-stranded endogenous non-coding RNAs with a length of 18–25 nt (11). miRNAs are involved in gene expression, cell development, differentiation and other processes. miRNAs also play an important role in autoimmune diseases (12,13). Feng et al (14) found that miRNA plays an important role in the development of liver fibrosis. miR-133a-3p belongs to the myocyte-specific miR-206 family and inhibits the proliferation and differentiation of myocytes (15). In addition, miR-133a has been identified as a tumor suppressor gene in several tumors, such as colorectal cancer, ovarian cancer, breast cancer and bladder cancer (16–18). However, the role of miR-133a-3p in sepsis has been rarely reported. Aquaporin 1 (AQP1) belongs to a small-molecule transmembrane protein family, which is involved in the rapid transmembrane transport of water (19). AQP1 is one of the earliest identified members and expressed in erythrocyte membrane and vascular endothelial cells (20). AQP1 plays an important role in cell migration, differentiation, proliferation and ion transport (21). AQP1 is a channel for the exchange of intracellular and extracellular water and the transport and exchange of oxygen in erythrocytes (22). The function of AQP1 is not only limited to the membrane aquaporin, and its abnormal expression is closely associated with the occurrence and development of a variety of common diseases (23–25).

In the present study, LPS was used to establish animal and cell models of sepsis, and the expression levels of FGD5-AS1, miR-133a-3p and AQP1 in cells were detected; their effect on sepsis and mechanism of action were also explored. The present study lays a theoretical foundation for further revealing the molecular mechanism of sepsis occurrence and development.

Materials and methods

Establishment of animal models of sepsis

A total of 36 female BALB/C mice (weight, 25–30 g; age, 4–6 weeks old) were purchased from Charles River Co., Ltd. Six mice were included in each group. The mice were raised in a sterile environment at a room temperature of 26–28°C, a humidity of 50–60%, and illumination time of 10 h. The mice had free access to autoclaved water and sterile food. The experiment was conducted after 1 week of adaptive feeding. The control group was intraperitoneally injected with 5 ml/kg sterile saline. The model group was intraperitoneally injected with 15 mg/kg LPS (a bacterial endotoxin, Escherichia coli LPS serotype 0111:B4; Sigma-Aldrich; Merck KGaA). Len-NC and Len-FGD5-AS1 (1×109 PFU/ml), were injected into the tail vein after 24 h of modeling. The control group was given an equal volume of sterilized saline. All indexes were detected 24 h after caudal vein administration. A 0.3% pentobarbital sodium solution was given at a dose of 50 mg/kg to anesthetize the mice. Then, 0.2 ml of blood was collected from the orbital vein, and the upper serum was collected after centrifugation (4,000 × g, 10 min, 4°C). The mice were euthanized with carbon dioxide immediately after blood extraction while still under anesthesia. The euthanasia chamber was filled with CO2 at a rate of 20% of the volume of the euthanasia chamber per minute after the mice were placed in the chamber. The mice were not moving or breathing, and their pupils were dilated when the administration of CO2 was stopped. The mice were watched for another 2 min to confirm their death. The animal experiments were approved by the Ethics Committee of Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital (Tianjin, China; approval no. 20190135). The animal experiments were conducted between May 25 and June 14, 2019.

Lentiviral vector packaging

RNAi lentiviral recombinant vector system, including Len-FGD5-AS1 vector with green fluorescent protein (GFP), pHelper 1.0 vector and pHelper 2.0 vector and negative control lentivirus (Len-NC). All plasmid vectors were purchased from Shanghai GeneChem Co., Ltd. 293T cells in logarithmic growth phase were inoculated into the culture dish at a cell number of 6×106/ml, and placed in an incubator for 24 h (CO2, 37°C). When the cell confluence reached 70–80%, each DNA solution (plasmid vector, helper plasmid vector pHelper 1.0, pHelper 2.0) and Lipofectamine® 2000 liposome were added for co-transfection. After 48 h, the supernatant of 293T cells was collected and centrifuged at 4,000 × g at 4°C for 10 min. The supernatant was then filtered using a 0.45-µm diameter filter and packed. The 96-well plate was inoculated with 4×104 cells per well. The virus was added into 8 experimental wells at a total volume of 100 µl by multiple dilution. Then, 24 h later, puromycin was added for screening, and the final concentration was 2.7 µg/ml. When the cell density reached 30%, lentivirus-transfected cells were transfected at a MOI of 30. A total of 16 h after lentivirus transfection, the solution was changed, and the downstream experiment was carried out after 96 h.

Cardiac function detection in mice

Cardiac function was evaluated using transthoracic Doppler ultrasound. Mice were prematurely fasted and weighed using a GE Vivid E9 ultrasound system (GE Healthcare). Mice were anesthetized to ideal depth and maintained with isoflurane (3% induction and 1–2% maintenance). The mice were fixed in supine position on a heating mat to maintain their body temperature, and the skin was prepared on the chest area. Measurement data: B-mode ultrasound was selected, and the left ventricle short axis image and the left ventricle long axis image were obtained at the left ventricle middle level using a high-frequency probe. Ejection fraction (EF%) and left ventricular fraction shorting rate (FS%) were measured under M-mode ultrasound. The data were measured three times, averaged and recorded.

Cell culture

Mice HL-1 cardiac muscle cells were purchased from the American Type Culture Collection. The cells were cultured in Dulbeccos modified Eagles medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher scientific, Inc.) with 100 U/ml penicillin and 100 µg/ml streptomycin (Thermo Fisher Scientific, Inc.). The cells were precultured at 37°C with 5% CO2 for 24 h. LPS intervention was performed in some cells. These cells were plated in a 6-well plate (2×106) and cultured for 48 h, and then 1 g/ml LPS was added to the medium. Normal saline was added to the control group. The cells were cultured for 12 h under the same conditions. The harvested cells were used in subsequent experiments.

Cell transfection

The FGD5-AS1-pcDNA3.1-overexpression plasmid [vector-FGD5-AS1; OBiO Technology (Shanghai) Corp., Ltd.] was constructed in strict accordance with the manufacturers instructions. A total of 4 µg vector-FGD5-AS1 and its negative control (vector-NC) were transfected into LPS-induced cells using Lipofectamine® 3000 (Thermo Fisher Scientific, Inc.) under 37°C. The cells were collected after 48 h of transfection, and transfection efficiency was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). After 48 h of transfection, follow-up experiments were performed. Small interfering RNAs (siRNAs) targeting FGD5-AS1 (100 nM, 5′-CAUUUGUAAUAGUGUUCAAUA-3′) and si-NC (100 nM, 5′-UUCUCCGAACGUGUCACGUTT-3′) were synthesized by Shanghai GenePharma Co., Ltd., and transfected using Lipofectamine® 3000 (Thermo Fisher Scientific, Inc.). Vector-FGD5-AS1 (1 µg) and miR-133a-3p (100 nM) or sh-AQP1 (1 µg; Shanghai GenePharma Co., Ltd.) were co-transfected using Lipofectamine 3000. After 48 h of transfection, follow-up experiments were performed. Cells were treated with 100 nM mimic control (5′-CAGCUGGUUGAAGGGGACCAAA-3′) and 100 nM miR-133a-3p mimic (5′-UUUGGUCCCCUUCAACCAGCUG-3′). miRNAs were purchased from Shanghai GenePharma Co., Ltd.

Enzyme-linked immunosorbent assay (ELISA)

Blood was drawn from the orbital venous plexus and centrifuged at 4°C for 10 min (4,000 × g). The serum was separated and stored in the refrigerator at 80°C for later use. The contents of TNF-α (cat. no. BMS607-3; Thermo Fisher Scientific, Inc.), interleukin (IL)-6 (cat. no. BMS603-2; Thermo Fisher Scientific, Inc.), and IL-1β (cat. no. BMS6002; Thermo Fisher Scientific, Inc.) in serum were detected by ELISA. ELISA was performed strictly in accordance with the manufacturers instructions of the kits.

RT-qPCR

RNA from tissue or cell samples was isolated using TRIzol® reagent (Thermo Fisher Scientific, Inc.). RNA was reverse transcribed into cDNA using the PrimeScript One Step RT-PCR kit (Takara Biotechnology Co., Ltd.), according to the manufacturers protocol. The reaction conditions for RT were as follows: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min. The following primers were used in the present study: miR-133a-3p forward, 5′-ACACTCCAGCTGGGTTGGTCCCCTTCAACC-3′ and reverse, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACAGCTGG-3′; AQP1 forward, 5′-ACCTCCTGGCTATTGACTAC-3′ and reverse, 5′-CCAGGATGAAGTCGTAGATG-3′; Bcl-2 forward, 5′-ATGCCTTTGTGGAACTATATGGC-3′ and reverse, 5′-GGTATGCACCCAGAGTGATGC-3′; and Bax forward, 5′-TGAAGACAGGGGCCTTTTTG-3′ and reverse, 5′-AATTCGCCGGAGACACTCG-3′. U6 RNA and GAPDH were used as the internal references. The primer sequences were as follows: U6 forward, 5′-CTCGCTTCGGCAgcacA-3′ and reverse, 5′-aACGCttcacgaatttGCGT-3′; and GAPDH forward, 5′-GAGTCAACGGATTTGGTCGT-3′ and reverse, 5′-TTgatttTGGATCTCG-3′. The fluorescence quantitative detection conditions were as follows: Pre-denaturation at 95°C for 30 sec, amplification at 95°C with extension for 15 sec, and 40 cycles of annealing at 60°C for 30 sec. The 2−ΔΔCq (26) was calculated as follows: ΔCq (experimental group)=Cq (experimental group target genes)-Cq (experimental group internal genes); ΔCq (control group)=Cq (control target gene)-Cq (control group).

Western blotting

HL-1 cells were collected after different treatments and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology), 100 µM PMSF (X100; Sigma-Aldrich; Merck KGaA) and a protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). The lysed solution was centrifuged (12,000 × g) at 4°C for 60 min and then at 12,000 × g for 20 min. PBS washing and precipitation were performed twice. Protein concentration was determined with the Bradford method. Then, 10 µg protein was added into 10% sodium dodecyl sulfate-polyacrylamide gel and subjected to electrophoresis. Subsequently, the gel was transferred to a polyvinylidene difluoride membrane, the membrane was then blocked with 5% skimmed milk powder at room temperature for 2 h. Following which, the membranes were incubated at 4°C overnight with primary antibodies against the following: Bax (1:1,000; cat. no. 89477; Cell Signaling Technology, Inc.), Bcl-2 (1:1,000; cat. no. 15071; Cell Signaling Technology, Inc.), GAPDH (1:2,000; cat. no. 97166; Cell Signaling Technology, Inc.) and AQP1 (1:1,000; cat. no. ab9566; Abcam). Then, horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. nos. 31430 and 31460; Thermo Fisher Scientific, Inc.) were added to the membrane and incubated at room temperature for 2 h. Chemiluminescence (ECL kit; Cytiva) was used to detect the target bands. After the strips were scanned, the optical density of the strips was determined using QuantityOne version 4.3.0 software (Bio-Rad Laboratories, Inc.). The relative expression level of each sample was calculated using GADPH as the internal reference.

Cell counting Kit-8 (CCK-8) experiments

Cell viability was detected by the CCK-8 method. The cells were made into a single cell suspension and inoculated at a density of 1×105/well to a 6-well plate. The cells were randomly grouped when cell confluence reached 60–70%. The control group was added with the corresponding volume of solvent. The LPS group was treated with 10 µg/ml LPS for 12 h. Lipofectamine® 2000 was used to transfect the target gene plasmid in the transfection group. The cells were incubated at 37°C with 5% CO2 for 48 h. CCK-8 experiment was performed after 48 h of cell treatment. CCK-8 reagent (Beyotime Institute of Biotechnology) was added to each well, and culture was continued for 4 h. The optical density at 490 nm was measured with a microplate reader.

Dual-luciferase reporter gene assay

StarBase version 2.0 (http://starbase.sysu.edu.cn/) online prediction software was used to predict the lncRNA-targeted miRNAs. TargetScan version 7.1 (http://www.targetscan.org/) online prediction software was used to predict the miRNA target genes. The interaction of the FGD5-AS1, miR-133a-3p and AQP1 cascade reaction was detected using a Dual-Luciferase Reporter Assay System (Promega Corporation). Wild-type (WT) and mutant (MT) 3UTR were designed and amplified using Primer Premier 5.0 primer design software (Premier Biosoft International). XhoI and NotI were introduced into the 5 end of the WT forward primer and reverse primer, respectively. The WT and MT recombinant plasmids were constructed by ligating the vector psiCHECK™-2 with XhoI and NotI. Human 293T cells (American Type Culture Collection) were transfected with 100 nmol/µl miR-133a-3p mimics (5′-UUUGGUCCCCUUCAACCAGCUG-3′) and its negative control (miR-NC; 5′-UUGUACUACACAAAAGUACUG-3′), 20 ng WT FGD5-AS1 (FGD5-AS1-WT) and MT FGD5-AS1 (FGD5-AS1-MT), or 20 ng 3-untranslated region (UTR) of WT AQP1 (AQP1-WT) and MT AQP1 (AQP1-MT) with Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Luciferase activity was detected 48 h after transfection (Promega Corporation). According to the requirements of the kit instructions, the ratio of Firefly/Renilla luciferase activity was calculated. The unit of the control group ratio was one. The relative luciferase activity of different treatment groups was obtained.

miRNA pull-down experiment

miR-133a-3p mimic (biotinylated miR-133a-3p or miR-NC probe) with biotinylated modification was synthesized at the 3 ends. A random sequence was used as a control. Transfection with miR-133a-3p probe (1 µg; Sangon Biotech Co., Ltd.) was performed, and cells were harvested after 48 h. The cells were washed with PBS and added with lytic extract (20 mM Tris, pH 7.5; 100 mM KCl; 5 mM MgCl2; 0.5% NP-40; and 1 U/µl recombinant RNAse inhibitor). Cell fragments were removed by centrifugation after lysis (4°C, 12,000 × g, 10 min). DNase l was added to the lysate to digest the DNA. Afterward, the lysates were heated to 65°C in a metal bath for 5 min and then quickly plunged into ice to cool. The lysates and 25 µl avidin-coated magnetic beads (New England BioLabs, Inc.) were mixed and incubated at 4°C for 4 h with gentle shaking. After incubation, the beads were washed twice with the lysis buffer. TRIzol was used to extract the RNA bound to the magnetic beads for RT-qPCR analysis. Cell lysate was used as a control (input group).

Statistical analysis

All data are expressed as the mean ± standard deviation. Comparison between two groups was performed using an unpaired Students t-test. Differences between two groups were calculated using one-way analysis of variance (ANOVA) followed by Tukeys multiple comparison test. All data were statistically analyzed using SPSS 19.0 (SPSS, Inc.). Results were obtained from three independent experiments. P<0.05 was used to indicate a statistically significant difference.

Results

FGD5-AS1 is downregulated in sepsis

A septic animal model was established to investigate the role of FGD-AS1 in sepsis. Experimental results showed that FGD5-AS1 was downregulated in the septic animal model (Fig. 1A). Furthermore, a septic cardiomyocyte model was established using LPS. It was found that FGD5-AS1 was downregulated in the septic cell model (mouse HL-1 cells, Fig. 1B).

Figure 1. FGD5-AS1 is downregulated in sepsis. (A) FGD5-AS1 was downregulated in animal models of sepsis (arterial tissue of septic mice) (n=6). (B) FGD5-AS1 was downregulated in the sepsis cell model (mouse cardiomyocyte UL-1 cells). Results were obtained from three independent experiments, each performed in triplicate, the error bars represent SD. **P<0.01. LPS, lipopolysaccharide.

Lentivirus overexpression of FGD5-AS1 can inhibit sepsis

It was investigated whether FGD5-AS1 overexpression has a protective effect on sepsis. FGD5-AS1 was overexpressed in an animal model of sepsis by the lentivirus technique. The experimental results showed that the lentivirus overexpression system could upregulate the expression of FGD5-AS1 (Fig. 2A). Inflammatory factor detection results showed that the concentrations of TNF-α, IL-1β and IL-6 in the sepsis model group were significantly higher compared with those in the control group (P<0.01). The concentrations of TNF-α, IL-1β and IL-6 in the sepsis model + Len-FGD5-AS1 group was significantly decreased compared with the sepsis model + Len-NC group (P<0.01; Fig. 2B-D). Furthermore, it was found that FGD5-AS1 overexpression protected heart functions in septic mice. The ejection fraction, fractional shortening and the maximum [LVdP/dt (max)] and minimum rates of the rise in left ventricular pressure [LVdP/dt (min)] were significantly decreased in the sepsis model group compared with the control group (P<0.01; Fig. 2E-H). Ejection fraction, fractional shortening, LVdP/dt (max), and LVdP/dt (min) were significantly higher in the sepsis model + Len-FGD5-AS1 group compared with the sepsis model + Len-NC group (P<0.01; Fig. 2E-H).

Figure 2. Lentiviral overexpression of FGD5-AS1 can inhibit sepsis. (A) Lentiviral overexpression of FGD5-AS1 could inhibit FGD5-AS1 expression in sepsis model mice. Detection of (B) IL-6, (C) TNF-α and (D) IL-1β activity in serum. (E) Detection of changes in mouse heart index (EF%). (F) Detection of changes in FS% of mouse heart index. (G) Detection of changes in mouse heart index LVdP/dt(max)(mmHg/sec). (H) Detection of changes in mouse heart indicators LVdP/dt(min)(mmHg/sec). n=6. Data are represented as the means ± SD. **P<0.01. Len-, lentivirus; IL, interleukin; TNF, tumor necrosis factor; EF, ejection fraction; FS, fractional shortening; NC, negative control; max, maximum; min, minimum; LVdP, left ventricular pressure.

FGD5-AS1 overexpression decreases LPS-induced HL-1 cell injury in vitro

A cell model was established by LPS, and the transfection efficiency of vector-FGD5-AS1 was first detected. Experimental results showed that vector-FGD5-AS1 could upregulate the expression of FGD5-AS1 (Fig. 3A). Subsequently, the effect of vector-FGD5-AS1 on cell viability was examined. The experimental results showed that transfection with vector-FGD5-AS1 could upregulate the viability of HL-1 cells compared with the vector-NC group (Fig. 3B). In addition, vector-FGD5-AS1 could inhibit Bax expression and upregulate Bcl-2 expression (Fig. 3C and D). The influence of FGD5-AS1 on the protein expression levels of Bax and Bcl-2 was further analyzed. The western blot analysis results were consistent with the RT-qPCR results; that is, vector-FGD5-AS1 transfection could inhibit Bax expression and upregulate Bcl-2 expression (Fig. 3C and D). Previous studies have shown that LPS treatment increases the levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β (27–29). Transfection with vector-FGD5-AS1 in the cell models reverted LPS-induced changes in TNF-α, IL-6 and IL-1β levels (Fig. 3E-G).

Figure 3. Overexpression of FGD5-AS1 decreases LPS-induced HL-1 cell damage. (A) Vector-FGD5-AS1 transfection efficiency test under LPS treatment conditions. (B) Cell Counting Kit-8 was performed to detect cell viability. Reverse transcription-quantitative PCR and western blotting was conducted to detect changes in (C) Bax expression and (D) Bcl-2 expression in cells after different treatments. Detection of (E) IL-6 expression, (F) TNF-α expression and (G) IL-1β activity in cells. Results were obtained from three independent experiments, each performed in triplicate, the error bars represent SD. *P<0.05, **P<0.01. NC, negative control; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor.

Regulation and interaction of FGD5-AS1 on miR-133a-3p

lncRNA-targeted miRNA prediction analysis using StarBase showed that FGD5-AS1 has binding sites with miR-133a-3p (Fig. 4A). Dual-luciferase reporter gene validation results showed that miR-133a-3p significantly inhibited the luciferase activity of FGD5-AS1-WT (P<0.01). However, miR-133A-3p had no effect on the luciferase activity of FGD5-AS1-MT (Fig. 4B). miRNA pull-down also verified the interaction between FGD5-AS1 and miR-133a-3p (Fig. 4C). Subsequently, the effect of FGD5-AS1 on the expression of miR-133a-3p was detected. Following transfection with si-FGD5-AS1, FGD5-AS1 expression was found to be downregulated compared with the si-NC group, whereas transfection with vector-FGD5-AS1 increased the expression levels of FGD5-AS1 compared with the vector-NC group (Fig. 4D). Furthermore, the experimental results showed that transfection with vector-FGD5-AS1 inhibited miR-133a-3p expression, whereas si-FGD5-AS1 transfection led to the upregulation of miR-133a-3p (Fig. 4E). miR-133a-3p was also upregulated in septic animal models compared with the control group (Fig. 4F).

Figure 4. FGD5-AS1 acts as the competitive endogenous RNA of miR-133a-3p. (A) Schematic diagram of the binding site of FGD5-AS1 and miR-133a-3p. (B) The dual-luciferase reporter gene detection experiment verified the binding of FGD5-AS1 and miR-133a-3p. (C) miRNA pull-down assay verified the binding of FGD5-AS1 and miR-133a-3p. (D) Verification of transfection efficiency of overexpression and knockdown of FGD5-AS1. (E) Effects of FGD5-AS1 knockdown and overexpression on miR-133a-3p expression. (F) miR-133a-3p expression was upregulated in animal models of sepsis. Results were obtained from three independent experiments, each performed in triplicate, the error bars represent SD. **P<0.01. miRNA/miR, microRNA; WT, wild-type; MT, mutant; NC, negative control; si-, small interfering RNA.

miR-133a-3p overexpression reverses the protective effect of FGD5-AS1 on HL-1 cells

After confirming the regulatory effect of FGD5-AS1 on miR-133a-3p, the effect of miR-133a-3p on the function of FGD5-AS1 was detected. The viability of HL-1 cells was measured by CCK-8 assay. The results showed that the viability of HL-1 cells was decreased in the LPS-stimulated group compared with the control group. Transfection with vector-FGD5-AS1 could upregulate the viability of HL-1 cells compared with the LPS group. However, the viability of HL-1 cells was decreased after the co-transfection of vector-FGD5-AS1 and miR-133a-3p mimics (Fig. 5A). The Bax expression detection results showed that, compared with the LPS group, transfection with vector-FGD5-AS1 could inhibit Bax expression. However, Bax expression was upregulated after the co-transfection of vector-FGD5-AS1 and miR-133a-3p mimics (Fig. 5B). The trend in Bcl-2 expression changes was opposite to that of Bax. FGD5-AS1-vector transfection upregulated the expression of Bcl-2 (Fig. 5C). LPS stimulation upregulated the expression of inflammatory cytokines IL-6, TNF and IL-1β compared with the control group. Transfection with vector-FGD5-AS1 decreased the levels of inflammatory factors compared with the LPS group. However, the inflammatory factors were upregulated after the co-transfection of vector-FGD5-AS1 and miR-133a-3p mimics (Fig. 5D-F).

Figure 5. Overexpression of miR-133a-3p reverses the protective effect of FGD5-AS1 on HL-1 cells. (A) Cell Counting Kit-8 detection of cell viability. Reverse transcription-quantitative PCR was performed to detect the expression of (B) Bax, (C) Bcl-2, (D) IL-6, (E) TNF-α and (F) IL-1β. Results were obtained from three independent experiments, each performed in triplicate, the error bars represent SD. **P<0.01. miR, microRNA; IL, interleukin; TNF, tumor necrosis factor; NC, negative control; LPS, lipopolysaccharide.

AQP1 acts as the target gene mediating miR-133a-3p expression

The complementary binding sites of miR-133a-3p and AQP1-3′-UTR-WT were predicted by TargetScan (Fig. 6A). Dual-luciferase reporter gene detection results showed that the relative luciferase activity of AQP1-WT + miR-133a-3p mimics group was significantly decreased compared with the AQP1-WT + miR-NC group (Fig. 6B). These results indicated that miR-133a-3p could inhibit luciferase activity by binding to AQP13-UTR. However, the relative luciferase activity of the AQP1-MT + miR-133a-3p mimics group was not significantly different from that of the AQP1-MT + miR-NC group. These results indicated that miR-133a-3p and miR-NC could not inhibit the luciferase activity of the mutant plasmid. miRNA pull-down also verified the binding of miR-133a-3p to AQP1 (Fig. 6C). Subsequently, the effect of miR-133a-3p on the expression level of AQP1 was detected. Transfection with the miR-133a-3p mimics upregulated the expression of miR-133a-3p compared with the mimics-NC group, whereas transfection with the miR-133a-3p inhibitor led to the downregulation of miR-133a-3p expression compared with the inhibitor-NC (Fig. 6D). The experimental results showed that transfection with the miR-133a-3p mimics inhibited AQP1 expression, whereas AQP1 expression was upregulated in the miR-133a-3p inhibitor group (Fig. 6E). Transfection with vector-FGD5-AS1 upregulated AQP1 expression compared with the vector-NC group, whereas transfection with si-FGD5-AS1 led to the downregulation of AQP1 expression compared with the si-NC group (Fig. 6F). The western blotting results were consistent with the RT-qPCR results (Fig. 6F). AQP1 was downregulated in septic animal models (Fig. 6G).

Figure 6. AQP1 as a target gene that mediates miR-133a-3p. (A) Schematic diagram of the binding between miR-133a-3p and AQP1. (B) The dual-luciferase reporter assay was performed to detect the binding of miR-133a-3p to AQP1. (C) miRNA pull-down assay verified the binding of miR-133a-3p to AQP1. (D) Verification of transfection efficiency of miR-133a-3p mimics and inhibitors. (E) Effects of miR-133a-3p knockdown and overexpression on the expression of AQP1. (F) Effects of FGD5-AS1 overexpression and knockdown on the expression of AQP1. (G) AQP1 was downregulated in the sepsis model. Results were obtained from three independent experiments, each performed in triplicate, the error bars represent SD. **P<0.01. AQP1, aquaporin 1; UTR, untranslated region; miR/miRNA, microRNA; WT, wild-type; MT, mutant; NC, negative control; si-, small interfering RNA.

AQP1 knockdown reverses the protective effect of FGD5-AS1 on HL-1 cells

RT-qPCR was used to detect the change in AQP1 expression (Fig. S1). FGD5-AS1-vector transfection could upregulate AQP1 expression, but the expression of AQP1 was decreased after the addition of sh-AQP1 (Fig. 7A). The viability of HL-1 cells was measured by CCK-8 assay. The results showed that the viability of HL-1 cells was decreased in the LPS-stimulated group compared with the control group. Transfection with vector-FGD5-AS1 could upregulate the viability of HL-1 cells compared with the LPS group. However, the viability of HL-1 cells decreased after the co-transfection of vector-FGD5-AS1 + sh-AQP1 (Fig. 7B). The Bax expression detection results showed that, compared with the LPS group, vector-FGD5-AS1 transfection could inhibit Bax expression. However, Bax expression was upregulated after the co-transfection of vector-FGD5-AS1 + sh-AQP1 (Fig. 7C). The change in Bcl-2 expression was opposite to that of Bax. Vector-FGD5-AS1 transfection upregulated Bcl-2 expression (Fig. 7D). LPS stimulation upregulated the expression of inflammatory cytokines IL-6, TNF and IL-1β compared with the control group. Transfection with vector-FGD5-AS1 decreased the levels of inflammatory factors compared with the LPS group. However, the inflammatory factors were upregulated after the co-transfection of vector-FGD5-AS1 + sh-AQP1 (Fig. 7E-G). Fig. 7H illustrates the proposed underlying mechanism for the signaling pathway that involves FGD-AS1-miR-133a-3p-AQP1 in sepsis.

Figure 7. Knockdown of AQP1 reverses the protective effect of FGD5-AS1 on HL-1 cells. (A) Detection of AQP1 expression level in HL-1 cells under different treatment conditions. (B) Cell Counting Kit-8 cell viability detection. Reverse transcription-quantitative PCR was performed to detect the expression of (C) Bax, (D) Bcl-2, (E) IL-6, (F) TNF-α and (G) IL-1β. (H) Schematic diagram of the proposed mechanisms of the signaling pathway involving FGD-AS1-miR-133a-3p-AQP1 in sepsis. Results were obtained from three independent experiments, each performed in triplicate, the error bars represent SD. *P<0.05, **P<0.01. AQP1, aquaporin 1; IL, interleukin; TNF, tumor necrosis factor; miR, microRNA; NC, negative control; sh-, short hairpin RNA; LPS, lipopolysaccharide; lncRNA, long non-coding RNA.

Discussion

Sepsis is a systemic inflammatory response caused by infection that leads to multiple organ failure, in which the heart is one of the most vulnerable organs (30,31). Current treatments, such as antimicrobial therapy and supportive therapies, are still at the early stages (32,33). The study of non-coding RNA provides a novel idea for the treatment of myocardial inflammation caused by sepsis (4,34).

The role of non-coding RNA in physiological and pathological processes, including cell proliferation, apoptosis, inflammatory response and immunity, has received extensive attention (35). Studies have shown that lncRNAs play a crucial role in a number of inflammatory diseases, such as sepsis (36,37). For example, lncRNA growth arrest specific transcript 5 is involved in regulating sepsis-induced podocyte injury by inhibiting the expression of phosphatase and tension protein homologous gene (38). In addition, miRNAs are valuable markers for the diagnosis and prognosis of sepsis. Lan et al (39) showed that the expression of serum miR-155-5p and miR-133a-3p could be used as a specific indicator for the diagnosis of sepsis. Further results showed that the expression of miR-155-5p could be an independent influencing factor for the prognosis of sepsis (39).

In the present study, in vitro cell models were used to investigate the expression and mechanism of FGD5-AS1 in sepsis inflammatory response. Experiments confirmed that plasma FGD5-AS1 expression decreased, miR-133a-3p expression increased, AQP1 expression was downregulated, and the release of serum proinflammatory cytokines was increased in the sepsis model. Inflammatory cytokines are involved in sepsis-induced myocardial damage. Studies have shown that TNF-α and IL-1β initiate an inflammatory response that has a negative inotropic effect on the myocardium (40,41). The negative regulation of IL-6 on myocardial systolic performance is associated through the protein kinase pathway (42,43). In the present study, FGD5-AS1 overexpression inhibited the expression of inflammatory cytokines TNF-α, IL-1β and IL-6 in the sepsis models. Thus, these results indicated that FGD5-AS1 is involved in the regulation of the expression of inflammatory cytokines.

In the present study, miR-133a-3p was upregulated in the blood samples of LPS mice. These results suggested that miR-133a-3p plays a regulatory role in sepsis-induced inflammatory response. Further results confirmed that miR-133a-3p and its predicted target gene AQP1 were remarkably upregulated in plasma and downregulated in the LPS-induced cells, respectively. AQP1 plays an important regulatory role in the inflammatory response caused by neonatal toxic erythema, rheumatoid arthritis and pulmonary edema (44–46). Thus, AQP1 can regulate inflammatory response. In the present study, FGD5-AS1 overexpression decreased LPS-induced upregulation of TNF-α, IL-6 and IL-1β by upregulating AQP1. This result suggested that FGD5-AS1 and miR-133a-3p are antagonistic regulators of AQP1 expression and inflammatory response. FGD5-AS1 was indicated to have a protective effect on sepsis-induced inflammatory response.

In summary, the present study focused on the mechanisms and signaling pathways that regulate inflammatory responses in sepsis and investigated whether FGD5-AS1 is a potential therapeutic target for sepsis and complications. In the present study, FGD5-AS1 and AQP1 expression was decreased in animal models of sepsis, and LPS-induced cells; the expression of miR-133a-3p was increased. Pro-inflammatory cytokines TNF-α, IL-6 and IL-1β were remarkably elevated in LPS-induced cells. FGD5-AS1 overexpression could reverse LPS-induced changes in the levels of miR-133a-3p, AQP1 and pro-inflammatory factors. Therefore, these results indicated that FGD5-AS1 is the competing endogenous RNA of miR-133a-3p on AQP1.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed in the current study are available from the corresponding author on reasonable request.

Authors contributions

JK designed the experiments. YC and XW performed the experiments and data analysis. YC, NY and YL performed data analysis and wrote the manuscript, with contributions from all authors. YC and JK confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The animal experiments were approved by the Ethics Committee of Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital (Tianjin, China; grant no. 20190135).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References