The effect of STAT1, miR-99b, and MAP2K1 in alcoholic liver disease (ALD) mouse model and hepatocyte

Animal model

All experiments were approved by the Animal Ethic Committee of Dongzhimen Hospital, Beijing University of Chinese Medicine. Female C57BL/6 mouse aged 6-weeks old were purchased from The Jackson Laboratory (USA). To establish ALD, mouse received a Lieber-DeCarli diet that contains 5% (v/v) alcohol or an isocaloric dextrin maltose diet for three months as per previous report [12]. The use of animals has ethical approval in addition to following guidelines All animal experiments were performed in compliance with Animal Use and Care Committee directives of Dongzhimen Hospital, Beijing University of Chinese Medicine. After treatment, the mice were sacrificed, liver samples and serum were collected for subsequent study. The serum concentration of alanine aminotransferase (ALT) and aspartate transaminase (AST) was detected using CheKine™ Alanine Aminotransferase (ALT/GPT) Activity Colorimetric Assay Kit (Abbkine, China) and Aspartate Aminotransferase Activity Assay kit (Abcam, USA) in line with manufacture’s description, respectively.

Microarray analysis

Total RNA was extracted from the liver tissues that collected from ALD mouse and control mouse by using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s description. The RNA was then quantified and approximately 5 μg of total RNA was used to construct cDNA library following a standard protocol. The differentially expressed miRNAs (DEmi) and differentially expressed genes (DEGs) were determined by using the “limma” package in R software (version 4.0.0). The |log2 fold change (FC)| > 0.5 and P-value < 0.05 were set as the cut-off criteria to determine DEmis and DEGs. The WGCNA R package was used to cluster the DEmis into differently colored modules using the dissimilarity measure (1-topological overlap measure (TOM), TOM ≥ 0.15) [19].

Cell line and treatment

The mouse hepatocyte cell line Alpha mouse liver (AML)-12 was brought from American Type Culture Collection (ATCC, USA), and maintained in DMEM/F-12 medium (Hyclone, USA), that contains 10% (v/v) fetal bovine serum (Life technologies, 10099-141), 5 mg/ml insulin (Sigma-Aldrich, I9278), 5 μg/ml transferrin (Sigma-Aldrich, USA), 5 ng/ml selenium (Sigma-Aldrich, USA), 40 ng/ml dexamethasone (Sigma-Aldrich, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, USA) at 37°C humidified atmosphere with 5% CO₂. To establish ALD cell model, AML-12 cells were treated with 100 mM EtOH for 48 hours, and collected for further experiments.

Cell transfection

The sequence of STAT1 and MAP2K1 CDSs were obtained from NCBI database and synthesized by GenePharma (Shanghai, China). The overexpressing vectors of MAP2K1 (MAP2K1 OE) and STAT1 (STAT1 OE) were constructed by inserting the CDS sequences of targeted genes into the pcDNA 3.1 vectors. The miR-99b mimics and negative control [20] mimics were synthesized by GenePharma. Transient cells transfection was performed by using the Lipofectamine 2000 according to the manufacturer’s instructions.

Cell apoptosis

Cell apoptosis was assessed by the Annexin V/PI apoptosis detection kit (Beyotime, China). In short, cells were collected and suspended in binding buffer, stained with Annexin V (5 μg/ml) and PI (5 μg/ml) in dark for 30 minutes. The cells were then analyzed by using flow cytometry (BD Biosciences, USA).

Quantitative real-time PCR

The total RNA extraction from liver tissues and cells were conducted by using the Trizol reagent (Invitrogen, USA). The RNA was then quantified, reverse transcribed to cDNA using a First strand synthesis kit (Transgen, China). The real time PCR was performed to quantify the gene levels using the SYBR Green PCR Master Mix (Takara, China). Relative gene expression was determined by normalization to U6 or Tublin.

Primers: pri-miR-99b: F: 5′-ctcctgggtcctggcacccac-3′; R: 5′-cccgacacggacccatagaca-3′; miR-99b: F: 5′-cacccgtagaaccgac-3′; R: 5′-GTGCAGGGTCCGAGGT-3′; U6: F:5′-CTCGCTTCGGCACATA-3′; R:5′-CGAATTTGCGTGTCATCCT-3′; STAT1: F: 5′-TCACAGTGGTTCGAGCTTCAG-3′; R:5′-GCAAACGAGACATCATAGGCA-3′; MAP2K1: F: 5′-AAGGTGGGGGAACTGAAGGAT-3′; R: 5′-CGGATTGCGGGTTTGATCTC-3′.

Western blotting

Cells were collected and were lysed in an ice-cold RIPA lysis buffer (Thermo, USA). The total proteins were quantified using a BCA Protein Assay Kit (Beyotime, China). Equal amounts of proteins (40 μg) were separated on an 8 to 12% SDS-PAGE gel and shifted to nitrocellulose [20] membrane. The membranes were blocked with 5% skim milk for 2 hours and were probed with primary antibodies against Stat1 (1;1000, Abcam, USA), MAP2K1 (1;1000, Abcam, USA), Tubulin (1;1000, Abcam, USA) at 4°C overnight. The protein bands were then hatched with horseradish peroxidase (HRP)-conjugated IgG secondary antibodies, and visualized by enhanced chemiluminescence (ECL, Millipore, USA).

Luciferase reporter gene assay

The sequence of the MAP2K1 3’UTR region was cloned into the pmirGLO vectors (Promega, USA) to generate MAP2K1-WT vectors. The site-specific mutation was performed to obtain MAP2K1-mut vectors. Cells were co-transfected with miR-99b-5p mimics or NC mimics and MAP2K1-WT or MAP2K1-mut for 36 hours, and harvested to test the luciferase activity by using a dual luciferase reporter gene assay kit (Promega, USA). The pRLTK renilla luciferase reporter vector (Promega, USA) was adopted as the internal control.

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was performed by using the EZ-ChIP kit (Millipore, USA) following the manufacturer’s instruction. In short, cells were fixed with 1% formaldehyde for 10 min to generate DNA-protein crosslinks and homogenized to obtain 800 μl of cell lysates. The cell lysates were sonicated to generate short chromatin fragments of 200 to 500 bp. Half of the lysates were stored as input. And 100 μl of cell lysates were immunoprecipitated with 1 μl of STAT1 antibody or IgG as control. The precipitation was then subjected to qRT-PCR assay to analyze the level of miR-99b.

Statistical analysis

All data were presented as mean ± SD for at least three independent tests. Statistical differences between two or multiple groups were evaluated using a student’s t test or one-way analysis of variance (ANOVA). Differences were regarded as statistically significant when p < 0.05.

The upregulation of miR-99b is identified by microarray analysis in ALD mouse model

To explore the abnormally expressed miRNAs in ALD, we performed microarray analysis in ALD mouse model. And multiple miRNAs were identified in the model (Figure 1A, 1B). In addition, we identified that the expression of miR-99b was elevated in the ALD mouse model (Figure 1C). Meanwhile, the AML-12 cells were treated with EtOH and the expression of miR-99b was enhanced in the cells (Figure 1D). Significantly, the expression of miR-99b was positively correlated with ALT levels in the ALD mice (Figure 1E). The elevated level of AST confirmed the liver damage and successful establishment of ALD model (Figure 1F).

Figure 1. The upregulation of miR-99b is identified by microarray analysis in ALD mouse model. (A, B) The microarray analysis was performed in ALD mice. The miRNA heatmap (A) and WGCNA (B) were shown. (C) The expression of miR-99b was detected by qPCR in ALD mice. (D) The expression of miR-99b was measured by qPCR in AML-12 cells treated with 100 mM EtOH. (E) The correlation of miR-99b with ALT levels was analyzed in ALD mice. (F) The serum AST level. ^*P < 0.05.

STAT1 is found as an upstream factor of miR-99b by microarray analysis in ALD mouse model

Next, we tried to identify the upstream factors of miR-99b in ALD. The microarray analysis identified the abnormally expressed mRNAs in ALD mice and the overlap analysis was performed based on the differently expressed mRNAs and the transcriptional factors of miR-99b, in which STAT1 was identified (Figure 2A). We next validated these findings using in vitro and in vivo models. The elevated expression of STAT1 was validated in ALD mice (Figure 2B). Meanwhile, the treatment of EtOH induced the expression of STAT1 in the AML-12 cells (Figure 2C). The expression of STAT1 was positively correlated with ALT levels in the ALD mice (Figure 2D). These data indicated that miR-99b and STAT1 potentially participate in the ALD generation.

Figure 2. STAT1 is found as an upstream factor of miR-99b by microarray analysis in ALD mouse model. (A) The microarray analysis was performed in ALD mice. The mRNA heatmap was shown. The overlap was performed using microarray analysis and TransmiR v2.0 database. (B) The expression of STAT1 was measured by qPCR in ALD mice. (C) The expression of STAT1 was measured by qPCR in AML-12 cells treated with 100 mM EtOH. (D) The correlation of STAT1 with ALT levels was analyzed in ALD mice. ^*P < 0.05.

STAT1 enhances miR-99b expression in ALD model

The positive correlation of STAT1 and miR-99b expression was identified by the bioinformatics analysis (Figure 3A). Consistently, we then performed qPCR assay and identified this positive correlation (Figure 3B). Meanwhile, the protein levels of STAT1 were upregulated in ALD mice (Figure 3C, 3D). We next performed STAT1 overexpression in vitro to assess the corresponding alteration of miR-99b. Overexpression of STAT1 successfully upregulated the STAT1 expression in hepatocytes (Supplementary Figure 1A). Significantly, the expression of miR-99b (Figure 3E) and pri-miR-99b (Figure 3F) was promoted by the overexpression of STAT1 in AML-12 cells. Moreover, previous study indicated that STAT1 usually interact with the gamma-activated site (GAS) region [21], which is content on the upstream of miR-99b. Prediction of STAT1 binding site on miR-99b promoter region was shown (Figure 3G). The Further ChIP analysis confirmed the enrichment of STAT1 on miR-99b promoter in AML-12 cells (Figure 3H).

Figure 3. STAT1 enhances miR-99b expression in ALD model. (A) The correlation of STAT1 and miR-99b was analyzed using bioinformatic analysis. (B) The correlation of STAT1 and miR-99b was measured by qPCR in ALD mice. (C, D) The expression of STAT1 was detected by Western blot analysis in ALD mice. (E, F) The AML-12 cells were transfected with STAT1 overexpressing plasmid and the expression of miR-99b (E) and pri-miR-99b (F) was determined in the cells. (G) Prediction of STAT1 binding site on miR-99b promoter region. (H) The enrichment of STAT1 on miR-99b promoter was identified by ChIP analysis in AML-12 cells. ^*P < 0.05.

MAP2K1 is negatively correlated with miR-99b and downregulated in ALD model

Next, the miRWalk database predicted the target genes of miR-99b and then intersected with the genes downregulated in the mRNA microarray, in which 32 genes were negatively correlated with miR-99b in the bioinformatic analysis using TCGA database, including MAP2K1 and GADD45G. We validated that the expression of MAP2K1 was negatively associated with miR-99b (Figure 4A). The expression of MAP2K1 was downregulated in ALD mice (Figure 4B, 4C). Consistently, the expression of MAP2K1 was reduced by the treatment of EtOH in AML-12 cells (Figure 4D). The expression of MAP2K1 was negative correlated with ALT levels in the ALD mice (Figure 4E).

Figure 4. MAP2K1 is negatively correlated with miR-99b and downregulated in ALD model. (A) The correlation of MAP2K1 and miR-99b was analyzed using bioinformatic analysis. (B) The expression of MAP2K1 was detected by Western blot analysis in ALD mice. (C) The expression of MAP2K1 was detected by qPCR in ALD mice. (D) The expression of MAP2K1 was measured by qPCR in AML-12 cells treated with 100 mM EtOH. (E) The correlation of MAP2K1 with ALT levels was analyzed in ALD mice. ^*P < 0.05.

miR-99b targets MAP2K1 in AML-12 cells

Then, we identified the binding site of MAP2K1 and miR-99b (Figure 5A). Meanwhile, the treatment of miR-99b mimic repressed the luciferase activity of MAP2K1 in AML-12 cells (Figure 5B) and primary hepatocytes (Figure 5C). The expression of MAP2K1 was suppressed by miR-99b in AML-12 cells and primary hepatocytes (Figure 5D).

Figure 5. mir-99b targets MAP2K1 in AML-12 cells. (A) The binding analysis between MAP2K1 and miR-99b. (B, C) The dual luciferase reporter assays of MAP2K1 were conducted in (B) AML-12 cells and (C) primary hepatocytes treated with control mimic or miR-99b mimic. (D) The Western blot analysis of MAP2K1 was carried out in the AML-12 cells treated with control mimic or miR-99b mimic. ^*P < 0.05.

STAT1/miR-99b/MAP2K1 axis regulates apoptosis of AML-12 cells

Next, we investigated the STAT1/miR-99b/MAP2K1 axis in hepatocytes. We observed that the expression of MAP2K1 was inhibited by the overexpression of STAT1 in AML-12 cells (Figure 6A). The apoptosis of AML-12 cells was induced by the treatment of EtOH, and miR-99b mimic promoted these effects. Meanwhile, the MAP2K1 overexpression vectors effectively elevated the expression of MAP2K1 expression (Supplementary Figure 1B) and abolished the promoting effects of miR-99b mimics on cell apoptosis (Figure 6B). The hypothesis model of this study was shown (Figure 6C).

Figure 6. STAT1/miR-99b/MAP2K1 axis regulates apoptosis of AML-12 cells. (A) The AML-12 cells were transfected with STAT1 overexpressing plasmid and the expression of MAP2K1 was determined in the cells. (B) The apoptosis was analyzed in AML-12 cells treated with EtOH, or cotreated with EtOH and miR-99b mimic or MAP2K1 overexpressing plasmid. (C) A hypothesis model of this study was shown. ^*P < 0.05, ^**P < 0.01.