The LncRNA MIAT acts as a sponge for miR-942-5p to exacerbate the inflammation and oxidative stress in macrophages during sepsis-associated liver injury

Abstract

Background

Sepsis-associated liver injury (SALI) is a risk factor for high mortality in patients with sepsis. However, the pathological mechanism and treatment strategies of SALI remain unclear. This study aims to explore the influence of lncRNA MIAT expression on SALI.

Results

The MIAT and TXNIP levels in the serum of SALI patients, RAW264.7 stimulated by LPS, and AML-12 co-cultured with RAW264.7 were increased, while miR-942-3p expression was decreased. The secretion level of inflammatory factors, the ROS positive rate, and the MDA content were increased, while the GSH levels and SOD enzymatic activity were reduced in LPS-stimulated RAW264.7. The activity of AML-12 was decreased, and the LDH level was increased. When the MIAT was inhibited, the above results showed opposite trends. MIAT was a molecular sponge for miR-942-5p and represses its activity. After inhibiting miR-942-5p, the inflammatory response and the oxidative stress level were enhanced in LPS-stimulated RAW264.7 and the damage of AML-12 was worsened.

Conclusions

The silence of MIAT alleviated the inflammatory response and oxidative stress in LPS-induced RAW264.7 and alleviated the damage of AML-12 cells by adsorbing miR-942-5p.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12865-026-00860-6.

Introduction

Sepsis is a syndrome that occurs when the body responds to a severe infection [1]. It has resulted in a huge economic burden. The liver is the main organ that regulates immunity and inflammation, so sepsis causes significant damage to the liver [2–4]. Approximately 34% to 46% of sepsis patients develop liver damage, and the mortality rate is as high as 54% to 68%, which is significantly higher than the mortality rate caused by other organ damage associated with sepsis [5, 6].

The liver-gut axis and the inflammatory-immune response in sepsis have attracted the attention of researchers [5]. During the progression of sepsis, disruption of the intestinal barrier, dysregulation of the intestinal microbiota, and translocation of lipopolysaccharide (LPS) led to liver cell damage and liver dysfunction [7]. The impact of sepsis-related liver injury (SALI) on patients is manifested in two aspects. On one hand, the immune system is abnormally activated, promoting the production of inflammatory factors, which aggravates tissue inflammation. On the other hand, the redox imbalance progresses towards a pro-oxygen state [8, 9]. The intense inflammatory response and oxidative stress damage the liver cells, leading to liver dysfunction [2, 6]. The Kupffer cells and hepatic macrophages within the hepatic sinusoids are the first line of defense for the liver against bacteria, endotoxins, and intestinal microorganisms, and are the main guardians protecting against pathogens that enter the liver through the portal vein [10, 11]. However, under the stimulation of harmful factors, Kupffer cells are activated and exhibit a pro-inflammatory phenotype. The inflammatory mediators and oxygen-providing products produced by activated Kupffer cells can exacerbate liver damage [12, 13]. Effective inhibition of inflammation and slowing down the oxidative response of macrophages may be potential targets for controlling the progression of SALI.

Research reports indicate that different lncRNAs are either upregulated or downregulated in various inflammatory conditions, which is expected to become a biomarker for inflammation [14]. Myocardial Infarction Associated Transcript (MIAT) is a type of long non-coding RNA. It has been discovered that MIAT can increase the risk of myocardial infarction [15]. As a competitive endogenous RNA (ceRNA) molecule, MIAT can regulate post-transcriptional regulation. MIAT was involved in the pathological process of inflammatory diseases [16]. It was reported that MIAT was upregulated in the septic mouse model, and inhibition of MIAT alleviated the inflammatory response and oxidative stress in septic cardiomyopathy [17]. Min L et al. found that inhibition of MIAT can alleviate lung injury induced by LPS [18]. In liver-related diseases, MIAT mainly promoted the invasion, migration, and proliferation of liver cancer cells [19, 20]. However, the role of MIAT in SALI is unknown.

As a competitive endogenous RNA (ceRNA), MIAT exerts regulatory effects by adsorbing multiple miRNA molecules. MIAT can act as a molecular sponge for several miRNAs, such as miR-411-5p [21], miR-362-3p [22], miR-10b-5p [23]. miR-942-5p was selected as the downstream molecule of MIAT in this study. The main reason is that previous studies have confirmed that miR-942-5p can inhibit the inflammatory response induced by lipopolysaccharide (LPS) [24, 25], and miR-942-5p was closely related to the core pathological process of SALI, which was characterized by excessive inflammation. Furthermore, existing studies have shown that miR-942-5p also had a protective effect in sepsis-related lung injury and kidney injury [26, 27], further indicating that miR-942-5p played an important regulatory role in sepsis-related organ damage. However, it is unknown whether MIAT regulates the pathological process of SALI by exerting its ceRNA function to sponge miR-942-5p. This study aims to investigate the combined effect of long non-coding RNA MIAT and miR-924-5p on SALI.

Methods and materials

Subjects

A total of 150 non-SALI volunteers and 150 SALI patients were included in this study at Qiannan Buyi and Miao Autonomous Prefecture People’s Hospital from 2023 to 2024. All participants and their family members agreed to participate in the experiment and signed the informed consent form. After being admitted to the hospital and diagnosed with sepsis, all the volunteers had their blood drawn immediately before the antibiotic treatment, for blood routine tests and RNA extraction. The trial protocol of this study was approved by the ethics review committee of Qiannan Buyi and Miao Autonomous Prefecture People’s Hospital. The bioethical committee approval number is:2023-024.

All patients met the criteria of the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) [28]: (1) Bacterial blood culture is positive. (2) Sequential Organ Failure Assessment (SOFA) score ≥ 2 points. According to the guidelines of the “Surviving Sepsis Campaign” (SSC), the diagnostic criteria for sepsis-associated liver injury are in line with the definitions of the “Surviving Sepsis Campaign (SSC)” [29, 30]: Bilirubin ≥ 34.2 µmol/L. 2. The international normalized ratio (INR) > 1.5. Patient exclusion criteria: (1) Having other chronic liver diseases; (2) Infected with HIV; (3) Having malignant tumors; (4) Discharged from the ICU within 24 hours of admission; (5) Those who did not undergo coagulation function tests within 24 hours of admission.

Cell culture and co-culture

Normal mouse liver cells (AML-12) and mouse macrophages (RAW264.7) were purchased from the Chinese Academy of Sciences. AML-12 and RAW264.7 were cultured in DMEM (10569-010, Thermo, USA) added with 10% fetal bovine serum (10099-141, Thermo, USA) and 1% penicillin-streptomycin (15140-122, Thermo, USA). The RAW264.7 was routinely cultured in a cell incubator at a temperature of 37 °C and containing 5% CO₂. Functional experiments were conducted using RAW264.7 cells and AML-12 cells that were passaged 3 to 6 times. The number of cells detected in each experiment was 1 × 10⁵ cells.

RAW264.7 cells were treated with 1 µg/ml LPS (L2630, Sigma-Aldrich, Shanghai) for 24 h to detect inflammatory responses, oxidative stress [31, 32].

Co-culture of RAW264.7 and AML-12: The Transwell method was used for cell co-culture. The pore size of the septum of the Transwell was chosen to be 0.4 μm (3470, Corning, USA) to ensure that the cells could only communicate through secreted factors. The LPS-treated RAW264.7 was used to co-culture with AML-12 cell. RAW264.7 was inoculated in the lower chamber of the Transwell, and AML-12 in the upper chamber. RAW264.7 and AML-12 were co-cultured for 48 h.

Cell transfection

RAW264.7 cells were uniformly distributed in 6-well plates. After the cell fusion rate reached 70%, the transfection experiment was conducted. 1 µg of the MIAT knockdown plasmid (si-MIAT) and negative control plasmid (si-NC) was transfected per well, and 50 nM of miR-942-5p-related oligonucleotide was transfected per well. The transfection medium was Lipofectamine 3000 (L3000015, Invitrogen, USA). Six hours after transfection, the cells were replaced with fresh culture medium and continued to be cultured for 24 h. The si-MIAT plasmid and si-NC plasmid were obtained from Generalbiol (AnHui, China). The oligonucleotide sequences of the miR-942-5p mimics and inhibitors were:

miR-942-5p inhibitor: CACAUGGCCAAAACAGAGAAGA.

miR-942-5p inhibitor NC: CAGUCCUUUUGUGUAGUACAA.

miR-942-5p mimic: UCUUCUCUGUUUUGGCCAUGUG.

miR-942-5p mimic NC: UUCUCCGAACGUGUCACGUUU.

RT-qPCR

RNA was extracted using 100 µL of serum. The total RNA in the serum was extracted using a specialized kit (DP424, TIANGEN, Beijing). The total RNA within the cells was extracted using the TRIzol reagent (15596018CN, Invitrogen, USA). LncRNA and miRNA were respectively synthesized into cDNA for the RT-qPCR using specific reverse transcription kits (lncRNA: 18091200, miRNA: 4366597, Invitrogen, USA). 1 µg of total RNA was used for the synthesis of cDNA. PCR amplification conditions: Synthesis of the first strand of cDNA: 42℃ for 60 min. Inactivation of the enzyme: 95℃ for 3 min. Store at 4℃. The experimental procedures should be carried out following the instructions provided in the manual. The lncRNA and miRNA detection results were standardized using GAPDH, while the relative expression levels of serum miRNAs were calculated using 5 S as the calibration standard, and the cell miRNA was used as U6 calibration. The 2^{⁻^ΔΔCt} method was employed to calculate the relative expression levels of MIAT, miR-942-5p, and thioredoxin-interacting protein (TXNIP). The primers were obtained by Ribo Biotechnology (Guangzhou, China). The downstream primer of miRNA was the universal downstream primer provided by the kit. Primer sequences were as follows:

MIAT-F: GCAGATACAAGTGTGGAGTAAGC.

MIAT-R: ACAACCATCGCCAATCTCTATG.

Human-TXNIP-F: CCTCCTATTTCCGTTCCACA.

Human-TXNIP-R: TCAGGCCTCATTGTGTGTGT.

Mouse-TXNIP-F: ATATCGGGTGGGCTCTTTCT.

Mouse-TXNIP-F: ATTTCATGACTCGCCTGAGC.

Human-GAPDH-F: TGCACCACCAACTGCTTAGC.

Human-GAPDH-R: GGCATGGACTGTGGTCATGAG.

Mouse-GAPDH-F: AACGACCCCTTCATTGAC.

Mouse-GAPDH-R: TCCACGACATACTCAGCA.

miR-942-5p: GCCGAGUCUUCUCUGUUUUG.

5 S: GCGGTCTCCCATCCAAGTAC.

U6: CTCGCTTCGGCAGCACA.

Detection of inflammatory factor secretion levels

The secretion levels of interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α) were detected by ELISA in the supernatant of RAW264.7 cells. The ELISA kit was obtained from Jian Cheng Biology Company (H002-1-2, H007-1-2, H052-1-2, Nanjing, China). The experimental procedures were carried out in accordance with the instructions provided in the manual. The results were read at a wavelength of 450 nm. Each sample was tested three times.

Detection of oxidative stress-related products

ROS was detected using the dichlorofluorescein diacetate (DCFH-DA) fluorescence probe method. After treating the cells with 10 µM DCFH-DA for 10 min, the cells were stained with 1X Hoechst at 37 °C for 5 min. The intracellular ROS was detected using a fluorescence spectrophotometer. The excitation wavelength was set at 480–530 nm. The ROS positive rate of the control group was manually set at 100%. MDA, SOD, and GSH were detected using specialized detection kits (A003-1-2, A001-3-2, A006-1-2, Jiancheng, Nanjing). The experimental operation steps were carried out according to the instructions in the manual.

Cell viability assay

The AML-12 cells that were co-cultured with RAW264.7 were prepared into cell suspensions and evenly distributed into 96-well plates. 10 µl of CCK-8 reagent was added to each well at 0 h, 24 h, 48 h, and 72 h, and the plates were incubated for 3–4 h in a cell incubator. The absorbance value was detected at a wavelength of 450 nm using an enzyme reader.

Verification of the relationship between MIAT and miR-942-5p

The MIAT and TXNIP reporter plasmids include the wild-type sequence reporter plasmids (WT-MIAT and WT-TXNIP) and the mutant sequence reporter plasmids (MUT-MIAT and MUT-TXNIP). The report plasmids were synthesized based on the genetic sequence by GenePharma (Shanghai, China). According to the experimental design, the miR-942-5p-related oligonucleotide sequences and the reporter plasmids were transfected into RAW264.7 cells and cultured for 24 h. After 24 h, the cells were collected, and the cell suspension was prepared. After adding the firefly luciferase substrate and the Renilla luciferase substrate, the results were read on the microplate reader. Renilla luciferase fluorescence was used as an internal control to normalize firefly luciferase level of samples, and the relative luciferase activity was subsequently calculated.

Data analysis

The general clinical information data of the volunteers included in this study were statistically analyzed using SPSS (USA). Prior to statistical analysis, all data were tested for normality of distribution using the Shapiro-Wilk test. Data conforming to a normal distribution were presented as the mean and the standard deviation (SD). Continuous variable data were analyzed using a T-test and one-way analysis of ANOVA or two-way analysis of ANOVA, followed by Tukey’s test for multiple comparisons correction to avoid type I errors; categorical variable data were analyzed using a chi-square test. The data from in vitro cell experiments were analyzed using Graphpad Prism (USA). P < 0.05 indicated statistical significance.

Result

The lncRNA MIAT expression was markedly increased in the serum of SALI patients

The general clinical information of the two groups of volunteers included in the clinical study was analyzed. The results showed significant differences in blood routine indicators and liver function indicators. In the SALI patients, the number of WBC and neutrophils significantly increased while the number of monocytes decreased, and the enzyme activities of ALT and AST significantly increased (Table 1). These results indicate that patients with SALI have a strong inflammatory response, decreased immune function, and significant liver damage. The MIAT was detected by RT-qPCR, and it was found that the level of MIAT in the serum of SALI patients was significantly higher than that of non-SALI patients (Fig. 1A). The ROC curve showed that MIAT is a promising diagnostic biomarker for SALI (AUC = 0.880) (Fig. 1B).

Table 1.

Comparison of general clinical information of the participants

Clinical features	non-SALI	SALI	P-value
Age (years)	56.48 ± 2.11	56.53 ± 2.06	0.790
Gender (male, n)	84	87	0.726
Hyperglycemia (yes, n)	28	31	0.435
Hypertension (yes, n)	22	27	0.423
Blood routine index
WBC (×109/L)	10.17 ± 3.41	12.96 ± 4.27	< 0.001
Neutrophil (×109/L)	7.68 ± 2.59	10.32 ± 3.14	< 0.001
Lymphocyte (×109/L)	1.32 ± 0.48	1.19 ± 0.41	0.002
liver function index
ALT (U/L)	38.21 ± 7.42	112.82 ± 21.46	< 0.001
AST (U/L)	43.57 ± 9.26	161.35 ± 49.71	< 0.001

SALI Sepsis-associated liver injury, WBC White blood cell, ALT Alanine aminotransferase, AST Aspartate aminotransferase

The statistical methods employed were t-test and chi-square test

Fig. 1.

Expression level of lncRNA MIAT and ROC curve. A The serum MIAT level in SALI patients was significantly elevated. B The ROC curve indicated that the MIAT served as a promising diagnostic marker for SALI. ***P < 0.001. The statistical tests were t-test and ROC curve analysis. n ≥ 3

The correlation between the clinical characteristics of SALI and the expression of MIAT

Using the median expression level of serum MIAT in the SALI group as the cutoff value, the SALI group was divided into the high-expression sub-group of MIAT and the low-expression sub-group of MIAT. The chi-square test was used to analyze the correlation between the clinical characteristics of SALI and the expression of MIAT. The results showed that WBC, neutrophils, lymphocytes, ALT, and AST were significantly correlated with the expression of MIAT. In the MIAT high-expression group, there were more SALI patients with increased WBC and neutrophil counts and decreased lymphocyte counts, as well as more patients with high levels of ALT and AST enzyme activities (Table 2).

Table 2.

Correlation between lncRNA MIAT expression and clinical features in SALI

Clinical features	LncRNA MIAT high expression(n = 76)	LncRNA MIAT low expression(n = 74)	P-value
Age (years)			0.599
> 56	33	29
< 56	43	45
Gender (male, n)			0.761
Male	45	42
Female	31	32
Hyperglycemia (yes, n)			0.602
Yes	17	14
No	59	60
Hypertension (yes, n)			0.892
Yes	14	13
No	62	61
Blood routine index
WBC (×109/L)			< 0.001
> 13.0	57	22
< 13.0	19	52
Neutrophil (×109/L)			< 0.001
> 10.5	55	32
< 10.5	21	42
Lymphocyte (×109/L)			< 0.001
> 1.2	22	48
< 1.2	54	26
Liver function index
ALT (U/L)			< 0.001
> 112.0	55	20
< 112.0	21	54
AST (U/L)			< 0.001
> 161.0	58	21
< 161.0	18	53

SALI Sepsis-associated liver injury, WBC White blood cell, ALT Alanine aminotransferase, AST

Aspartate aminotransferase

Using the median expression level of serum MIAT in the SALI group as the cutoff value, the SALI group was divided into the high-expression sub-group of MIAT and the low-expression sub-group of MIAT. The statistical method employed the chi-square test

Inhibition of MIAT expression weakens the inflammatory response, oxidative stress levels, and cytotoxicity

For the stimulation concentration of LPS, the preliminary concentration gradient pre-experiment was conducted. After LPS stimulation, the expression level of MIAT in RAW264.7 cells and the levels of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α showed a concentration-dependent significant upregulation (Supplementary Fig. 1A-B). Based on the results of the above pre-experiment, we finally chose 1 µg/mL as the optimal working concentration for the subsequent in vitro experiments. In RAW264.7 cells treated with LPS, the level of MIAT was significantly increased (Fig. 2A). It was consistent with the serum test results of SALI patients. The ELISA results showed that the levels of inflammatory factors IL-1β, IL-6, and TNF-α were significantly increased (Fig. 2B). The positive rate of ROS and the content of MDA were significantly increased (Fig. 2C-D), while the contents of antioxidants GSH and SOD were significantly decreased (Fig. 2E-F). AML-12 was co-cultured with RAW264.7 cells, and the MIAT level, cell viability, and LDH were detected. The results indicated that the MIAT level was upregulated (Fig. 3A), the cell viability was decreased (Fig. 3B), and the LDH level was significantly increased (Fig. 3C). After RAW264.7 cells were transfected with si-MIAT plasmid, the level of MIAT was decreased, and the secretion levels of inflammatory factors were significantly decreased; the content of oxidative products was reduced, and the content of antioxidants increased. The MIAT level in AML-12 cells was downregulated, cell viability increased, and the LDH level decreased. This indicates that the MIAT level affects the inflammatory response and oxidative stress levels of RAW264.7 cells.

Fig. 2.

The secretion levels of inflammatory factors and oxidative stress-related factors in RAW264.7 cells were measured. A The MIAT level in LPS-stimulated RAW264.7 was elevated. B The secretion of inflammatory factors was increased, but was decreased after inhibition of MIAT in LPS-stimulated RAW264.7. C-D The ROS positive rate and MDA level were increased, but were decreased after inhibition of MIAT in LPS-stimulated RAW264.7. E-F The levels of GSH and SOD were decreased, but were increased after inhibition of MIAT in LPS-stimulated RAW264.7. ***P < 0.001. The statistical tests were one-way ANOVA followed by Tukey’s post-hoc test and two-way ANOVA followed by Tukey’s post-hoc test. n ≥ 3

Fig. 3.

The cell viability and LDH levels of AML-12 cells were measured. A-C The MIAT level was increased, cell viability was decreased, and the LDH level was increased in AML-12 cells co-cultured with RAW264.7 cells. However, after the MIAT level of RAW264.7 was inhibited, the MIAT expression was decreased, cell viability was increased, and LDH levels were decreased in AML-12. ***P < 0.001. The statistical tests were one-way ANOVA followed by Tukey’s post-hoc test and two-way ANOVA followed by Tukey’s post-hoc test. n ≥ 3

MIAT exerted the ceRNA function to adsorb miR-942-5p

The ENCORI data showed that MIAT and miR-942-5p have complementary sequences (Fig. 4A). The dual-luciferase reporter gene assay revealed that the fluorescence intensity in the WT-MIAT group was weakened by miR-942-5p mimic but enhanced by miR-942-5p inhibitor. However, in the MUT-MIAT group, the fluorescence intensity was not affected by the miR-942-5p mimic and miR-942-5p inhibitor (Fig. 4B). Therefore, as a molecular sponge for miR-942-5p, MIAT can bind to miR-942-5p. The level of miR-942-5p in the serum of SALI patients was significantly lower compared to that of non-SALI patients (Fig. 4C). The expression of MIAT in SALI patients was negatively correlated with the miR-942-5p expression (Fig. 4D). The level of miR-942-5p was also decreased in RAW264.7 cells stimulated by LPS and in AML-12 cells co-cultured with RAW264.7 cells, but the miR-942-5p expression was upregulated after inhibiting the level of MIAT (Fig. 4E-F). This indicates that MIAT can sponge miR-942-5p.

Fig. 4.

The targeted relationship between MIAT and miR-942-5p and the level of miR-942-5p were detected. A The complementary sequence between MIAT and miR-942-5p. B Dual luciferase reporter gene assay verified that MIAT specifically binds to miR-942-5p. C The miR-942-5p level in the serum of SALI patients was decreased. D The expression of miR-942-5p was negatively correlated with the expression of MIAT in patients with SALI. E-F The miR-942-5p level was decreased in LPS-stimulated RAW264.7 and in AML-12 cells co-cultured with RAW264.7. After inhibiting MIAT, the level of miR-942-5p increased. ns indicates P > 0.05. ***P < 0.001. The statistical tests were t-test, one-way ANOVA followed by Tukey’s post-hoc test, two-way ANOVA followed by Tukey’s post-hoc test, Pearson correlation analysis, and one-way ANOVA followed by Tukey’s post-hoc test. n ≥ 3

After inhibiting miR-942-5p, the inflammatory response and oxidative stress in RAW264.7 cells were aggravated, and the damage to AML-12 cells was increased

After miR-942-5p transfection inhibitor in RAW264.7 cells stimulated by LPS, in the LPS + MIAT + miR-942-5p inhibitor group, the miR-942-5p level was downregulated (Fig. 5A), the levels of inflammatory factors IL-1β, IL-6, and TNF-α were markedly increased (Fig. 5B), the positive rate of ROS and the level of MDA were elevated (Fig. 5C-D), and the GSH and SOD levels were decreased compared with the LPS + MIAT + inhibitor NC group (Fig. 5E-F). The miR-942-5p level of AML-12 cells was also downregulated in the LPS + MIAT + miR-942-5p inhibitor group (Fig. 6A), and the cell viability was decreased, while the LDH level was increased (Fig. 6B-C). This indicates that inhibiting the expression of miR-942-5p could reverse the effect of reducing damage after inhibiting MIAT.

Fig. 5.

After inhibiting miR-942-5p, the inflammatory response and oxidative stress in RAW264.7 cells were intensified. A After inhibiting miR-942-5p, the miR-942-5p level was decreased. B After inhibiting miR-942-5p, the inflammatory factors were increased. C-D After inhibiting miR-942-5p level, the ROS positive rate and the MDA level were increased. E-F After inhibiting miR-942-5p levels, the GSH and SOD levels were decreased. ***P < 0.001. The statistical tests were one-way ANOVA followed by Tukey’s post-hoc test and two-way ANOVA followed by Tukey’s post-hoc test. n ≥ 3

Fig. 6.

RAW264.7 cells were treated with miR-942-5p inhibitor, and the miR-942-5p level, cell viability, and LDH were detected in AML-12. A-C After RAW264.7 was treated with miR-942-5p inhibitor, the miR-942-5p expression and the cell viability were decreased, but the LDH level increased in AML-12 cells. ***P < 0.001. The statistical tests were one-way ANOVA followed by Tukey’s post-hoc test and two-way ANOVA followed by Tukey’s post-hoc test. n ≥ 3

TXNIP was a target gene for miR-942-5p

The ENCORI data showed that TXNIP and miR-942-5p have complementary sequences (Fig. 7A). The dual-luciferase reporter gene assay revealed that the fluorescence intensity in the WT- TXNIP group was weakened by miR-942-5p mimic but enhanced by miR-942-5p inhibitor. However, in the MUT- TXNIP group, the fluorescence intensity was not affected by the miR-942-5p mimic and miR-942-5p inhibitor (Fig. 7B). The level of TXNIP was upregulated in RAW264.7 cells stimulated by LPS. However, after MIAT was knocked down, the level of TXNIP was downregulated (Fig. 7C). In AML-12 cells, the expression trend of TXNIP was consistent with that in RAW264.7 cells (Fig. 7D). However, when the level of miR-942-5p was also knocked down, the level of TXNIP was upregulated in RAW264.7 and AML-12 cells (Fig. 7E-F). This indicates that MIAT and miR-942-5p can regulate the expression of TXNIP mRNA.

Fig. 7.

TXNIP was the target gene of miR-942-5p. A The complementary sequence between TXNIP and miR-942-5p. B Dual luciferase reporter gene assay verified that TXNIP was a target gene of miR-942-5p. C-D The TXNIP level was decreased in LPS-stimulated RAW264.7 and in AML-12 cells co-cultured with RAW264.7 after MIAT expression was inhibited. E-F The TXNIP level was increased in LPS-stimulated RAW264.7 and in AML-12 cells co-cultured with RAW264.7 after miR-942-5p expression was inhibited. ***P < 0.001. The statistical tests were one-way ANOVA followed by Tukey’s post-hoc test and two-way ANOVA followed by Tukey’s post-hoc test. n ≥ 3

Discussion

Sepsis remains a major clinical challenge in the medical field. The activation of the systemic inflammatory response in sepsis patients leads to a cascade release of inflammatory factors, ultimately resulting in multiple organ dysfunction syndrome (MODS) [33, 34]. SALI is an important component of sepsis-related organ dysfunction syndrome, which often indicates a poor prognosis for the patient [35, 36]. This study found that the MIAT expression was markedly increased in the serum of SALI patients and in RAW264.7 cells stimulated by LPS. Inhibiting the level of MIAT effectively alleviated the inflammatory response and oxidative stress in LPS-stimulated RAW264.7 cells.

LncRNA MIAT was associated with an increased risk of myocardial infarction [15, 37]. Subsequently, numerous studies have reported that MIAT has pro-inflammatory and oxidative stress-enhancing effects. For instance, MIAT promotes the release of pro-inflammatory factors to exacerbate the symptoms of allergic rhinitis in mice [23]. Inhibiting MIAT can effectively alleviate the inflammatory response and ferroptosis in myocardial injury induced by sepsis [16]. MIAT can downregulate miR-330-5p and enhance inflammation and oxidative stress in myocardial injury [17]. This study found that knockdown of MIAT effectively alleviated inflammation and oxidative stress in RAW264.7 cells. After co-culture of AML-12 with LPS-treated RAW264.7 cells, cell viability was decreased, and LDH release was increased. However, after inhibiting the expression of MIAT, inflammation and oxidative stress in RAW264.7 cells were reduced, and the cell damage in AML-12 was alleviated. This indicates that reducing the level of MIAT has a protective effect on liver damage caused by sepsis. This is consistent with the content reported in the aforementioned literature. It is worth noting that lncRNA MIAT may exhibit different or even opposite biological functions in different cell types and disease models. Although our study indicates that MIAT exacerbates the inflammatory response and oxidative stress of SALI, Sun et al. reported that Overexpression of MIAT alleviates the inflammatory response mediated by J774A.1 macrophages in rheumatoid arthritis [38]. This demonstrates that MIAT function may have spatial specificity and disease specificity.

miRNA plays a significant role in regulating the inflammatory phenotype of macrophages [39, 40]. miR-942-5p functions as a protective microRNA that attenuates sepsis-associated inflammatory response and oxidative stress. It has been reported that the inhibition of miR-942-5p aggravated the inflammatory response and oxidative damage in LPS-stimulated HK-2 cells [25]. Overexpression of miR-942-5p significantly inhibited the inflammatory response and oxidative stress in sepsis-related kidney injury and lung injury [26, 27]. This study found that MIAT can sponge miR-942-5p by exerting the function of ceRNA. The miR-942-5p level was markedly reduced in RAW264.7 cells induced by LPS and in AML-12 cells co-cultured with LPS-stimulated RAW264.7. After inhibiting MIAT, the miR-942-5p level was increased. When the miR-942-5p inhibitor was transfected into RAW264.7, the inflammatory response and oxidative stress in RAW264.7 were aggravated, the cell viability of AML-12 was weakened, and the LDH release was increased. This indicates that inhibition of miR-942-5p can reverse the anti-inflammatory and anti-oxidative effects of silence of MIAT.

Previous literature indicated that under oxidative stress conditions, TXNIP can participate in regulating the process of cell apoptosis [41]. Multiple studies have confirmed that TXNIP was involved in apoptosis and inflammatory responses, and played a significant role in organ damage related to sepsis, including sepsis-related myocardial injury, renal injury, and brain injury [42–45]. Li et al. discovered that overexpression of peroxisome proliferator-activated receptor gamma (PPARγ) reduced the release of reactive oxygen species (ROS) during sepsis, thereby inhibiting the expression of the TXNIP/nod-like receptor protein 3 (NLRP3) signaling pathway, and subsequently alleviating liver damage caused by sepsis [46]. This study found that the level of TXNIP was upregulated in LPS-induced RAW264.7 cells as well as in AML-12 cells co-cultured with RAW264.7 cells. This was consistent with the reports in the literature. The dual-luciferase reporter gene assay revealed that TXNIP was the target gene of miR-942-5p. After inhibiting MIAT, the mRNA level of TXNIP was downregulated. However, when the level of miR-942-5p was further inhibited, the expression trend of TXNIP showed an opposite pattern. This indicates that TXNIP may be regulated by MIAT and miR-942-5p to participate in the pathological process of SALI. The specific mechanism needs to be further explored through experiments.

In this research framework, knockdown of MIAT can significantly alleviate inflammation, oxidative stress and liver cell damage induced by LPS. The main reason is that MIAT exerts a ceRNA function by adsorbing miR-942-5p to regulate multiple downstream inflammatory and oxidative stress pathways. Although LPS is a potent stimulant, after knocking down MIAT, miR-942-5p was upregulated, which might be involved in the convergence of multiple key inflammatory and oxidative signaling pathways. Therefore, this research may hold significant clinical value. Clinically, MIAT can serve as a specific biomarker for the diagnosis of SALI. More importantly, MIAT provides potential targets for drug development. Targeted drugs that inhibit the expression of MIAT may specifically intervene in the MIAT/miR-942-5p regulatory axis, effectively alleviating liver damage in patients with sepsis, and offering a more precise treatment option compared to traditional therapies.

However, these results were obtained from in vitro cell experiments. Without the support of in vivo experiments, the experimental results may be biased. In the future, we will further conduct in vivo experiments to verify these results. Proteins are the executors of various functions in life activities. However, this study did not investigate the changes in the downstream target genes of miR-942-5p in SALI. According to previous studies, miR-942-5p can alleviate LPS-induced inflammation and sepsis-related kidney or lung damage by targeting TXNIP, OXSR1, TRIM37 and FOXO3 [24–27]. After verification by the miRDB database, TXNIP, OXSR1, TRIM37 and FOXO3 were predicted to be the target genes of miR-942-5p. In our future research, we plan to conduct high-throughput sequencing and bioinformatics analysis to identify the downstream target genes of miR-942-5p in SALI. In addition, this study still has some limitations. In the present study, RAW264.7 cells were stimulated with LPS at a single concentration and duration to establish an in vitro inflammatory model. However, time‑gradient and dose‑dependent effects were not investigated, which may limit the generalizability of our findings. Therefore, further experiments will be performed using multiple LPS concentrations and time points to validate the current results and improve the reliability and robustness of our conclusions.

Conclusion

Overall, in the serum of SALI patients and RAW264.7 cells stimulated by LPS, the MIAT expression was increased, while the miR-942-5p expression was decreased. After inhibiting MIAT, the miR-942-5p expression was upregulated, effectively alleviating the inflammatory response and oxidative stress in RAW264.7 cells stimulated by LPS.

Abbreviations

SALI

Sepsis-associated liver injury

LPS

Lipopolysaccharide

MIAT

Myocardial Infarction Associated Transcript

ceRNA

Competitive endogenous RNA

SOFA

Sequential Organ Failure Assessment

SSC

Surviving Sepsis Campaign”

INR

International normalized ratio

si-MIAT

MIAT knockdown plasmid

si-NC

Negative control plasmid

IL-1β

Interleukin 1β

TNF-α

Tumor necrosis factor α

DCFH-DA

Dichlorofluorescein diacetate

WT-MIAT

Wild-type sequence reporter plasmid

MUT-MIAT

Mutant sequence reporter plasmid

Authors’ contributions

Conceptualization, J.S., E.S.; Data curation, J.S., Z.L., J.L.; Formal analysis, J.S., Z.L., J.L.; Funding acquisition, J.S.; Investigation, J.S., Z.L., J.L., E.S.; Methodology, J.S., Z.L., J.L.; Project administration, E.S.; Resources, J.S., Z.L., J.L., E.S.; Software, Z.L., J.L.; Supervision, E.S.; Validation, Z.L., J.L.; Visualization, J.S., Z.L., J.L.; Roles/Writing - original draft, J.S.; Writing - review & editing, E.S.

Funding

None.

Data availability

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

Declarations

Ethics approval and consent to participate

Approval was obtained from the ethics committee of Qiannan Buyi and Miao Autonomous Prefecture People’s Hospital. The procedures used in this study adhere to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all individual participants included in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.