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JHEP Reports logoLink to JHEP Reports
. 2026 Jan 29;8(5):101755. doi: 10.1016/j.jhepr.2026.101755

The endonuclease MCPIP1 protects against liver cancer development in a sex-dependent manner by modulating β-catenin and CREB1

Oliwia Kwapisz 1,2,†, Paulina Marona 1,†, Judyta Gorka 1, Rafał Myrczek 1,2, Ester Gonzalez-Sanchez 3,4,&, Esther Bertran 3,4, Jerzy Kotlinowski 1, Maciej Głuc 1, Ania Alay 5,6, Natalia Pydyn 1, Monika Kujdowicz 7, Emilio Ramos 3,4,8, Isabel Fabregat 3,4, Katarzyna Miekus 1,⁎
PMCID: PMC13091202  PMID: 41955682

Abstract

Background & Aims

Monocyte chemoattractant protein-induced protein 1 (MCPIP1), encoded by ZC3H12A, is a negative regulator of inflammation and tumorigenesis. While its role has been implicated in various cancers, the function of MCPIP1 in hepatocellular carcinoma (HCC) remains poorly understood. This study explored the contribution of hepatocyte-specific MCPIP1 loss to HCC pathogenesis, highlighting its role in overcoming the inherent tumor resistance observed in female mice.

Methods

Liver tissues (n ≥5 per group) and primary hepatocytes (n ≥3 per group) were evaluated using western blotting, immunohistochemistry, immunofluorescence, RNA sequencing and pathway-enrichment analysis. The expression levels of MCPIP1 in HCC were measured by quantitative reverse-transcription PCR. The results are presented as mean ± SD, Student's t or Mann‒Whitney U tests were used for statistical analysis of two groups. For more than two groups, ordinary two-way ANOVA was used.

Results

The hepatocyte-specific loss of MCPIP1 markedly promoted fibrosis and tumorigenesis, particularly in female mice, disrupting the normal sex-related protection observed in the diethylnitrosamine model. As determined by next-generation sequencing and bioinformatics analysis, oncogenic and fibrotic programs, including the EMT, Wnt/β-catenin, and JAK/STAT3 pathways, were activated in MCPIP1 knockout livers. These molecular events activated β-catenin, c-Met, and IL-6/STAT3/NF-κB signaling, and they enhanced fibrotic remodeling. In MCPIP1-deficient hepatocytes, active β-catenin and CREB1 accumulated in the nucleus, and the expression of protumorigenic targets, such as Spp1, Tgfb2, and Adam17 increased. Moreover, MCPIP1 expression was significantly downregulated in human HCC tissues and correlated with tumor progression.

Conclusions

MCPIP1 plays a protective role against inflammation-driven hepatocarcinogenesis, particularly in females, by restraining fibrotic remodeling and oncogenic signaling. The downregulation of MCPIP1 expression promotes a tumor-promoting microenvironment through the coordinated activation of the β-catenin, STAT3, and CREB1 pathways.

Keywords: MCPIP1, Zc3h12a, HCC, fibrosis, EMT, β-catenin, CREB1

Graphical abstract

Image 1

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Highlights

  • •

    MCPIP1 deficiency triggers liver fibrosis, inflammation, and immune cell infiltration.

  • •

    Loss of MCPIP1 promotes hepatocellular carcinoma development in female mice.

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    MCPIP1 regulates Wnt/β-catenin, STAT3, and NF-κB signaling in hepatocytes.

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    MCPIP1 knockout activates β-catenin and CREB1, promoting malignant transformation.

Introduction

Hepatocellular carcinoma (HCC) is among the most prevalent and clinically challenging malignancies worldwide, with the annual number of cases anticipated to surpass one million by 2025.1 In approximately 90% of patients, HCC is diagnosed at an advanced stage, making curative surgery difficult and contributing to a consistently poor prognosis, with a 5-year survival rate of only 18%.2,3 Therapeutic options are limited, with the sorafenib multikinase inhibitor showing a survival benefit of only 3 months.3 Therefore, there is a critical need for earlier diagnosis and more effective treatment options for HCC.

Compared with women, men are at a significantly greater risk of developing HCC, with an approximately three to five times increased risk. This sex disparity is also evident in rodent models of HCC. Estrogen administration in male mice has been shown to suppress the development of chemically induced HCC.4 However, the mechanisms underlying this sex difference and the anticancer effects of estrogen remain poorly understood. The administration of diethylnitrosamine (DEN) to mice leads to increased IL-6 concentrations in males, this response is suppressed in females by estrogens.4

Interleukin 6 (IL-6) is among the main inducers of inflammation and is a catalyst for DNA damage, mutagenesis, hepatic cell death, and compensatory proliferation.[4], [5], [6] This inflammatory environment is closely associated with the hyperactivation of key signaling pathways, including the NF-κB, MAPK, STAT3, and AKT pathways, which are essential for HCC development.[6], [7], [8], [9] Moreover, tumor promotion depends on interactions between initiated cells and their microenvironment, which exerts constant evolutionary pressure on early neoplastic cells through the production of proinflammatory cytokines, chemokines, and reactive oxygen species.7 Understanding the molecular mechanisms underlying the malignant conversion of premalignant lesions during HCC development is critical for delaying or preventing HCC development.

Monocyte chemoattractant protein-induced protein 1 (MCPIP1 or ZC3H12A, also known as Regnase-1) has emerged as a key regulator of the inflammatory response and negatively modulates cellular inflammation. The primary function of MCPIP1 involves binding to stem‒loop structures in the 3′ untranslated regions of major proinflammatory cytokines (IL-6, IL-1β, and IL-12b).10 Additionally, MCPIP1 negatively regulates JNK and NF-κB activity,11 indicating its potential to regulate cancer-associated features, including proliferation, angiogenesis, growth arrest, and the modulation of a proinflammatory microenvironment.

Moreover, we have previously reported that MCPIP1 expression is reduced in patients with MASLD (metabolic dysfunction-associated steatotic liver disease) and that MCPIP1 inhibits hepatic stellate cell activation.12,13 However, the mechanism through which MCPIP1 regulates HCC development remains unexplored.

For the first time, the present study demonstrated that MCPIP1 protects against HCC development in females, acting as a necessary second hit for HCC occurrence. A time-dependent transcriptomic analysis of tumor initiation and development revealed that the absence of MCPIP1 first changes the set of genes important for K-Ras signaling, angiogenesis and epithelial-to-mesenchymal transition (EMT), as well as the genes important for β-catenin signaling. Moreover, the absence of MCPIP1 in hepatocytes triggers the expression of Ctgf, Mmp2, Hgf, Tgfb2, Spp1 and Il6, which are integral to tumor development and immune cell activation, thus promoting significant fibrotic alterations and enhanced tissue remodeling in the liver. Moreover, a lack of MCPIP1 leads to β-catenin and cAMP-responsive element-binding protein 1 (CREB1) activation, which is crucial for HCC development. The present study revealed that liver-specific MCPIP1 knockout has notable molecular consequences, indicating that MCPIP1 plays a protective role during hepatocarcinogenesis.

Materials and methods

Patient samples

Samples from tumor and adjacent non-tumor liver tissues were obtained from patients during surgical procedures at Bellvitge University Hospital (HUB). The samples were of histological grade 1 or 2. Human tissues were collected after receiving the required written informed consent from each patient and with the approval of the Institutional Review Board (Comité Ético de Investigación Clínica-CEIC, University Hospital of Bellvitge; approval number PR202/22). Patients provided written consent, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki.

Animal studies

Animal experiments were conducted in accordance with the Institutional Animal Care and II Local Ethics Committee of the Institute of Pharmacology, Polish Academy of Sciences (approval numbers 254/2018, 112/2023 and 935A/2024). The mice were handled in accordance with the regulations of national and local animal welfare under specific pathogen-free conditions, and they were provided water and food ad libitum. Two-week-old mice were intraperitoneally administered diethylnitrosamine (DEN; Sigma‒Aldrich, St. Louis, MO, USA) dissolved in NaCl at a concentration of 25 mg/kg body weight. Tissues were collected 40 weeks after birth and 12, 24, or 40 weeks after DEN administration. Liver lobes were divided for RNA and protein isolation (lobus hepatis sinister medialis and lobus hepatis caudatus for reserve) and histology (lobus hepatis sinister lateralis and dexter lateralis).

Primary hepatocyte isolation and culture

Primary hepatocytes were isolated from Zc3h12afl/fl and Zc3h12afl/flAlbCre mice via collagenase perfusion as described previously.14 Briefly, the animals were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally. Next, the livers were perfused via the inferior vena cava with 20 ml of Krebs-Ringer buffer supplemented with 0.1 mM EGTA, followed by 25 ml of digestion solution (Krebs-Ringer containing 4.76 mM CaCl2 and 200 U/ml collagenase IV (Gibco)). After the liver was excised, it was disrupted in a Petri dish containing 10 ml of complete medium and filtered through a 100 μm cell strainer. The cells were subsequently centrifuged (50 × g, 2 min, 4 °C), after which the pellet was resuspended in 10 ml of culture medium. The cells were added to Percoll solution, mixed thoroughly, and centrifuged (50 × g, 4 °C). The viability of isolated hepatocytes, as estimated by trypan blue staining, was usually 80–90%. The cells were seeded onto collagen I-coated 12-well plates (50 μg/ml; Becton Dickinson) at 150,000 viable cells/well in Williams E medium (Lonza) supplemented with 10% FBS, 2 mM l-glutamine (Lonza), 1% penicillin‒streptomycin (Lonza), 1 mM sodium pyruvate solution (Lonza), 1% ITS Liquid Media Supplement (Sigma), 7.5 μg/ml hydrocortisone (Sigma), and 20 ng/ml epidermal growth factor (Sigma), and the cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. After 5 h of attachment, the cultures were washed with phosphate-buffered saline and maintained in medium for the duration of the experiment.

Statistical analysis

Student's t tests or Mann‒Whitney U tests were used for statistical analysis of two groups. For more than two groups, ordinary two-way ANOVA was used. The number of animals or patient samples is indicated in the figure legends. All the results are presented as the mean ± SD. For graph preparation and statistical analysis, GraphPad Prism 10 (San Diego, CA, USA) was used. The p values are marked with asterisks in the charts (∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 and ∗∗∗∗p <0.0001 vs. control).

The methods used for transduction, western blot analysis, mRNA extraction, real-time PCR analysis and staining are described in the supplementary materials and methods.

Results

MCPIP1 expression is downregulated in human HCC, and hepatocyte-specific Zc3h12a knockdown induces fibrosis and increases tumor growth in vivo

Previous research has demonstrated reduced levels of MCPIP1 in various tumors, including breast cancer,15 clear cell renal cell carcinoma (ccRCC),16,17 and melanoma.18 In this study, the role of MCPIP1 in HCC development was investigated. Analysis of HCC patient samples revealed a significant decrease in MCPIP1 expression in HCC tissue (Fig. 1A–D). Compared with adjacent non-tumor tissue samples, stage 1 and 2 HCC tissue samples presented significantly lower ZC3H12A expression (Fig. 1A–C). Across individual cancerous tissues, a decrease in the MCPIP1 level was observed in most samples regardless of tumor stage compared with non-cancerous tissues (Fig. 1A–C). Moreover, analysis of HCC patient databases revealed that the protein level of MCPIP1 decreased with HCC progression (p = 5.70E-03) (Fig. 1D).

Fig. 1.

Fig. 1

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ZC3H12A expression decreases in tumor samples and promotes HCC development in mice after DEN administration.

(A) mRNA expression of MCPIP1 (ZC3H12A) in stage 1 and 2 HCC patient samples compared to non-tumor tissue, quantified with real-time PCR, EF2 was used as the reference gene. Non-tumor stage 1 n = 32, non-tumor stage 2 n = 31, tumor stage 1 n = 34, tumor stage 2, n = 31. p values were estimated using one-way ANOVA, ∗p <0.05. (B) Correlation between non-tumor stage 1 and tumor stage 1 samples, n = 19. (C) Correlation between non-tumor stage 2 and tumor stage 2 samples, n = 18. p values were estimated using unpaired Student’s t test, ∗p <0.05. (D) Analysis of HCC patient databases proteomic expression of ZC3H12A gene (p = 5.70E-03). (E) Schematic representation of the in vivo model for Zc3h12afl/flAlbCre mice with Zc3h12a gene knockout in hepatocytes and cholangiocytes and Zc3h12afl/fl control mice. (F,G) Tumor number in Zc3h12afl/flAlbCre and Zc3h12afl/fl mice n = 15, 40 weeks after DEN administration. The results are presented as the mean ± SD with dot plot. p values were estimated using unpaired Student’s t test, ∗∗p <0.01, ∗∗∗∗p <0.0001. (H,I) Representative images of Masson's trichrome and Picrosirius red staining for Zc3h12afl/flAlbCre and Zc3h12afl/fl mice, 40 weeks after DEN administration. DEN, diethylnitrosamine; HCC, hepatocellular carcinoma.

To determine whether the lack of MCPIP1 in hepatocytes and the resulting inflammation are involved in chemical hepatocarcinogenesis, we used a DEN-induced HCC model. For the present study, we generated an in vivo mouse model with liver-specific Zc3h12a gene knockout by crossing Zc3h12alox/lox mice with liver-specific Cre-expressing AlbCre transgenic mice (Fig. S1A). The mice were designated Zc3h12afl/flAlbCre mice (Fig. 1E).19

HCC predominantly affects men, and this sex discrepancy is also observed in mice exposed to the chemical carcinogen DEN. A single dose of DEN given to 2-week-old mice is sufficient to induce HCC, similar to human HCC, in 100% of male mice; however, females are largely resistant to carcinogenesis.20 Surprisingly, a lack of MCPIP1 in female hepatocytes significantly induced tumorigenesis. Almost all the females in the Zc3h12afl/flAlbCre group developed tumors, whereas in the control group, only 2/15 females presented one small nodule (Fig. 1F). In the present study, all male mice developed tumors after 40 weeks of DEN administration. Compared with wild-type mice, Zc3h12afl/flAlbCre mice developed more tumors (Fig. 1G). These results indicated that MCPIP1 expression in the hepatocyte compartment is important for protection against HCC development, especially in female mice.

Masson’s trichrome staining revealed collagen deposition, indicating fibrotic changes, with a more pronounced effect in Zc3h12afl/flAlbCre mice (Fig. 1H). Picrosirius red staining revealed the greatest degree of collagen deposition in hepatocytes from mice lacking MCPIP1 (Fig. 1I), suggesting enhanced fibrotic processes in addition to neoplastic changes.

Zc3h12a knockout mice that were not treated with DEN did not develop tumors (data not shown), and no macroscopic differences were observed between the sexes. However, a lack of MCPIP1 in hepatocytes induced intrahepatic bile duct pathology in the liver parenchyma and collagen deposition in Zc3h12afl/flAlbCre mice, as indicated by Masson's trichrome and Picrosirius red staining (Fig. S1C and D). Moreover, there was a slight increase in the influx of CD45-positive cells into the livers of Zc3h12afl/flAlbCre mice (Fig. S1E). Analysis of fibrosis-associated transcripts revealed that the absence of MCPIP1 significantly upregulated expression of Ctgf, a key mediator of tissue remodeling and fibrosis, as well as the expression of the mesenchymal markers Vim and Fn1. Additionally, Mgl2 transcript levels were increased in Zc3h12afl/flAlbCre mice (Fig. S1F). Further, increased protein levels of Yes1, RhoA and Yap were detected in the livers of Zc3h12afl/flAlbCre mice (Fig. S1G). These results suggested that the fibrotic changes are induced by a lack of MCPIP1 expression in hepatocytes (Fig. S1).

MCPIP1 liver knockout changes hepatocyte metabolism and increases fibrosis and inflammation

Glutamine synthetase in the liver is expressed in a small, perivenous population of highly specialized hepatocytes, and it plays a key role in nitrogen metabolism and ammonia detoxification. The number of glutamine synthetase-positive cells was increased in the livers of Zc3h12a (which encodes MCPIP1) knockout mice and after DEN administration (Fig. 2A, B). In addition, the absence of Zc3h12a in hepatocytes increased the expression of a-SMA, which is positively correlated with the degree of fibrosis. a-SMA staining revealed enhanced fibrotic processes in female mice lacking MCPIP1 in hepatocytes (Fig. 2C). Immunofluorescence staining for the leukocyte marker CD45 and the monocyte marker CD68 revealed increased staining intensity in Zc3h12afl/flAlbCre mice (Fig. 2D,E), especially in areas with tumor cells (Fig. 2F), indicating the activation of the microenvironment in MCPIP1-deficient livers. The transcript levels of macrophage galactose-type C-type lectin 2 (Mgl2), Cd3e (T cell marker), and Cd14 (monocyte/macrophage marker) were evaluated. Treatment of Zc3h12afl/flAlbCre mice with DEN resulted in the highest levels of Mgl2 and Cd14, and Zc3h12afl/flAlbCre mice that did not receive DEN exhibited the highest transcript levels of Cd3e (Fig. 2G). Moreover, the expression of the proinflammatory markers Tnfa, Ifng, Csf2 and Casp1 was increased in Zc3h12afl/flAlbCre mice 12 and 24 weeks after DEN administration (Fig. 2H).

Fig. 2.

Fig. 2

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MCPIP1 liver knockout changes hepatocyte metabolism, increases fibrosis and inflammation.

(A) Representative images of glutamine synthetase immunofluorescent staining of liver Zc3h12afl/flAlbCre and Zc3h12afl/fl female mice 40 weeks after DEN administration (Hoechst for nuclei). (B) Calculation of mean thickness of GS-positive hepatocytes layer around central vein. (C- E) Representative images of α-SMA, CD45 and CD68 immunofluorescent staining of liver Zc3h12afl/flAlbCre and Zc3h12afl/fl female mice 40 weeks after DEN administration (Hoechst for nuclei). (F) Representative images of α-SMA and CD45 immunofluorescent staining of liver Zc3h12afl/flAlbCre and Zc3h12afl/fl male mice (Hoechst for nuclei). (G) mRNA expression level of Mgl2, Cd3e, Cd14. EF2 was used as the reference gene. Females; n = 5 per group, males; n = 4-5 per group. (H) mRNA expression level of Tnfa, Ifng, Csf2, Casp1. Actb was used as the reference gene. The results are presented as the mean ± SD with dot plot. p values were estimated using two-way ANOVA, ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001. DEN, diethylnitrosamine; GS, glutamine synthetase.

MCPIP1 knockout leads to increased activation of the c-met and β-catenin signaling pathways and drives malignant HCC phenotypes by activating the STAT3/NFκB pathway in female mice

Among the principal pathways involved in HCC development, the Wnt/β-catenin pathway, c-Met protooncogene activation, and EMT play prominent roles at 40 weeks after DEN administration, Zc3h12afl/flAlbCre mice exhibited stronger β-catenin and c-Met signals, indicating that a lack of MCPIP1 may enhance changes that lead to liver damage (Fig. 3A,B). In MCPIP1-deficient livers, immunohistochemical analyses revealed increased β-catenin activation in both the membrane and the cytoplasm of hepatocytes (Fig. 6A). Additionally, the expression of Ctnnb1, Myc, and Mmp2 significantly increased, suggesting that fibrosis progression occurred in females (Fig. 2C,D).

Fig. 3.

Fig. 3

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MCPIP1 knockout leads to the increased activation of the c-Met and β-catenin signaling pathways and drives HCC malignant phenotypes by activating the STAT3/NFκB pathway in female mice.

(A,B) Representative images of β-catenin and c-Met IHC staining of liver 42 weeks old Zc3h12afl/flAlbCre and Zc3h12afl/fl mice males/females after DEN administration. (C,D) mRNA expression level of Ctnnb1, Mmp2 and Myc. EF2 was used as the reference gene. 42-weeks old females N = 5-9, males = 4-6 per group. (E-H) Western blot and densitometric analysis of STAT3, phospho-STAT3 (Y705), NF-κB, phospho-NF-κB (S536), p38 and phospho-p38 (T180/Y182), ERK protein level in Zc3h12afl/flAlbCre and Zc3h12afl/fl female and male mice 40 weeks after DEN administration, n = 5 per group, densitometric quantification with β-actin as the loading control. The results are presented as the mean ± SD with dot plot. p values were estimated using unpaired Student’s t test or Mann-Whitney U test, ∗p <0.05, ∗∗p <0.01. DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; IHC, immunohistochemistry.

Fig. 6.

Fig. 6

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CREB1 as key player in liver changes after Zc3h12a knockout.

(A) Confocal staining of β-catenin phosphorylated on S552 and S675 in hepatocytes of females Zc3h12afl/flAlbCre mice and Zc3h12afl/fl control mice (DAPI for nuclei). (B) Analysis of β-catenin (S675) and non-phosphorylated (S45) β-catenin protein level in cytoplasmic and nuclear fraction in hepatocytes of Zc3h12afl/flAlbCre mice and Zc3h12afl/fl control mice. Histon H3 was used as nuclear loading control and α-tubulin was used as cytoplasmic loading control. (C) mRNA expression level of Creb1, Tgfb2, Spp1, Adam17 and Il-6. EF2 was used as the reference gene. 8-week-old females, n = 3 per group. (D) Analysis of NF-kB, phospho-NF-kB (S536), Akt, phospho-Akt (S473), ERK1/2, phospho-ERK (T202/Y204), c-Met, phospho-c-Met (Y1234/1235), STAT3, phospho-STAT3 (Y705), with β-actin as the loading control in Zc3h12afl/flAlbCre and Zc3h12afl/fl hepatocytes isolated from females and males. (E,F) Analysis of CREB1 (S133) protein level in Zc3h12afl/flAlbCre and Zc3h12afl/fl hepatocytes isolated from females and males (indicated on the graph), n = 2-4 per group, densitometric quantification with β-actin as the loading control. (G) Confocal staining of Creb1 phosphorylated on Ser133 in hepatocytes of Zc3h12afl/flAlbCre and Zc3h12afl/fl control mice (DAPI for nuclei). The results are presented as the mean ± SD with dot plot. P values were estimated using unpaired Student’s t test, Mann-Whitney U test, ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001, ∗∗∗∗p <0.0001.

Immunohistochemistry analysis of β-catenin revealed that a lack of MCPIP1 led to strong β-catenin activation, even in the absence of DEN (Fig. S2A). In mice that did not receive DEN, the lack of MCPIP1 in the livers of mice of both sexes led to a significant increase in the level and activation of c-Met (Fig. S2B–D). Western blot analysis confirmed that the level of the active c-Met receptor phosphorylated at Tyr1234/1235 increased (Fig. S2C and D). Furthermore, the loss of MCPIP1 led to increased transcript levels of the fibrosis and the inflammatory response markers Mmp2, Il1b, Ctnnb1, Hgf, Cxcl12, Cxcr4, Zeb1 and Twist (Fig. S2E and F). Furthermore, females presented more pronounced differences (Fig. S2E and F).

MCPIP1 plays a pivotal role as a negative regulator of NF-κB, which is important for tumor promotion in inflammation-associated liver cancer.7,11 Compared with that in the control group, the total and active phosphorylated forms of NF-κB p65 in the female liver and the total NF-κB p65 protein level in the male liver significantly increased 40 weeks after DEN administration (Fig. 3E–H). Another key player in liver inflammation and tumorigenesis is signal transducer and activator of transcription 3 (STAT3), an oncogenic transcription factor that is critical for HCC development.7 The levels and activation of STAT3 were increased in the livers of female mice with MCPIP1-deficient hepatocytes. Moreover, there were increased levels of p38 (T180/Y182) and ERK MAPK activation (Fig. 3E,F); however, these pathways were not activated in the livers of male mice (Fig. 3G,H). These results indicated that the development of HCC in female Zc3h12afl/flAlbCre mice is linked to the pronounced activation of the NF-κB and STAT3 signaling pathways, which are involved in inflammation-mediated hepatocarcinogenesis.7

MCPIP1 deficiency activates transcriptomic changes that lead to HCC development

To investigate the importance of MCPIP1 at different stages of HCC development, we investigated DEN-induced hepatocarcinogenesis over time (Fig. 4A). Because MCPIP1 deficiency markedly affects HCC development in female mice, subsequent analyses focused on the livers of female animals. To identify transcriptional programs associated with a lack of MCPIP1, livers isolated from Zc3h12afl/flAlbCre mice (Zc3h12a gene knockout mice) and control mice after DEN administration were subjected to gene expression profiling via RNA sequencing (RNA-seq) and pathway analysis. The genes were dysregulated in the livers of Zc3h12afl/flAlbCre and control mice at 12 and 24 weeks after DEN administration (Fig. 4B). A total of 332 genes were upregulated and 341 were downregulated in the livers of Zc3h12afl/flAlbCre mice 12 weeks after DEN administration. Twenty-four weeks after DEN administration, 2,474 genes were upregulated and 1,593 were downregulated in livers lacking MCPIP1 compared with control livers (Fig. 4B). Twelve weeks after DEN administration, we assessed changes between wild-type and knockout mice (Figs 4C–F and S3). In Zc3h12afl/flAlbCre mice, there was significant enrichment of several cellular pathways, such as those related to KRAS signaling, angiogenesis, EMT and the inflammatory response (Figs 4F and S3). Global transcriptome analysis of liver samples 24 weeks after DEN administration revealed profound differences in gene expression between the livers of control and Zc3h12a knockout mice treated with DEN (Figs 4G–J and S3). A comparison of the tissues revealed hepatocyte MCPIP1-dependent effects on large sets of genes, mainly key genes for Wnt/β-catenin signaling, EMT, the inflammatory response, and IL6/JAK/STAT3 signaling (Figs 4J and S3). We subsequently analyzed changes in the expression of genes related to several pathways, such as liver fibrosis, oncogenes and EMT (Figs 4K and S3). Functional profiling and quantitative reverse-transcription PCR revealed that the transcript levels of factors associated primarily with fibrosis, EMT and cytokine-mediated signaling pathways, including Spp1, Ctnnb1, Ctgf, Src, Zeb1, Vim, Tgfb2 and Adam17, were significantly increased in the livers of Zc3h12afl/flAlbCre female mice 12 weeks after DEN administration (Fig. 5A). Moreover, the active β-catenin levels were increased, and β-catenin translocated from the cell membrane to the cytoplasm (Fig. 5B). Analysis of liver samples collected 24 weeks after DEN administration revealed increased expression of multiple factors involved in the Wnt signaling pathway, including Wnt4, Wnt5, Wnt6, Wnt7, Wnt11, Itpr3, Tbx3, Fzd8, Dvl2, and Axin (Fig. 5C, D). MCPIP1 directly regulated the half-life of the Il6 transcript, which activates the IL-6/JAK/STAT3 pathway. Liver samples collected 24 weeks after DEN administration exhibited elevated IL-6 secretion (Fig. 5E). Additionally, MCPIP1 deficiency increased the STAT3 levels in knockout mice, suggesting that a lack of MCPIP1 may be involved in amplifying destructive changes in hepatocytes after DEN administration (Fig. 5F).

Fig. 4.

Fig. 4

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Lack of MCPIP1 activates transcriptomic changes leading to HCC development.

(A) Timelines for the study. (B) Diagram showing number of upregulated and downregulated genes in Zc3h12afl/flAlbCre female mice 12 and 24 weeks after DEN administration (p value <0.05). (C) PCA chart - comparison of Zc3h12afl/fl control mice (green) and Zc3h12afl/flAlbCre mice (blue) 12 weeks after DEN administration. (D) Volcano plot shows the downregulated (green) and upregulated (blue) genes in Zc3h12afl/flAlbCre females compared to controls 12 weeks after DEN administration, padj ≤0.05. (E) Heatmap of the downregulated and upregulated genes in Zc3h12afl/flAlbCre mice (green) compared to Zc3h12afl/fl control female mice (blue) 12 weeks after DEN administration, padj ≤0.05. (F) Dotplot with significant enrichment in several cellular pathways in Zc3h12afl/flAlbCre female mice 12 weeks after DEN administration compared to control mice prepared in GSEApy Python package, FDR = 0,25. (G) PCA chart - comparison of Zc3h12afl/fl control mice (green) and Zc3h12afl/flAlbCre mice (blue) 24 weeks after DEN administration. (H) Volcano plot shows the downregulated (green) and upregulated (blue) genes in Zc3h12afl/flAlbCre female mice compared to controls 24 weeks after DEN administration, padj ≤0.05. (I) Heatmap of the downregulated and upregulated genes in Zc3h12afl/flAlbCre mice (green) compared to Zc3h12afl/fl control female mice (blue) 24 weeks after DEN administration, padj ≤0.05. (J) Dotplot with significant enrichment in several cellular pathways in Zc3h12afl/flAlbCre female mice 24 weeks after DEN administration compared to control mice prepared in GSEApy Python package, FDR = 0.25. (K) Heatmap of changed genes in several pathways - liver fibrosis, oncogene and EMT. DEN, diethylnitrosamine; EMT, epithelial-to-mesenchymal transition; FDR, false discovery rate; GSEApy, gene set enrichment analysis in Python; HCC, hepatocellular carcinoma; PCA, principal component analysis.

Fig. 5.

Fig. 5

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Zc3h12a knock out in the liver affects phenotype changes and IL-6/STAT signaling.

(A) mRNA expression level of Creb1, Ctgf, Ctnnb1, Hgf, Spp1, Tgfb2, Vim, Zeb1, Adam17 and Src. EF2 was used as the reference gene. 12-week-old females after DEN administration, n = 5 per group. (B) Representative images of β-catenin (S45) IHC staining of liver Zc3h12afl/flAlbCre and Zc3h12afl/fl female mice 12 weeks after DEN administration. (C) Heatmap of changed genes in Wnt signaling pathway. (D) mRNA expression level of transcripts involved in Wnt signaling pathway in 24-weeks old females and males after DEN administration. Actb was used as the reference gene. (E) Analysis of IL-6 protein level in Zc3h12afl/flAlbCre and Zc3h12afl/fl female mice 24 weeks after DEN administration, n = 3 per group. (F) Analysis of STAT3 in Zc3h12afl/flAlbCre and Zc3h12afl/fl female mice 24 weeks after DEN administration. The results are presented as the mean ± SD with dot plot. P values were estimated using unpaired Student’s t test or Mann-Whitney U test, ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001. DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; IHC, immunohistochemistry.

MCPIP1 knockout leads to the activation and nuclear translocation of β-catenin and the CREB1 transcription factor

To investigate the principal pathways involved in HCC development and to understand the mechanism underlying the observed phenotypic and genotypic changes in Zc3h12afl/flAlbCre mice, we analyzed primary hepatocytes isolated from 8-week-old Zc3h12afl/fl and Zc3h12afl/flAlbCre mice. β-catenin activation involves a series of phosphorylation events that direct β-catenin to the nucleus. MCPIP1 deficiency in hepatocytes led to increased levels of the transcriptionally active form of β-catenin phosphorylated at S675 and S552 (Fig. 6A). In addition, active β-catenin (not phosphorylated at S45 but phosphorylated at S675) accumulated in the nuclei of hepatocytes (Fig. 6B). β-catenin phosphorylation at S675 induces the expression of SPP1, which encodes osteopontin, in hepatocytes, inducing their transdifferentiation to early hepatocyte progenitors and thus promoting a malignant cell fate.21 Osteopontin induces β-catenin activation and contributes to maintaining the stem-like properties and tumorigenicity of hepatic progenitor cells in the liver.22 In the present study, MCPIP1 deficiency and β-catenin S675 phosphorylation induced the expression of Spp1 in hepatocytes (Fig. 6C).

IL-6 regulates the JAK/PI3K/Akt/CREB signaling pathway in hepatocytes.23 In the present study, MCPIP1 deficiency increased the hepatic expression of Creb1 and Tgfb2 (Fig. 6C), and high levels of these factors were maintained in 12- and 24-week-old mice (Fig. 5A). Moreover, MCPIP1 deficiency in hepatocytes increased the level of its direct target, Il6, which directly affects hepatocyte dedifferentiation24 (Fig. 6C). In addition, MCPIP1-deficient hepatocytes exhibited increased levels of protumorigenic Adam17 (Fig. 6C), which regulates IL-6 trans-signaling.25 Further, NF-kB, Akt, Erk, c-Met and STAT3 were phosphorylated in MCPIP1-defcient hepatocytes (Fig. 6D). Deletion of Zc3h12a induced the activation (phosphorylation at S133) and translocation of CREB1 into the cell nucleus (Fig. 6E–G).

Analysis of HCC patient databases from the CPTAC (Clinical Proteomic Tumor Analysis Consortium) and the ICPC (International Cancer Proteogenome Consortium) confirmed a notable increase in the protein expression of SPP1 (p = 6.58E-14), CREB1 (p = 1.10E-15), TGF-β2 (p = 3.98E-11), CTGF (p = 7.67E-15), and ADAM17 (p = 1.19E-13) (Fig. 7A).26 The gene expression levels of SPP1 (p = 1.90E-12), CREB1 (p <1E-12), TGF-β2 (p = 7.61E-05), CTGF (p = 9.97E-01), ADAM17 (p <1E-12), and MET (p = 1.32E-10) were also significantly increased (Fig. 7B).26 Analysis of samples from patients with stage I and II HCC revealed higher CTNNB1, TGFb2 and SPP1 expression in tumors than in adjacent non-tumor tissue (Fig. 7C). These results may, at least partially, explain the protective role of MCPIP1 and the sex disparities associated with a higher level of MCPIP1 in females.

Fig. 7.

Fig. 7

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CTNNB1 expression is lower in tumor samples compared to adjacent non tumor samples.

(A) Protein expression analysis option using data from the CPTAC and the ICPC datasets - https://ualcan.path.uab.edu/analysis-prot.html. SPP1 (p = 6.58E-14), CREB1 (p = 1.10E-15), TGFβ2 (p = 3.98E-11), CTGF (p = 7.67E-15) and ADAM17 (p = 1.19E-13). Normal n = 165, Primary tumor n = 165. Z-values represent standard deviations from the median across samples for the given cancer type. Log2 Spectral count ratio values from CPTAC were first normalized within each sample profile, then normalized across samples. (B) Expression analysis using data from TCGA. SPP1 (p = 1.90E-12), CREB1 (p <1E-12), TGFβ2 (p = 7.61E-05), CTGF (p = 9.97E-01), ADAM17 (p <1E -12) and MET (p = 1.32E-10). Normal n = 50, Primary tumor n = 371. (C) mRNA expression level of CTNNB1, TGFB2 and SPP1 in tumor (T) and non-tumor (NT) samples (n = 20 per group; Stage 1 n = 11; Stage 2 n = 9). EF2 was used as the reference gene. The results are presented as the mean ± SD with dot plot. P values were estimated using unpaired Student’s t test, ∗∗p <0.01, ∗∗∗p <0.001. CPTAC, Clinical Proteomic Tumor Analysis Consortium; ICPC, International Cancer Proteogenome Consortium; TCGA, The Cancer Genome Atlas.

Discussion

HCC is a complex multistep process that involves the early transformation of hepatocytes and the subsequent development of HCC. Although progress has been made, the pathophysiology of HCC remains incompletely understood, and most patients with HCC are not eligible for curative surgery because of the advanced stage of the disease at the time of diagnosis.

MCPIP1 has been reported to be a potential tumor suppressor in ccRCC that regulates tumor cell proliferation, tumor cell survival, tumor growth, vascularization and metastasis.16 The present study revealed that MCPIP1 (ZC3H12A) expression was significantly lower in tumor tissue than in adjacent healthy liver tissue from patients with HCC, suggesting a potential regulatory role for MCPIP1 in hepatocarcinogenesis. However, larger public datasets do not consistently report significant reductions in ZC3H12A mRNA levels. This discrepancy may reflect the limitations of bulk RNA-seq in heterogeneous tumor samples, where signals from tumor cells can be diluted by surrounding stromal and immune components. Additionally, normalization methods commonly used in RNA-seq analyses may mask moderate but biologically relevant differences between tumor and non-tumor tissues. In contrast, the present quantitative reverse-transcription PCR approach allowed more sensitive detection of gene-specific changes in expression, supporting a potential tumor-suppressive role for MCPIP1 in liver cancer.

The present findings revealed that MCPIP1 deficiency in hepatocytes did not induce spontaneous tumor formation or a further increase in protumorigenic pathway activation in the livers of MCPIP1 knockout mice; instead, fibrotic changes were activated in the liver. The absence of MCPIP1 in hepatocytes increased YES1–YAP signaling, contributing to the downstream expression of profibrotic Ctgf, a key mediator of tissue remodeling and fibrosis. Additionally, the expression of EMT inducers (such as Zeb1, Twist, and Ctnnb1) and mesenchymal markers (such as Fn1, Vim, and Mmp2) increased, creating a profibrotic environment. Consequently, the liver tissue was fibrotic, with abundant collagen deposition. Hepatocyte-specific MCPIP1 knockout mice exhibited increased levels of the proinflammatory cytokines Il1b and Il6, which are directly regulated by MCPIP1,13 as well as increased levels of Hgf and Cxcl12, which are involved in tumor growth and progression. Approximately 80% to 90% of HCC cases are associated with underlying cirrhosis, resulting from chronic liver inflammation, making cirrhosis a significant risk factor.1 Furthermore, we have previously demonstrated reduced MCPIP1 protein levels in patients with MASLD, a condition associated with potential progression to end-stage liver disease, including HCC.13

Glutamine synthetase is an important liver enzyme responsible for catalyzing the ATP-dependent conversion of glutamate and ammonia to glutamine. Glutamine synthetase is localized primarily in perivenous hepatocytes and is a target of the Wnt/β-catenin pathway in the liver.27 In MCPIP1-deficient livers, the expression and activity of glutamine synthetase were increased. This upregulation is often associated with early hepatocellular changes and is considered a marker of preneoplastic lesions, reflecting metabolic reprogramming and altered nitrogen handling.28 Although the absence of MCPIP1 in the liver leads to pathological alterations, it is not sufficient to induce tumor development and must be accompanied by an additional oncogenic stimulus.

Sex dimorphism represents a prominent feature of HCC, with men being predominantly affected. This sex disparity is also observed in mice exposed to the DEN chemical carcinogen; DEN induces HCC in 100% of male mice, whereas female littermates are largely resistant to carcinogenesis.4 Additionally, the administration of DEN leads to higher levels of circulating IL-6 in males than in females.4 Unexpectedly, 40 weeks after DEN administration in the present study, almost all the female mice in the Zc3h12afl/flAlbCre group developed tumors. Although estrogens have been proposed to be general suppressors of HCC by reducing the proinflammatory effects of MyD88-mediated IL-6 secretion,4 changes in the levels of estradiol, the expression of Esr1, or the expression of the Foxa1/Foxa2 transcription factors that regulate sex hormones were not observed (data not shown). However, there were increased levels of IL-6 in Zc3h12afl/flAlbCre mice, which induced the compensatory proliferation of hepatocytes and the accumulation of DNA damage due to DEN.4 The hepatocyte-specific MCPIP1 knockout model lacked the protective effect of MCPIP1, which is a key negative regulator of IL-6. MCPIP1 physically interacts with stem‒loop structures in the 3′ untranslated region of Il6 transcripts through its PIN domain, leading to mRNA destabilization and degradation.29 These findings confirmed that increased levels of IL-6 play important roles in hepatocarcinogenesis and indicated that the level of MCPIP1 is important in protecting against HCC development in female mice.

The effects of MCPIP1 deficiency in hepatocytes may influence the hepatocyte phenotype and the microenvironment within the liver.19 The influx of CD45+ and CD68+ immune cells was increased after DEN administration, suggesting macrophage activation. Moreover, the expression levels of the monocyte/macrophage markers Cd14 and Mgl2, as well as the T cell marker Cd3e, were increased in the livers of Zc3h12a knockout mice after DEN administration. α-SMA staining revealed increased inflammation together with fibrosis, highlighting the importance of MCPIP1 in tissue microenvironment changes.

Early signaling events in HCC pathogenesis include the activation of Wnt/β-catenin signaling.30 Studies have reported that mutation of CTNNB1, which encodes β-catenin, is among the key genetic events in human HCC.31 Furthermore, Wnt/β-catenin has been implicated in HCC stemness, progression, metastasis, and drug resistance.32,33 Up to 30–50% of HCC cases exhibit upregulated Wnt/β-catenin expression and CTNNB1 gene mutations.34 β-catenin plays dual roles, functioning as a component of the adherens junction complex at the membrane associated with cadherins and as a critical effector of the Wnt signaling pathway in the nucleus. Activation of β-catenin involves its phosphorylation at two sites, namely, S552 and S675, promoting its transcriptional activity as a co-factor for the TCF/LEF transcription factor.35 However, the molecular mechanisms governing β-catenin activation and nuclear translocation remain largely unknown. Additionally, the transgenic expression of degradation-resistant β-catenin in the liver is insufficient to drive HCC initiation in vivo.36,37 Hence, whether β-catenin activation is a driving mechanism or a cooperative event that supports HCC progression initiated by other oncogenic factors remains unclear.38 β-catenin is a male-biased HCC driver whose mutation frequency is relatively high in HCC specimens from males. The present study revealed a notable increase in the level of the active form of β-catenin in female mice lacking MCPIP1 in hepatocytes, even shortly after DEN administration. Furthermore, active β-catenin translocated into the hepatocyte nucleus. Previous findings have demonstrated that MCPIP1 regulates the level, localization, and activity of β-catenin in ccRCC cells by modulating the expression of negative regulators of the Wnt pathway. The absence of MCPIP1 increases the transcriptionally active form of β-catenin, influencing the acquisition of mesenchymal features.17 The present data indicated that in addition to β-catenin activation, the expression of other components of the Wnt signaling pathway was concomitantly upregulated in hepatocytes lacking MCPIP1. In the present study, inflammation-driven Wnt signaling activated β-catenin in hepatocytes, promoting proliferation and oncogenic transformation, as shown in liver injury and cancer models.39 We hypothesize that MCPIP1, through its involvement in β-catenin and Wnt signaling activation, may contribute to HCC initiation in female mice.

The present results indicate that the development of HCC in female Zc3h12afl/fl AlbCre mice was linked to the pronounced activation of the NF-κB and STAT3 signaling pathways, which are involved in inflammation-mediated hepatocarcinogenesis.7 Increased levels of IL-6 and increased STAT3 activity have been reported in patients with HCC.40 STAT3 may increase the nuclear localization of β-catenin in colorectal cancer through potential crosstalk between the Wnt/β-catenin pathway and the IL6/gp130/STAT3 pathway.41 Thus, we hypothesized that the regulation of IL-6 levels and the activation of β-catenin by MCPIP1, as in the case of ccRCC,17 may be missing. Another activator of β-catenin in hepatocytes lacking MCPIP1 may be osteopontin, which is encoded by SPP1 and promotes hepatic progenitor cell expansion and tumorigenicity.22 In the present study, the expression of Spp1 was increased in mice lacking MCPIP1 and in patients with HCC.

IL-6 also activates the JAK/PI3K/Akt/CREB signaling pathway in hepatocytes.23 CREB1 belongs to the CREB/activating transcription factor family of transcription factors and is activated by S133 phosphorylation, which is mediated by AKT. CREB factors promote tumorigenesis in many cancers. Patients with HCC and increased expression and phosphorylation of CREB1 at S133 have decreased overall survival and a greater risk of tumor relapse.42 The present study revealed that a lack of MCPIP1 in hepatocytes increased CREB1 expression and activation, i.e. S133 phosphorylation. CREB1 binds to the identified CRE in the proximal region of the TGFB2 promoter. The present results revealed increased TGFB2 expression in both mice lacking MCPIP1 and patients with HCC. In support of their importance in cancer biology, CREB1 and TGFB2 mRNA levels are positively correlated in human glioblastoma samples.43 Moreover, recent evidence has indicated that phosphorylated CREB1 (S133) binds to a Ctnnb1 enhancer and regulates β-catenin transcription. In the present MCPIP1-deficient hepatocyte model, CREB1 activation served as a significant mediator of HCC development through its influence on β-catenin expression.44

Fate-tracing studies have demonstrated that HCC originates primarily from hepatocytes. In the initial stages of HCC, transformed hepatocytes, regardless of their proliferation status, promote the expansion of hepatic progenitor cells. However, the precise mechanisms initiated by injured hepatocytes in this process remain unclear.45 The present study highlighted the crucial role of MCPIP1 in hepatocytes, preserving liver homeostasis, preventing fibrosis development, and acting as a potent suppressor of liver tumor development, particularly in female mice resistant to tumorigenesis, revealing the importance of MCPIP1 as a guardian/cell protector. While the present research highlighted the role of MCPIP1 in tumor biology and the intricate regulation of the development of HCC, the full extent of the actions of MCPIP1 and the complexities involved remain poorly understood.

A limitation of the present study was the use of the Alb-cre transgenic line for Zc3h12a deletion. Alb-cre becomes active around embryonic Day 18, a time when hepatoblasts are still bipotential and capable of giving rise to both hepatocytes and cholangiocytes. Although the Alb promoter drives predominantly hepatocyte-specific recombination postnatally, recombination during late embryogenesis may influence both lineages, especially under pathological conditions where cell plasticity and transdifferentiation may occur. This is particularly relevant considering the ductular reaction and biliary alterations observed in the present model. Moreover, differences between constitutive (Alb-cre) and inducible (AAV8-Tbg-Cre) models have been previously noted, including in Ctnnb1 knockout mice, in which early deletion induces compensatory mechanisms that alter adult phenotypes.46 These factors should be considered when interpreting the phenotypic spectrum observed in the present study, as they may partially explain some of the biliary features and suggest a developmental component to the observed pathogenesis. While acknowledging the limitations associated with the developmental timing of Alb-cre, the present model also offers several important strengths. Chronic, hepatocyte-targeted deletion of Zc3h12a allows investigation of liver tumorigenesis in the context of progressive inflammation and fibrosis, closely mimicking many clinical cases of HCC. Finally, the present transcriptomic analysis was performed on female livers, in which the inflammatory and tumor phenotypes were most robust. While male MCPIP1-deficient mice were included in other parts of the study, a direct male vs. female transcriptomic comparison remains an important next step to better understand sex-specific disease mechanisms.

Abbreviations

ccRCC, clear cell renal cell carcinoma; CREB1, cAMP-responsive element-binding protein 1; DEN, diethylnitrosamine; EMT, epithelial-to-mesenchymal transition; HCC, hepatocellular carcinoma; IL-6, interleukin 6; MASLD, metabolic dysfunction-associated steatotic liver disease; MCPIP1, monocyte chemoattractant protein-induced protein 1; Mgl2, macrophage galactose C-type lectin 2; RNA-seq, RNA sequencing; STAT3, signal transducer and activator of transcription 3.

Authors’ contributions

Conceptualization and design: OK, PM, KM; Formal Analysis: OK, PM, JG, RM, KM; Funding Acquisition: OK, PM, KM, JG; Investigation: PM, OK, RM, JG, E G-S, EB, JK, MG, AA, NP, MK, ER, KM; Methodology: OK, PM, JG, RM, KM; Analysis: OK, PM, JG, RM, IF, KM; Project Administration: OK, PM, KM; Data interpretation: OK, PM, IF, KM; Supervision: KM; Validation: OK, PM, RM, KM; Visualization: OK, PM, JG, RM; Writing: OK, PM, KM; All authors read and approved the final manuscript.

Data availability

The authors declare that all the data supporting the findings of this study are available within the paper in the main text or the Supplementary Materials. Raw and processed data from next-generation sequencing were deposited at https://www.ncbi.nlm.nih.gov/bioproject/1198596 (accession number: PRJNA1198596).

Acknowledgment and financial support

This work was supported in part by research grants from National Science Centre no. 2017/26/E/NZ5/00691 and 2022/45/B/NZ5/01973 to K.M., 2021/41/N/NZ4/04187 to O.K., 2022/47/B/NZ5/02724 to P.M. and by research task under IDUJA – funded from the scientific subsidy of the Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University no. 19000082_N_25_47 to J.G. Part of the optical microscopy experiments: confocal imaging of the β-catenin and CREB presented in the Figure 6A and G were performed at the Bioimaging Laboratory which serves as an imaging core facility at the Faculty of Biochemistry, Biophysics and Biotechnology JU. We would like to thank Beata Rysiewicz, PhD from the Bioimaging Laboratory for her technical support.

Conflict of interest

The authors declare no potential conflicts of interest.

Please refer to the accompanying ICMJE disclosure forms for further details.

Footnotes

Author names in bold designate shared co-first authorship.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jhepr.2026.101755.

Supplementary data

The following are the Supplementary data to this article:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
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Multimedia component 2
mmc2.docx (65.8KB, docx)
Multimedia component 3
mmc3.pdf (2.9MB, pdf)
Multimedia component 4
mmc4.pdf (132.3MB, pdf)

Data Availability Statement

The authors declare that all the data supporting the findings of this study are available within the paper in the main text or the Supplementary Materials. Raw and processed data from next-generation sequencing were deposited at https://www.ncbi.nlm.nih.gov/bioproject/1198596 (accession number: PRJNA1198596).

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