MiRNA-29b accelerates the PDGF in exosomes and stimulates hepatic stellate cells to promote liver fibrosis in biliary atresia

doi:10.21203/rs.3.rs-5233237/v1

Background

Biliary atresia (BA) is one of the most common liver diseases in infants and young children. Its etiology and pathogenesis are still unclear. The purpose was to investigate the mechanism by which the highly expressed miRNA-29b in exosomes regulates the high expression of PDGF through methylation, hepatic stellate cell activation, and participation in the hepatic fibrosis of BA.

Methods

In previous experiments, peripheral blood exosomes of children with biliary atresia and cholangiectasia (CC) were analyzed, and it was found that miRNA-29b was highly expressed in the exosomes of children with BA. Meanwhile, PDGF was highly expressed.Jurkat cells were infected with lentivirus overexpressing miRNA-29b, and the exosomes of Jurkat cells were extracted and cocultured with hepatic stellate cells (LX-2).

Results

DNMT3a and DNMT3b expression was downregulated and PDGFA expression was upregulated in the miR-29b overexpression group, which activated hepatic stellate cells. DNMT3a/3b was identified as the target gene of miR-29b by luciferase assays. Downregulation of DNMTs resulted in hypomethylation of the PDGF promoter region and high expression of PDGFA. Western blot analysis showed increased expression of COL1A1 and α-SMA, and CCK-8 analysis showed increased proliferation of LX-2 cells.

Conclusions

Our study showed that miRNA-29b was highly expressed in peripheral blood exosomes and that PDGFA was highly expressed in liver tissues in children with BA. Exosomes overexpressing miRNA-29b caused hypomethylation of the DNA promoter region of PDGFA in hepatic stellate cells, promoted the high expression of PDGFA, activated hepatic stellate cells, and participated in BA-associated liver fibrosis, elucidating the pathogenesis of BA.

Biliary atresia

exosomes

liver fibrosis

miRNA-29b

Platelet-derived growth factor

Biliary atresia (BA) is a serious neonatal hepatobiliary disease. This condition is caused by intrahepatic and external bile duct obstruction leading to cholestasis, progressive liver injury and liver fibrosis, which seriously endanger the life of newborns [1]. At present, the pathogenesis of BA is still unclear; in particular, children with BA after Kasai surgery still have progressive liver fibrosis [2] and ultimately must undergo liver transplantation [3, 4]. Therefore, the pathogenesis of liver fibrosis in children with BA should be further investigated.

Exosomes are small vesicles with a diameter of 30–150 nm that are secreted by a variety of cells and are widely found in blood, urine, saliva and other body fluids [5–7]. Exosomes carry cell-specific substances, such as nucleic acids, proteins and lipids [8], which can have biological effects on recipient cells [9]. Therefore, exosomes represent a novel mode of intercellular communication, and exosomal miRNAs can be transferred to recipient cells to regulate gene expression, which may play an important role in many biological processes and diseases [10–12]. However, there are few studies on exosomes in the pathogenesis of BA.

Recent studies have shown that epigenetics plays an important role in the occurrence and development of biliary atresia. MiRNA-29b is a member of the microRNA family and plays an important regulatory role in the process of acquired immunity [12]. Our previous experiments found that miRNA-29b in the peripheral blood of children with BA have abnormally high expression [13, 14]. Wang et al. found that by downregulating miRNA-29c, DNMT3a and DNMT3b could be targeted to inhibit biliary atresia-related liver fibrosis [15]. Therefore, the abnormal expression of miRNA-29b may be involved in the pathogenesis of BA.

Platelet-derived growth factor (PDGF) can stimulate the proliferation and migration of hepatic stellate cells and promote the generation and deposition of collagen in hepatic stellate cells, which plays a very important role in the occurrence and development of hepatic fibrosis [16]. Our study found that peripheral blood exosomal miR-29b was highly expressed in children with biliary atresia, and the expression of DNA methyltransferase (DNMT) 1 was significantly decreased. The hypomethylation of the PDGF promoter was negatively correlated with the overexpression of PDGF. The promoter region of the PDGF gene has many CpG islands as methylation sites, and the PDGF gene is a methylation-sensitive gene [17]. Studies have shown that inhibition of methylation by 5-Azac (DNA methyltransferase inhibitor) can induce biliary atresia [17–19]. These results suggest that DNA hypomethylation is involved in the pathogenesis of biliary atresia, possibly through upregulation of PDGFA.

Overexpression of miRNA-29b and targeted inhibition of DNMT in peripheral blood exosomes of children with BA led to reduced overall gene methylation and overexpression of the methylation-sensitive gene PDGFA, which may lead to biliary atresia. Therefore, we initiated this study to elucidate the regulation of PDGF by miRNA-29b in exosomes. Overexpression activates the mechanism of hepatic stellate cells promoting biliary atresia-related liver fibrosis.

Patient specimens and cells

Fifteen patients with biliary atresia (BA) and 15 patients with choledochal cysts (CCs) admitted to the Affiliated Hospital of Zunyi Medical University from September 2020 to October 2023 were selected for intraoperative liver biopsy specimens. All BA patients were diagnosed with biliary atresia by biliary angiography and underwent Kasai gate enterostomy. The choledochal cyst group with normal liver function was used as the control group. The study was approved by the Human Ethics Committee of the Affiliated Hospital of Zunyi Medical University (Ethics number: KLLY-2020-110). Tissue and blood samples were immediately rapidly frozen and stored in a − 80°C refrigerator for RNA and protein extraction prior to the initiation of the study procedure.

The Jurkat human leukemia T-cell line and LX-2 human hepatic stellate cell line were purchased from Shanghai Zhongqiao Xinzhou. The two kinds of cells were cultured in RPMI-1640 and DMEM with 10% fetal bovine serum, a 1% concentration of 100 IU/mL penicillin and 100 mg/mL streptomycin sulfate. Cells were treated with 5-azacytidine (MCE) at a final concentration of 10 µM. Jurkat and LX-2 cells were inoculated at a density of 1× 10-6cells/ml and 5× 10 − 5 cells/ml, respectively.

Transfection

The Jurkat cells were infected with lentivirus (provided by Shanghai GeneChem Co., Ltd.) and stable strains with miRNA-29b overexpression, miRNA-29b inhibition and a negative no-load control were obtained. After 72 hours of infection with Jurkat cells by lentivirus, the fluorescence expression of GFP was observed under an inverted fluorescence microscope. More than 85% of the cells in the three groups expressed green fluorescence.

Exosome extraction and identification:

The collected peripheral blood or cell culture solution was isolated using Invitrogen™. The peripheral blood serum was collected after centrifugation at 12000 rpm for 5 min. The exosome isolation reagent was added to the serum and incubated at a static temperature of 4 ℃ for 30 min.

The samples were centrifuged at 10000 rpm for 10 min, the upper layer solution was discarded, precipitation of exosomes was performed, the precipitate was resuspended with PBS, and the samples were stored in the refrigerator at -80℃ for future use.

Exosome morphology was observed by transmission electron microscopy (Hitachi), an exosome particle size analyzer (NanoFCM), exosome fluorescently labeled antibodies (CD9, CD63, IgG), and nanoflow detection, and the protein index was detected by a NanoFCM instrument.

RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT‒PCR):

Total RNA was extracted from cells or tissues using TRIzol reagent (Thermo Fisher Technology Co., Ltd.). Quantitative reverse transcription polymerase chain reaction (qRT‒PCR) for total miRNAs was performed using the PrimeScriptTM RT Reagent Kit (perfect real time) and TB Green Kit Polymerase Chain Reaction Kit (TaKaRa, Japan), and CFX96 Touch TM fluorescence quantitative PCR (Bio-Rad) was performed. U6 was used as the endogenous control. The gene expression levels of DNMT1, DNMT3a, DNMT3b, COL1A1, α-SMA and PDGFA were determined by the SYBR Green method, with Gapdh as the internal reference. The primer sequences are shown in Supplementary Table 1. The results of quantitative reverse transcription polymerase chain reaction were analyzed by the 2-ΔΔCT method.

Western blot analysis:

The rapidly frozen liver samples and cells were homogenized in RIPA lysis buffer (Beijing Solebao Company). The same amount of protein was transferred to a PVDF membrane after electrophoresis (Millipore, USA). The membrane was blocked with 5% skim milk and incubated with primary antibodies against DNMT1, DNMT3a, DNMT3b, PDGFA, COL1A1 and α-SMA (GeneTex, Abcam). After incubation with the corresponding horseradish peroxidase-conjugated secondary antibody (Abcam), the exposure solution was prepared and exposed by a gel imaging system (Shanghai Tianneng Technology Co., Ltd.). The western blotting results were analyzed with ImageJ software.

Luciferase reporting experiment:

DNMT (DNMT1, DNMT3a and DNMT3b) mRNA 3ʹ- UTR fragments contain the miR − 29b binding sequence. Genomic DNA extraction of Jurkat cells, double luciferase plasmid pcDNA3.1 _Luci transformation, extraction, and cloning of the DNMT gene 3'-UTR were performed. Primers used for the DNMT − 3ʹUTR cloning are shown in Supplement Table 2. The construct sspcDNA3.1_Luci_DNMT3a_3 'UTR_wt and pcDNA3.1_Luci_DNMT3a_3' UTR_mut with binding site mutations, pcDNA3.1_Luci_DNMT3b_3 'utr_WT and pcDNA3.1_Luci_DNMT3b_3 'uTR_mut were transfected into cells with miRNA mimic or miRNA mimic NC. Cell extracts were prepared 24 h after transfection. A dual luciferase reporting and analysis system (Promega, Madison, WI, USA) was used to measure luciferase activity according to the manufacturer's protocol.

DNA extraction and methylation analysis:

Exosome samples of 100 µl-200 µl were loaded into a 2 ml centrifuge tube, and genomic DNA was extracted from the exosomes by a DNA extraction kit (Shanghai Jizhen Biological Co., Ltd.).

The conversion rate of bisulfite was determined using the Qiagen sulfite kit. Methylation levels of PDGF were measured by methylation-specific polymerase chain reaction analysis. The reaction components of bisulfite were as follows: DNA 1 µg; bisulfite solution 85 µl; DNA protection buffer 35 µl; and a total volume of 140 µl. The adsorption column and collection tube were prepared on the test tube stand, and all 8 mixtures were transferred to the MinElute adsorption column. DNA was collected and modified with bisulfite by a thermal cycler.

CCK-8 analysis:

Cell count: A total of 2x103 cells were cultured in each 96-well plate with a volume of 100 µL.

Ten microliters of CCK-8 reagent was added at 24 h, 48 h and 72 h after plating and incubated in the incubator for 30 min. Cell-free medium was used as a blank control.

OD values were recorded at 450 nm by an enzyme-labeling instrument.

Statistical analysis:

SPSS 29.0 was used for statistical analysis of experimental data, and GraphPad Prism 6 was used for mapping. The units of measurement are expressed as the mean ± standard error (mean ± SEM). The difference between multiple groups was compared by one-way ANOVA, and the difference between two groups was compared by two-independent sample t test. p < 0.05 was considered statistically significant.

Supplement table 1

Primers		Sequences
miR-29b	Forward	5′-GCCCTAGCACCATTTGAAA-3′
miR-29b	Reverse	5′-TGGTATCCTTGAGGGATTGGTTC-3′
U6	Forward	5'-GCTTCGGCAGCACATATACTAAAAT-3'
U6	Reverse	5'-CGCTTCAGAATTTGCGTGTCAT-3'
DNMT3a	Forward	5'-CAGCTTCCACGTTGCCTTCT-3'
DNMT3a	Reverse	5'-CATCTGCAAGCTGTCTCCCTTT-3'
DNMT3b	Forward	5'-TACACAGACGTGTCCAACATGGGC-3'
DNMT3b	Reverse	5'-GGATGCCTTCAGGAATCACACCTC-3'
PDGFA	Forward	5'-GCAAGACCAGGACGGTCATTT-3'
PDGFA	Reverse	5'-GGCACTTGACACTGCTCGT-3'
α‐SMA	Forward	5'-CTGCTGAGCGTGAGATTGTC-3'
α‐SMA	Reverse	5'-CTCAAGGGAGGATGAGGATG-3'
Col1A1	Forward	5'-GATT CCCTGGACCTAAAGGTGC-3'
Col1A1	Reverse	5'-AGCCTCTCCATCTTTGCCAGCA-3'
GAPDH	Forward	5'-CGCTCTCTGCTCCTCCTGTTC-3'
GAPDH	Reverse	5'-ATCCGTTGACTCCGACCTTCAC-3'

Cloning the 3 '-UTR region of DNMTs(DNMT1, DNMT3a, DNMT3b) gene. According to the action sites of miRNA and corresponding target genes, the upper and downstream sequences of corresponding mirNA-29b binding sites in the 3 '-UTR region of DNMT3a and DNMT3b mRNA were amplified. Wild-type and mutant DNMTs 3 '-UTR primer sequences were designed using Primer5, as shown in the following table.(Supplementary Table 2)

Table 2

was added to construct the primer sequence of luciferase reporter gene vector
gene	sequence(5’-3’)	Length of product
DNMT3a-1F	TAG GAATTC AACCCAGTTAGCAGCAG	362bp
DNMT3a-1R	TAG TCTAGA GCTCCCAAGTTCTCCTC
DNMT3a-2R	CGCTGTTTGAA ACTATGT TTATG	221bp
DNMT3a-3F	CATAA ACATAGT TTCAAACAGCG	182bp
DNMT3b-1F	TAG GAATTC GACAGCAGTCAGGGACAG	408bp
DNMT3b-1R	TAG TCTAGA TCCGTCATCTTTCAGCC
DNMT3b-2R	TCTACAAAA TATGGAGT GTAAGAAG	237bp
DNMT3b-3F	CTTCTTAC ACTCCATA TTTTGTAGA	224bp
Note: 1F ,1R are wild-type forward and reverse primers; 2R ,3F were mutant reverse and forward primers; The color font is the site of action, the red base is the unmutated base, and the blue base is the mutated base. The structure of wild-type forward primer was protective base TAG + EcoRⅠ cleavage site + primer sequence. Wild-type reverse primers consist of protective base TAG + Xbal restriction site + primer sequence.

Identification of miR-29b overexpression and elevated PDGFA in children with biliary atresia

Exosomes in the peripheral blood of the children with BA and CC were collected and observed by transmission electron microscopy (Fig. 1a). The expression of miRNA-29b in the peripheral blood exosomes of the BA and CC groups was detected, and the expression of miRNA-29b in the exosomes of the BA group was significantly higher than that of the CC group (Fig. 1b). The PDGFA transcription and protein levels in liver tissues of the BA group were significantly higher than those of the CC group (Fig. 1c, d).

Construction of stable transmissible strains and verification of exosomes

Jurkat cells were infected with lentivirus to obtain stable strains with miRNA-29b overexpression, miRNA-29b inhibition and a negative no-load control. After 72 hours of infection with lentivirus, the fluorescence expression of GFP in Jurkat cells was observed under an inverted fluorescence microscope to confirm the infection efficiency. More than 85% of cells in all three groups expressed green fluorescence (Fig. 2a). Exosomes secreted by Jurkat cells in the lentivirus-infected group and the nonlentivirus-infected group were collected, and the exosomes were observed by transmission electron microscopy. The morphology of the exosomes was disk-like (Fig. 2b). The expression of miRNA-29b in the miRNA-29b overexpression group was significantly higher than that in the blank control group and the negative control group. The expression of miRNA-29b in the miRNA-29b low expression group was significantly lower than that in the blank control and negative control groups (Fig. 2c). The expression of miRNA-29b in exosomes obtained from Jurkat cells in the miRNA-29b overexpression group was significantly higher than that in the blank control and negative control groups. The expression of miRNA-29b in exosomes obtained from Jurkat cells in the miRNA-29b low expression group was significantly lower than that in the blank control and negative control groups (Fig. 2d). Nanoparticle tracking analysis showed that the average particle size of the exosomes collected in the lentivirus-infected group was 88.35 nm, and the concentration was 0.89×1010 particles/mL, while that in the nonlentivirus-infected group was 82.14 nm. The concentration was 1.02×1010 particles/mL (Fig. 2e). By nanoflow detection of exosome surface proteins (CD9 and CD63), the positive expression rates of the exosome surface proteins CD9 and CD63 were 10.6% and 9.1%, respectively, in the lentiviral-infected group and 9.9% in the FITC-IgG control group due to the presence of GFP fluorescence from lentivirus in exosomes. Therefore, the results showed interference. In the nonlentiviral infection group, the positive expression rate of the exosome surface protein CD9 was 5.1%, the positive expression rate of the surface protein CD63 was 3.5%, and that of the homologous antibody control FITC-IgG was 0.0% (Fig. 2f).

Effects of miRNA-29b overexpression and inhibition in exosomes on the expression of DNMT3a, DNMT3b, PDGFA, COL1A1 and α-SMA in LX-2 cells and verification of miRNA-29b target genes by double luciferase assays

After 72 h of coculture with 50 ng/ml miRNA-29b-overexpressing exosomes and LX-2 cells, the mRNA and protein levels of LX-2 cells in each group were determined. The transcription levels and protein expression levels of DNMT3a and DNMT3b in the Up group were significantly lower than those in the blank control and negative control groups (Fig. 3a). The transcription level of PDGFA in the Up group was significantly higher than that in the blank control and negative control groups. The protein expression of PDGFA in the Up group was significantly higher than that in the blank control group (p < 0.05), but there was no significant difference between the Up group and the negative control group (p > 0.05) (Fig. 3b). The transcription levels and protein expression levels of COL1A1 and α-SMA in the Up group were significantly higher than those in the blank and negative control groups (Fig. 3c).

After 72 h of coculture with 50 ng/ml miRNA-29b low-expression exosomes and LX-2 cells, the mRNA and protein levels of LX-2 cells in each group were determined. The transcription levels of DNMT3a and DNMT3b in the Down group were significantly higher than those in the blank control and negative control groups (p < 0.05). The protein expression of DNMT3a in the Down group was significantly higher than that in the blank control and negative control groups. However, there was no significant difference in the protein expression of DNMT3b in the Down group compared with the blank and negative control groups (p > 0.05) (Fig. 3d). The transcription level and protein expression of PDGFA in the Down group were significantly lower than those in the blank control and negative control groups (Fig. 3e). The transcription levels and protein expression levels of COL1A1 and α-SMA in the Down group were significantly lower than those in the blank and negative control groups (Fig. 3f). A dual luciferase assay showed that DNMT3a and DNMT3b were the direct target genes of miR-29b-3p (Fig. 3g).

Effects of exosomes on the methylation of the PDGFA DNA promoter region in LX-2 cells and cell proliferation

LX-2 cells were cocultured with 50 ng/ml exosomes with high expression of miRNA-29b and exosomes with low expression of miRNA-29b for 72 h, and the methylation level of the DNA promoter region of PDGFA in LX-2 cells was detected by the bisulfite method. The DNA methylation level of PDGFA in the Up group was significantly lower than that in the blank control and negative control groups. The DNA methylation level of PDGFA in the Down group was significantly higher than that in the blank control and negative control groups (Fig. 4a, b). The proliferative capacity of LX-2 cells in the Up group was significantly higher than that in the blank control group and the negative control group after 72 h. The proliferative capacity of LX-2 cells in the Down group was significantly lower than that in the blank control group and the negative control group after 72 h (Fig. 4c, d).

Effect of the DNA methyltransferase inhibitor 5-Azac on LX-2 cells cocultured with miRNA-29b-overexpressing exosomes

When 50 ng/ml miRNA-29b-overexpressing exosomes were cocultured with LX-2 cells and the DNA methyltransferase inhibitor 5-Azac was added at the same time, the transcription levels and protein expression levels of DNMT3a and DNMT3b in the Up + 5Azac group were significantly lower than those in the blank and negative control groups (Fig. 5a). The transcription level and protein expression of PDGFA in the Up + 5Azac group were significantly higher than those in the blank and negative control groups (Fig. 5b). The transcription levels and protein expression levels of COL1A1 and α-SMA in the Up + 5Azac group were significantly higher than those in the blank and negative control groups (Fig. 5c).

LX-2 cells were cocultured with 50 ng/ml exosomes with low expression of miRNA-29b, and 10 µM of the DNA methyltransferase inhibitor 5-Azac was added. The transcription levels and protein expression levels of DNMT3a and DNMT3b in the Down + 5Azac group were significantly lower than those in the blank and negative control groups (Fig. 5d). The transcription level and protein expression of PDGFA in the Down + 5Azac group were significantly higher than those in the blank and negative control groups (Fig. 5e). The transcription levels and protein expression levels of COL1A1 and α-SMA in the Down + 5Azac group were significantly higher than those in the blank and negative control groups (Fig. 5f).

Regulation of PDGFA DNA promoter methylation in LX-2 cells by exosomes + 5Azac

LX-2 cells were cocultured with 50 ng/ml exosomes with high miRNA-29b expression and exosomes with low miRNA-29b expression. Moreover, 10 µM of the DNA methyltransferase inhibitor 5-Azac was added. After 72 h of culture, the methylation level of the PDGFA DNA promoter region in LX-2 cells was detected by the bisulfite method. The methylation level of PDGFA DNA in the Up + 5-Azac group was significantly lower than that in the blank control and negative control groups. The DNA methylation level of PDGFA in the Down + 5-Azac group was significantly lower than that in the blank control and negative control groups (Fig. 6a, b). The proliferative capacity of LX-2 cells in the Up + 5Azac group was significantly higher than that in the blank and negative control groups after 72 h, and the proliferative capacity of LX-2 cells in the Down + 5Azac group was significantly higher than that in the blank and negative control groups after 72 h (Fig. 6c, d).

Exosomes are secreted by a variety of cells and widely exist in different body fluids [20, 21].

Exosomes have been extensively studied due to their functions, such as intercellular communication, genetic material exchange, antigen presentation, and adjustment of tissue metabolism [22]. Exosomes can be used not only as biomarkers for disease diagnosis but also for disease treatment due to their special functions [23]. By measuring lncRNA H19 levels in the liver and serum exosomes of BA patients, Li et al. [24] found that lncRNA H19 levels were positively correlated with the severity of liver fibrosis and found that lncRNA H19 deficiency could alleviate the proliferation of bile duct cells and the progression of liver fibrosis in biliary ligation model mice to a certain extent. LncRNA H19 is a key regulator of bile acid homeostasis [25] and has an important relationship with bile duct cell proliferation and cholestatic liver injury [26]. LncRNAH19-overexpressing exosomes, which may be derived from bile duct cells, are taken up by hepatocytes and regulated through different signaling pathways, leading to the proliferation of bile duct cells, cholestatic liver injury and the occurrence of liver fibrosis [27–30]. This finding suggests that exosomes may be involved in the pathogenesis of BA. Our team's previous experiments found that miRNA-29b was highly expressed in peripheral blood and liver tissues of children with BA [14], and the highly expressed miRNA-29b in peripheral blood came from exosomes. In this study, the expression of miRNA-29b in the peripheral blood exosomes of BA was detected, miRNA-29b was found to be highly expressed, and overexpressed miRNA-29b entered the liver tissue and was involved in the pathogenesis of BA.

MiRNAs are a class of noncoding RNAs with a length of approximately 22 nt that are mainly involved in the regulation of the post-transcriptional level of genes. These molecules can form an RNA-induced silencing complex in the cytoplasm [31] and can bind to the mRNA of specific genes to degrade the mRNA or inhibit its translation [32]. miRNAs are involved in a variety of biological processes, including development, viral defense, hematopoietic processes, organogenesis, cell proliferation and apoptosis, and lipid metabolism [33]. One research team [14] found that the liver tissue of children with BA was hypomethylated. Zhang et al. [34] also confirmed that miRNA-29b can target the expression of DNMT3a and DNMT3b and then regulate the level of DNA methylation.

MiRNA-29b can also lead to DNA hypomethylation of CD4 + T cells in systemic lupus erythematosus by indirectly targeting DNA methyltransferase 1 [35]. Chen et al. [36] found that DNMT1, DNMT3a and DNMT3b were the target genes of miRNA-29b through the analysis of dual luciferase reporter genes, and transfection of miRNA-29b could inhibit the expression of DNMT3a and DNMT3b in Kasumi-1 cells. In this study, miRNA-29b was found to be highly expressed in peripheral blood exosomes of children with BA. Therefore, Jurkat cells with abnormal expression of miRNA-29b were constructed, and their exosomes were collected and cocultured with LX-2 cells. In LX-2 cells with exosomes with abnormal expression of miRNA-29b, the expression of DNMT3a and DNMT3b was regulated by the abnormally expressed miRNA-29b, and DNMT3a and DNMT3b were directly inhibited by the DNA methyltransferase inhibitor 5-Azac. Therefore, exosomes with high expression of miRNA-29b may act on hepatic stellate cells, leading to decreased expression of DNMT3a and DNMT3b in hepatic stellate cells and participating in the pathogenesis of BA.

DNA methylation is an epigenetic mechanism that regulates gene expression by recruiting proteins involved in gene inhibition or by inhibiting the binding of transcription factors to DNA [37, 38]. In the current research on hepatic fibrosis, many teams focus on DNA methylation levels [39]. In studies of biliary atresia, Alu and LINE-1 elements were significantly hypomethylated in BA patients [13]. Some scholars have found that hypomethylation and overexpression of the autotoxin promoter may play a promoting role in the pathogenesis of BA liver fibrosis [40]. Randolph [18] et al. found that DNA hypomethylation caused bile duct defects in zebrafish and proposed epigenetic activation of IFN-γ signal transduction as a common etiological mechanism of BA intrahepatic bile duct defects. Li et al. [41] found that the abnormal methylation of the Foxp3 promoter in immunosuppressive regulatory T cells impaired the inhibitory function of the cells and aggravated the inflammatory damage of BA. Zenobia [17] et al. detected the bile duct cells of BA patients and showed decreased DNA methylation, indicating that PDGFA was hypomethylated in BA, which confirmed the increased transcription and protein expression of PDGFA in BA livers and indicated that PDGF was a new candidate in the pathogenesis of BA. This research team previously found that DNA hypomethylation occurred in BA liver tissue [14].

Epigenetics plays an important role in the occurrence and development of biliary atresia. When the liver is damaged, a large amount of PDGF is secreted to stimulate the proliferation of hepatic stellate cells, transform into muscle fibroblast-like cells, and promote the migration of stellate cells to accumulate in the damaged area of inflammation [42]. Studies have shown that PDGF plays an important role in liver fibrosis [43]. In this study, we found that the transcription level and protein expression of PDGFA were significantly increased in LX-2 cells cocultured with exosomes overexpressing miRNA-29b, and the PDGFA DNA was hypomethylated. DNA methylation occurred in the CG sequence-concentrated region of DNA, and after the methylation occurred at this location, it can cause transcription factors and RNA synthase to bind to this section of DNA, promoting DNA transcription and subsequent mRNA translation [44]. In this study, in LX-2 cells, miRNA-29b indirectly regulated the DNA methylation level of PDGFA by targeting the expression of DNMT3a and DNMT3b, resulting in a change in PDGFA expression, which was also affected by the DNA methyltransferase inhibitor 5-Azac. This result indicates that the high expression of miRNA-29b in exosomes of children with BA may act on hepatic stellate cells, resulting in the decreased expression of DNMT3a and DNMT3b in hepatic stellate cells, resulting in the hypomethylation of the DNA promoter region of PDGFA and the high expression of PDGFA.

PDGF is a core factor that can repair acute and chronic tissue damage, which can strongly stimulate the proliferation and migration of hepatic stellate cells and promote the production and deposition of collagen, playing a very important role in the occurrence and development of hepatic fibrosis [45, 46]. Some scholars have found that PDGF can improve cell proliferation [47], and the expression of α-SMA in the liver of transgenic mice overexpressing PDGFA was also significantly increased [48], indicating that PDGFA is involved in the activation of hepatic stellate cells and liver fibrosis. In this study, we found that overexpressed PDGFA existed in the liver tissue of children with BA, and we concluded that overexpressed PDGFA was involved in BA-related liver fibrosis. In LX-2 cells, miRNA-29b indirectly regulates the DNA methylation level of PDGFA by targeting the expression of DNMT3a and DNMT3b, which leads to changes in the expression of PDGFA, thus affecting the proliferative ability of LX-2 cells and altering their collagen-forming ability.

Therefore, in children with BA, exosomes with high expression of miRNA-29b may act on hepatic stellate cells in liver tissue to cause their high expression of PDGFA, activate hepatic stellate cells, produce a large number of collagen fibers and extracellular matrix, and cause hepatic fibrosis.

In BA, miRNA-29b was highly expressed in peripheral blood exosomes, and PDGFA was highly expressed in liver tissues. Exosomes overexpressing miRNA-29b cause the DNA promoter region of PDGFA in hepatic stellate cells to be hypomethylated, which promotes the high expression of PDGFA and activates hepatic stellate cells; these changes may be involved in BA hepatic fibrosis, providing a new idea for the diagnosis and treatment of BA hepatic fibrosis. This study provides a basis for further understanding biliary atresia-related liver fibrosis from the perspective of exosomes and epigenetics and explores the pathogenesis of BA-related liver fibrosis.

BA, Biliary atresia ; PDGF, Platelet-derived growth factor ; DNMT ,DNA methyltransferase; CC, choledochal cysts ; qRT‒PCR, RNA extraction and quantitative reverse transcription polymerase chain reaction; 5Azac, 5-azacytidine ;

AUTHOR CONTRIBUTIONS Dr. Xilin Liao, Rui Liu, Teng Zhou, prepared the manuscript equally and should be considered co-first authors. Dr. Xiaohuan Zhao, Xiangang Xiong, Kaizhi Zhang, Zebing Zheng for providing samples for this study, and Dr. Xingrong Xia, Chengyan Tang, Yuan Gong, Qing Du, Lu Huang, Daiwei Zhu, Wankang Zhou, Yuanmei Liu, Zhu Jin for technical assistance.

ETHICAL APPROVAL The study was approved by the Human Ethics Committee of the Affiliated Hospital of Zunyi Medical University (Ethics number: KLLY-2020-110). Informed consents to participate in the study have been obtained from participants and their legal guardians.

DATA AVAILABILITY The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

FUNDING This work was supported by the National Natural Science Foundation of China (81760099).

ACKNOWLEDGMENTS We thank Dr. Xilin Liao, Rui Liu, Teng Zhou, Xiaohuan Zhao, Xiangang Xiong,Kaizhi Zhang, Zebing Zheng for providing samples for this study, and Dr. Xingrong Xia，Chengyan Tang, Yuan Gong, Qing Du, Lu Huang, Daiwei Zhu, Wankang Zhou, Yuanmei Liu, Zhu Jin for technical assistance.

CONFLICTS OF INTEREST No fnancial or non-fnancial benefts have been received or will be received from any party related directly or indirectly to the subject of this article. The authors have no confict of interest to declare.

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No competing interests reported.

Supplementaryfile.docx

MiRNA-29b accelerates the PDGF in exosomes and stimulates hepatic stellate cells to promote liver fibrosis in biliary atresia

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

INTRODUCTION

METHODS

Patient specimens and cells

Transfection

Exosome extraction and identification:

RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT‒PCR):

Western blot analysis:

Luciferase reporting experiment:

DNA extraction and methylation analysis:

CCK-8 analysis:

Statistical analysis:

RESULTS

Identification of miR-29b overexpression and elevated PDGFA in children with biliary atresia

Construction of stable transmissible strains and verification of exosomes

Effect of the DNA methyltransferase inhibitor 5-Azac on LX-2 cells cocultured with miRNA-29b-overexpressing exosomes

Regulation of PDGFA DNA promoter methylation in LX-2 cells by exosomes + 5Azac

DISCUSSION

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1