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. 2025 Aug 29;8:1304. doi: 10.1038/s42003-025-08755-9

OTUB2/NR4A1 restrains the development of preeclampsia by suppressing macrophage M1 polarization

Yue Wang 1,#, Yang Wang 1,#, Fei Tong 2,#, Qianqian Gao 3, Baoxuan Li 1, Yan Zhao 1,✉
PMCID: PMC12397338  PMID: 40883596

Abstract

Preeclampsia is one of the most common pregnancy disorders, and characterized by insufficient trophoblast invasion and placental inflammation. Our RNA sequencing results showed that OTU deubiquitinase, ubiquitin aldehyde binding 2 (OTUB2) was downregulated in placenta from preeclampsia patients, compared with the healthy control. Clinical and experimental data demonstrated that OTUB2 was expressed in macrophages. Gain- and loss-of-function experiments revealed that OTUB2 overexpression blocked M1 polarization of macrophages, and attenuated macrophages-induced apoptosis, and impaired migration and invasion of trophoblasts. Moreover, OTUB2 alleviated LPS-induced preeclampsia-like symptoms in rats, including hypertension, proteinuria, inflammation in placenta and insufficient infiltration of trophoblasts. Additionally, OTUB2 mediated deubiquitination of nuclear receptor subfamily 4 group A member 1 (NR4A1) and suppressed its degradation, and the Cys51 is crucial for catalytic activity of OTUB2. The effects of OTUB2 were partially reversed by shNR4A1. In conclusion, OTUB2 suppressed macrophage M1 polarization, macrophages-induced trophoblast damage, and restrained experimental preeclampsia in rodents by mediating deubiquitination of NR4A1. These findings may provide novel insight for clinical prevention and diagnosis of preeclampsia.

Subject terms: Ubiquitylation, Ubiquitylation

Wang et al. found that OTUB2 is downregulated in preeclampsia and its overexpression reduces macrophage-induced trophoblast damage. It acts by stabilizing NR4A1, alleviating preeclampsia-like symptoms in a rat model.

Introduction

Preeclampsia is one of the most common pregnancy disorders, with 42000 maternal deaths annually1,2. Preeclampsia is a progressive disease involving multiple organ systems. The most common clinical manifestations include new onset hypertension (systolic pressure (SP) ≥ 140 mm Hg or diastolic pressure ≥90 mm Hg or both) and proteinuria (24 h urinary protein ≥300 mg) at ≥20 weeks of gestation3,4. In the absence of proteinuria, the diagnosis of preeclampsia requires the presence of hypertension together with evidence of systemic disease (such as thrombocytopenia, elevated levels of liver transaminases, renal insufficiency, pulmonary oedema and visual or cerebral disturbances)3–5. To date, no treatment has been identified to affect the progress of preeclampsia, except for delivery2. However, understanding the pathogenesis of preeclampsia is beneficial for preventing the disease and improving the maternal and infant outcomes as much as possible.

In a normal early pregnancy, the placenta remodels the local uterine vasculature, setting up optimal conditions for nutrient and oxygen exchange throughout pregnancy. Extravillous placental trophoblast cells invade through the inner third of myometrium, and replace the smooth muscle cells to recoin the maternal spiral artery. Consequently, the uterine spiral artery is widened to provide plentiful blood flow to the implantation site, allowing for efficient maternal–fetal nutrient exchange6,7. In preeclampsia or eclampsia, the spiral artery fails to undergo remodeling. The narrow vessels are unable to provide adequate perfusion, resulting in placental ischemia and oxidative stress, and leading to the villi damage and abnormal angiogenic protein levels in the maternal blood8–10. Subsequently, spontaneous abortion, intrauterine growth restriction (IUGR), fetal death, placental abruption or preterm labor may occur3.

Immunoregulation is also necessary for successful pregnancy. Macrophages are the second largest group of cells, accounting for 20–30% of all leukocytes, second only to the predominant uterine natural killer (uNK) cells11,12. Macrophages are divided into M1 and M2 subtypes based on their activation states13. The number and proportion of M1/M2 macrophages are changed during different gestation periods to protect the fetus from the maternal immune microenvironment. To sustain fetomaternal tolerance, polarization of macrophages towards to the anti-inflammation M2 phenotypes in a healthy pregnancy. Conversely, dysregulated macrophage polarization is associated with inadequate uterine remodeling and deficient trophoblast invasion, which lead to spontaneous abortion, preterm birth and preeclampsia11,14. M1 macrophages were found to be more abundant in the placenta, decidual and uterine spiral artery of preeclampsia women15,16. M1 macrophages were demonstrated to suppress the proliferation and migration of trophoblasts, and promote their apoptosis in vitro17. Inhibiting macrophage M1 polarization could alleviate experimental preeclampsia in rodent18.

In this study, we collected placenta from preeclampsia cases and healthy controls, and RNA sequencing was performed. More than 400 differentially expressed genes (DEGs) were presented in the preeclamptic placenta, compared with those from healthy volunteers. Among these, OTU deubiquitinase, ubiquitin aldehyde binding 2 (OTUB2) was downregulated in placenta of preeclampsia patients. OTUB2 was reported to facilitate the migration and invasion of several tumor cells19–21, but its effect on preeclampsia, trophoblasts or macrophages was unknown. As a deubiquitinase, OTUB2 mediated the deubiquitination of yes-associated protein (YAP) and stabilized its protein molecule20. YAP was inactive in placenta from preeclampsia and IUGR women, and its overexpression promoted the migration and invasion of trophoblasts22,23, suggesting that OTUB2 may be involved in development of preeclampsia.

The analysis from bioinformatic website BioGRID (https://thebiogrid.org/) showed that OTUB2 bound to nuclear receptor subfamily 4 group A member 1 (NR4A1), which was a transcription factor. NR4A1 was decreased in placenta of women with preeclampsia, and overexpression of NR4A1 accelerated the migration and invasion of trophoblasts24. Additionally, NR4A1 inhibited M1 while enhanced M2 polarization of RAW264.7 macrophages, and the NR4A1 deficiency exacerbated inflammation in central nervous system, spleen and colon of mice25,26. It is unclear if NR4A1 regulated the inflammation in preeclampsia.

In this study, we demonstrated that OTUB2/NR4A1 alleviated experimental preeclampsia and suppressed inflammation-induced trophoblast impairment.

Results

The transcriptome analysis of placenta from preeclampsia cases and healthy controls

We collected placentas from preeclampsia patients and healthy pregnant women, and performed RNA sequencing. Figure 1A showed the principal component of samples, and Fig. 1B, C displayed the DEGs. There were 419 DEGs with |log2FC| ≥1 and p < 0.05 in preeclamptic placentas, compared with healthy placentas. The enrichment analysis exhibited that the DEGs were enriched in immune-related and malignant tumor-associated terms (Fig. 1D, E). Several terms were crossed, resulting in 43 DEGs (Fig. 1F), among which OTUB2 was selected for subsequent investigation.

Fig. 1. The transcriptome analysis of placenta from preeclampsia cases and healthy controls.

Fig. 1

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Four placentas from preeclampsia patients and four placentas from healthy pregnant volunteers were applied for transcriptome analysis. A The principal component analysis (PCA) of all specimens. B The volcano plot of all genes in placental tissues. C The heatmap of differentially expressed genes (DEGs). D Gene Ontology (GO) enrichment analysis of DEGs. E Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of DEGs. F Chord analysis of DEGs. (PE, preeclampsia).

OTUB2 was downregulated in placenta of preeclampsia patients

We continued to collected another 30 placentas from preeclampsia cases and 30 placentas from healthy volunteers. Real-time PCR results showed that OTUB2 was decreased in preeclamptic placenta, compared with that from healthy controls (Fig. 2A). ELISA results revealed increased levels of TNF-α and IL-1β in preeclamptic placenta (Fig. 2B, C), suggesting inflammation in placenta of preeclampsia cases. The correlation analysis revealed that the expression of OTUB2 was negatively related with TNF-α and IL-1β levels in placentas from preeclampsia cases and controls (Fig. 2D, E). Immunofluorescence staining showed the colocalization of OTUB2 and M1 macrophage marker CD68 in placental tissues (Fig. 2F), suggesting that OTUB2 may regulate macrophage function in placenta. Subsequently, western blot showed that both OTUB2 and NR4A1 were decreased in preeclamptic placenta, and they possessed similar expression pattern (Fig. 2G–I). All original western blots were shown in supplementary Fig. 2, and original data shown in supplementary Fig. 3.

Fig. 2. OTUB2 was downregulated in placenta of preeclampsia patients.

Fig. 2

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A The OTUB2 mRNA level in placental tissues from preeclampsia patients or control cases was measured by real-time PCR. B, C The TNF-α and IL-1β levels in placenta were determined by ELISA. D, E The correlation between OTUB2 expression and TNF-α/IL-1β level in placenta was analyzed. F The colocalization of OTUB2 and CD68 in placenta (the scale bar represented 50 μm, and the arrows indicated the colocalization). G, H The expression of OTUB2 and NR4A1 was assessed by western blot. I Immunohistochemistry staining was performed to detect the expression and localization of OTUB2 and NR4A1 in placenta (the scale bar represented 50 μm). (PE, preeclampsia; ***p < 0.001).

OTUB2 suppressed M1 polarization of macrophages

Next, we cultured THP-1 monocytes, and induced their differentiation to macrophages, followed with M1 polarization stimulation. The results from real-time PCR and western blot showed that OTUB2 was lowly expressed in M1 macrophages, compared with M0 macrophages (Fig. 3A, B).

Fig. 3. OTUB2 suppressed M1 polarization of macrophages.

Fig. 3

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A, B THP-1 monocyte was induced to differentiate into macrophage (M0), and further to M1 polarization. The OTUB2 expression in M0 and M1 cells were determined by real-time PCR and western blot. C Adenovirus loaded with OTUB2 coding sequence or its knockdown fragment was infected into THP-1 cells, and the infection efficiency was measured with green fluorescence in cells (the scale bar represented as 200 μm). D–G OTUB2 expression efficiency was assessed by real-time PCR and western blot in THP-1 with adenovirus infection. H, I The content of inflammatory factors, TNF-α and IL-1β in supernatant of THP-1 was detected by ELISA. J The expression of macrophage M1 molecules, CD86 and iNOS in THP-1 after OTUB2 overexpression or silencing. (PE, preeclampsia; *p < 0.05, **p < 0.01, ***p < 0.001).

To investigate the role of OTUB2 in macrophages, adenovirus vector loaded with OTUB2 coding sequence or knockdown fragment was infected into THP-1 cells before differentiation, and green fluorescence in adenovirus showed that the infection efficiency was more that 90% (Fig. 3C). The real-time PCR and western blot demonstrated that the OTUB2 expression was increased by 8 folds at transcriptional level and three folds at translational level after OTUB2-overexpressed adenovirus infection, while decreased more than 80% in OTUB2-silenced cells (Fig. 3D–G). Then the expression of inflammatory molecules was determined. ELISA results showed that the secretion of TNF-α and IL-1β significantly elevated after M1 induction in macrophages, while this elevation was attenuated by OTUB2 overexpression, and aggravated by OTUB2 knockdown (Fig. 3H–I). Similarly, the expression of M1 markers, CD86 and iNOS, was upregulated in M1 macrophages. They were reduced in OTUB2-overexpressed M1 macrophages, and raised in OTUB2-silenced M1 macrophages (Fig. 3J). These results suggested that OTUB2 inhibited M1 polarization of macrophages.

OTUB2-overexpressed macrophages promoted migration and invasion of trophoblasts

To verify the effects of OTUB2 on trophoblasts, the co-culture of macrophages and trophoblasts was performed. As shown in Fig. 4A, the THP-1 monocytes were infected with adenovirus, then induced for differentiation to macrophages by PMA, and stimulated by IFN-γ + LPS for M1 polarization. The M1 macrophages were incubated with trophoblast medium, and the supernatant was collected as CM to incubate the trophoblasts (Fig. 4A). The primary trophoblasts were isolated from human placental tissues and identified by immunofluorescence staining of CK7 and vimentin. As shown in Fig. 4B, the primary trophoblasts were CK7-positive and vimentin-negative. Similar to macrophage culture alone, the inflammatory TNF-α and IL-1β levels in supernatant were decreased after OTUB2 overexpression and increased after OTUB2 knockdown (Fig. 4C, D). TUNEL, wound healing and transwell assay demonstrated that the supernatant from M1 macrophages induced apoptosis of trophoblasts, and interrupted their migration and invasion (Fig. 4E, F, K). While the effects of macrophages on apoptosis, migration and invasion of trophoblasts were restrained by OTUB2 upregulation and exacerbated by OTUB2 downregulation in macrophages (Fig. 4E, F, K). The activity of MMP2 and MMP9 in trophoblasts with treatment of M1 macrophage supernatant was significantly decreased, compared with M0 macrophages (Fig. 4I–J). MMP2/9 activity was promoted by supernatant from OTUB2-overexpressed macrophages, and inhibited by that from OTUB2-silenced macrophages (Fig. 4I, J). The results in this section suggested that OTUB2-overexpressed macrophages facilitated the migration and invasion of trophoblasts, and suppressed their apoptosis.

Fig. 4. OTUB2-overexpressed macrophages promoted migration and invasion of trophoblasts.

Fig. 4

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A THP-1 monocytes were infected with adenovirus, induced to macrophages, and further to M1 subtype. The M1 macrophages were incubated with trophoblast-specific medium, and the supernatant was collected as conditional medium (CM) for trophoblast incubation. B The primary trophoblasts were identified with immunofluorescence staining of CK7 and vimentin (the scale bar represented as 50 μm). C, D TNF-α and IL-1β levels in supernatant. E, F The migratory ability of trophoblasts was measured by wound healing assay (the scale bar represented as 200 μm). G, H The invasion of trophoblasts was assessed by transwell assay (the scale bar represented as 100 μm). I, J The enzyme activity of MMP2 and MMP9 in trophoblasts was determined. K The apoptosis was evaluated by TUNEL assay (the scale bar represented as 50 μm). (PE, preeclampsia; *p < 0.05, **p < 0.01, ***p < 0.001).

OTUB2 enhanced NR4A1 protein stability by intercepting its ubiquitination

OTUB2 was predicted to interact with NR4A1, and the clinical analysis revealed their similar expression (Fig. 2I). Therefore, we investigated whether OTUB2 regulated the expression of NR4A1. Western blot result showed that the NR4A1 protein level was increased after OTUB2 overexpression and decreased after OTUB2 knockdown in THP-1 cells (Fig. 5A). Next, the degradation of NR4A1 was determined in THP-1 cells with CHX treatment to block translation. The results revealed that the degradation of NR4A1 protein was delayed in OTUB2-overexpressed cells, and accelerated in OTUB2-silenced cells (Fig. 5B, C). With application of MG132, the OTUB2 silencing accelerated NR4A1 degradation was reversed by interception of proteasome degradation (Fig. 5C), suggesting that the NR4A1 was degraded via proteasome pathway. The interaction between OTUB2 and NR4A1 was verified by co-IP in 293 T cells (Fig. 5E). Considering that OTUB2 was a deubiquitinating enzyme, the ubiquitination of NR4A1 was assessed. The results showed that after enhanced expression of OTUB2, the ubiquitination level of NR4A1 was reduced (Fig. 5G). The Cys51 of OTUB2 was reported to be indispensable for its deubiquitinase enzyme activity20, and this amino acid was conservative in different species (Fig. 5K). Therefore, we constructed the deubiquitinase-deficient OTUB2 C51S mutant. The co-IP assay showed that both wild type (WT) and mutant OTUB2 bound to NR4A1 (Fig. 5G). After transfection of C51S OTUB2, the degradation of NR4A1 was accelerated, compared with WT OTUB2 transfection (Fig. 5H, I). Finally, the co-IP results revealed that the ubiquitination level of NR4A1 was increased in C51S OTUB2-transfected cells, by comparison with WT OTUB2 (Fig. 5J). These results proved that the C51S mutant impaired the deubiquitinating enzyme activity of OTUB2, but did not impair its binding to substrates.

Fig. 5. OTUB2 enhanced NR4A1 protein stability by intercepting its ubiquitination.

Fig. 5

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A The expression of NR4A1 in THP-1 cells with OTUB2 overexpression or knockdown. B, C The NR4A1 protein levels were assessed after OTUB2 dysregulation combined with CHX treatment for different times. D The NR4A1 expression after OTUB2 silencing combined with treatment of CHX and MG132. E The binding of OTUB2 and NR4A1 was verified by co-immunoprecipitation in 293 T cells. F The ubiquitination of NR4A1 after OTUB2 overexpression. G The Cys51 of human OTUB2 was mutated to Ser, and the binding between mutant OTUB2 and NR4A1 was verified. H, I The NR4A1 expression after transfection of mutant OTUB2 coding sequence. J The ubiquitination of NR4A1 after overexpression of mutant OTUB2. K The OTUB2 amino acid sequences of human, mouse and rats. (PE, preeclampsia).

The effects of OTUB2 were abolished by silencing of NR4A1

To verify the role of NR4A1, THP-1 cells were infected with NR4A1-silenced adenovirus, and induced for macrophage differentiation and M1 polarization. The ELISA results displayed that knockdown of NR4A1 significantly promoted the production of inflammatory TNF-α and IL-1β in macrophages (Fig. 6A, B). Then the supernatant from macrophages was applied for trophoblast culture. TUNEL results exhibited that NR4A1-silenced macrophages facilitated the apoptosis of trophoblasts (Fig. 6G). Wound healing and transwell assays revealed that NR4A1 knockdown delayed migration and invasion of trophoblasts (Fig. 6C–F).

Fig. 6. The effects of OTUB2 were abolished by silencing of NR4A1.

Fig. 6

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THP-1 cells were infected with adenovirus loaded with OTUB2 coding sequence or/and NR4A1 knockdown fragment, and the supernatant was collected as CM to culture trophoblasts. A, B The TNF-α and IL-1β content in supernatant of macrophages. C, D The migration of trophoblasts (the scale bar presented as 200 μm). E, F The invasion of trophoblasts (the scale bar presented as 100 μm). G Apoptosis was measured by TUNEL assay (the scale bar presented as 50 μm). (PE, preeclampsia; *p < 0.05, **p < 0.01, ***p < 0.001).

Subsequently, both OTUB2 overexpression and NR4A1 knockdown were performed at the same time in THP-1 cells. The ELISA data showed that OTUB2-reduced TNF-α and IL-1β secretion was promoted by shNR4A1 (Fig. 6A, B). With the incubation of supernatant from macrophages, OTUB2-suppressed apoptosis was facilitated by interference of NR4A1 (Fig. 6G), and OTUB2-induced increase of migration and invasion were partially reversed by NR4A1 downregulation (Fig. 6C–F).

OTUB2 restrained LPS-induced preeclampsia in rats

Finally, we explored the effects of OTUB2 on preeclamptic rats. As shown in Fig. 7A, preeclampsia was induced by intravenous injection of LPS at GD5 in pregnant rats, and the adenovirus loaded with rat OTUB2 coding sequence was delivered at GD4. LPS induced preeclamptic symptoms, including increased SP and proteinuria, which were effectively mitigated by injection of OTUB2-overexpressed adenovirus (Fig. 7B, C). ELISA demonstrated that LPS-induced inflammatory TNF-α and IL-1β in placenta was significantly reduced by OTUB2 (Fig. 7D). Western blot results proved that the expression of M1 macrophage markers, CD86 and iNOS, was facilitated by LPS stimulation and relieved by OTUB2 in placenta of pregnant rats (Fig. 7E). Immunofluorescence staining showed the colocalization of CD68 and OTUB2, confirming the expression of OTUB2 in macrophages of placenta from rats (Fig. 7F). The adenovirus infection in placenta was verified in by green fluorescence, and the OTUB2 expression was confirmed by western blot (Fig. 7G, H). Moreover, NR4A1 was decreased in the placenta of preeclampsia rats, and increased by OTUB2 overexpression (Fig. 7H). In addition, the vessels with large diameter and abundant CK7-positive trophoblasts were observed in healthy placenta, while the narrow vessels and decreased CK7 expression were shown in LPS-stimulated placenta (Fig. 7I). After OTUB2 overexpression, the situation of vascular stenosis and decreased CK7 expression was improved (Fig. 7I). The fetal rats were isolated at GD20, and shown in Fig. S1. The preeclampsia rats showed obvious decreased of fetal weight, as well as the small reduced fetus number in pregnant rat individuals. With the OTUB2 overexpression, the fetal weight was increased in some degree, and more fetus developed successfully (Fig. S1). The results in this section suggested that OTUB2 significantly alleviated LPS-induced preeclampsia symptoms in rats, including hypertension, proteinuria, placental inflammation, impaired spiral artery remodeling and IUGR.

Fig. 7. OTUB2 restrained LPS-induced preeclampsia in rats.

Fig. 7

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A The pregnant female SD rats were injected with LPS using infusion pump at GD5 to induce preeclampsia, intravenously injected with adenovirus at GD4, and suffered euthanasia at GD20. B The systolic pressure of rats in each group were measured at GD0, GD3, GD6, GD9, GD12, GD15 and GD18. C The urine protein content in 24 h of rats were assessed at GD3, GD10 and GD19. D The serum TNF-α and IL-1β of rats. E The expression of macrophage M1 markers, CD86 and iNOS in placenta of rats with LPS stimulation or/and adenovirus infection. F The co-localization of CD68 and OTUB2 in placenta of rats with or without LPS stimulation (the scale bar represented as 50 μm). G The distribution of adenovirus vector in placental tissue (the scale bar represented as 200 μm). H The expression of OTUB2 and NR4A1 in placental tissues. I The expression of CK7 in placenta was assessed by immunohistochemistry staining (the scale bar represented as 50 μm). (PE, preeclampsia; *p < 0.05, **p < 0.01, ***p < 0.001).

Discussion

In the present study, we found that OTUB2 was decreased in placental tissues from preeclampsia patients, and its expression was negatively correlated with the levels of inflammatory factors. Adenovirus-mediated OTUB2 overexpression ameliorated LPS-induced preeclampsia-like symptoms in rats. Gain- and loss-of-function experiments demonstrated that OTUB2 interrupted macrophage M1 polarization, and restrained macrophage-mediated apoptosis and migration and invasion impairment of trophoblasts in vitro. Additionally, OTUB2 acted as a deubiquitinase to blocked ubiquitination of NR4A1 and delayed its protein degradation (Fig. 8).

Fig. 8. The mechanism diagram of OTUB2 regulating inflammation and preeclampsia.

Fig. 8

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In normal pregnant women, OTUB2 bound to NR4A1 and stabilized its expression in macrophages. NR4A1 bound to p65 and intercepted its binding to DNA, deactivated canonical NF-κB signaling, and suppressed M1 polarization of macrophages. Trophoblasts invaded into uterus to ensure the normal placentation and embryonic development. In preeclampsia patients, OTUB2 was lowly expressed, NR4A1 was ubiquitinated and degraded, and NF-κB signaling was excessive activated in macrophages. A large number of M1 macrophages led to damage of trophoblasts, and placentation and embryonic development may be impaired.

Ubiquitination is a common post-translational modification, which controls location, activity, function and stability of proteins27. Through a three-step enzymatic cascade utilizing ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3), the C terminal of ubiquitin protein bind to lysine residue of substrate via a covalent linkage27. Ubiquitination is a dynamic modification, and could be removed by deubiquitinating enzymes (DUBs)28,29. Currently, DUBs are derived to seven evolutionarily conservative families: ovarian tumor proteases (OTUs), ubiquitin C-terminal hydrolases (UCHs), Machado-Joseph domain-containing proteases (MJDs), JAMM/MPN domain-associated Zn-dependent metallporoteases (JAMMs), motif-interacting with ubiquitin containing proteases (MINDYs) and zinc finger containing ubiquitin peptidase 1 (ZUP1)30. There has been found 16 members in OTU family in human, which were divided to four subfamilies: OTU domain-containing ubiquitin aldehyde-binding proteins (Otubains) subfamily (OTUB1 and OTUB2); ovarian tumor domain-containing proteins (OTUDs) (OTUD1, OTUD2, OTUD3, OTUD5, OTUD6A, OTUD6B, ALG13); A20-like subfamily (A20, OTUD7A, OTUD7B, TRABID, VCPIP); OTU DUB with linear linkage specificity (OTULIN)31.

OTUB1 has been demonstrated to act in catalytic activity-independent or -dependent manner. The N-terminal ubiquitin-binding domain in OTUB1 binds E2-ubiquitin complex and blocks ubiquitin transferring from E2 to substrates32. It also interacts with ubiquitinated substrate and remove its K48-linked ubiquitin chains33. The ubiquitin-binding domain is not found in OTUB2, and it removes K63 or K48 ubiquitin chains in a deubiquitinating activity-dependent manner21,34. K48 ubiquitination was a common protein-degrading modification, and OTUB2 was reported to delay the degradation of U2 small nuclear ribonucleoprotein auxiliary factor 2 (U2AF2) and receptor interacting serine/threonine kinase 2 (RIPK2) by inhibiting their K48 ubiquitination21,35. In our study, OTUB2 reduced ubiquitination of NR4A1 and stabilized its protein molecule. However, the ubiquitin linkage subtype was not clear. In a recent paper, the K48 ubiquitination and degradation of NR4A1 was suppressed by OTUB136, so we supposed that OTUB2 may remove K48-linked ubiquitin chains of NR4A1 in a similar manner.

Both OTUB1 and OTUB2 inhibited virus infection-induced activation of nuclear factor-kappa B (NF-κB) signaling in 293 cells37, suggesting their immunosuppression roles. Co-culture with OTUB1-overexpressed cancer cells inhibited M1 polarization and promoted M2 polarization of macrophages38. OTUB2 deficiency alleviated dextran sulfated sodium (DSS)-induced colitis and secretion of M1 cytokines in mice35. In our study, OTUB2 suppressed M1 polarization of macrophage, which may be realized by stabilizing NR4A1. The function of NR4A1 in inflammatory diseases has been well reported39. NR4A1 blocked M1 polarization of macrophages by deactivating NF-κB signaling in several tissues and cells26,40. NF-κB is a regulation center of inflammation. When extracellular signals bind to integral member receptors, inhibitor of κB (IκB) kinase (IKK) is activated and phosphorylated IκB proteins, and p65 is released from NF-κB complex. The activated p65 translocates into nucleus, recruits co-activators and binds to κB sites to activate gene expression41,42 s. Act as a nuclear receptor, NR4A1 prevents p65 from binding to the κB sites and intercepts canonical NF-κB signaling42,43. Therefore, we hypothesized that OTUB2 inhibited macrophage M1 polarization by deubiquitinating NR4A1 and deactivating NF-κB signaling.

NF-κB is involved in pathogenesis of preeclampsia. In pregnant early stage, M1 macrophages contribute to embryo implantation, placental formation and embryo development. After the placenta has fully developed, M2 macrophages predominate11. The M1/M2 ratio is sustainedly increased in preeclampsia placenta, in contrast to healthy pregnancies44. NF-κB-regulated granulocyte-macrophage colony stimulating factor (GM-CSF), M-CSF, TNF-α, IL-1, IL-6, IL-8, placental growth factor (PIGF), vascular endothelial growth factor (VEGF), Ang-1, Ang-2 and MMPs are involved in placental development, angiogenesis, spiral artery remodeling and inflammation. Excessively activated NF-κB leads to placental dysplasia and impaired spiral artery remodeling, which exacerbate inflammation and hypoxia. Therefore, NF-κB is sustainedly activated in this vicious circle45,46. Women with preeclampsia displayed up to 10-fold increase in NF-κB expression in the placenta and maternal circulation, compared to control pregnancies47–49. Our data showed increased inflammatory factors in placenta of preeclampsia cases and experimental preeclamptic rodents. The enrichment analysis also revealed the DEGs enriched in inflammatory and immune pathways. OTUB2 was located in macrophages, and its overexpression interrupted the excessive M1 polarization of macrophages in vivo and in vitro. In addition, OTUB2-overexpressed macrophages suppressed migration and invasion of trophoblasts.

The migration and invasion of trophoblasts is the basics of successful gestation. Impaired trophoblast invasion results in failure of spiral artery remodeling, placental dysplasia, hypoperfusion, ischemia and vascular endothelial injury7. The behaviors of trophoblasts are regulated by NF-κB. NF-κB-activated TNF-α inhibits migration and invasion of primary and immortalized trophoblasts50,51. The production of PIGF and Endoglin in trophoblasts is also regulated by NF-κB52,53. Our results demonstrated that OTUB2 promoted the migration and invasion of trophoblasts indirectly via impairing M1 polarization of macrophages. However, it is unclear whether OTUB2 affects trophoblasts directly. The colocalization in placenta from patients and animals exhibited that OTUB2 was not specifically expressed in macrophages and was also present in other cells. Previous reports revealed that OTUB2 controlled migration and invasion of several tumor cells19–21, so it was not impossible for OTUB2 to regulate behaviors of trophoblasts. Whether OTUB2 was expressed in trophoblasts and how it functioned in trophoblasts would be investigated in the future.

The enrichment analysis displayed that many DEGs in preeclampsia placenta were enriched in cancer pathways. The proliferation and invasion of trophoblasts in pregnant early stage were similar to growth and metastasis of cancer cells, and called “pseudo-tumorigenesis”54,55. As a semi-allogenic graft, the blastocyst adheres to uterus, and the cytotrophoblast cells rapidly invaded into myometrium. Trophoblasts secret enzymes to degrade extracellular matrix, and produce adhesion molecules to help blastocyst to attach. These behaviors were imitated by malignant tumors. We assumed that OTUB2 may regulate migration and invasion of trophoblasts because of its promoting role in tumor cells. However, it needs more evidences to verify. Moreover, the conclusions from gestation may be helpful for cancer investigation.

In conclusions, we found that OTUB2 was lowly expressed in placenta of preeclampsia patients, and it was partially located in macrophages. Gain- and loss-of-function experiments demonstrated that OTUB2 suppressed M1 polarization of macrophages, and attenuated macrophage-induced impairment of trophoblast migration, invasion and survival. Adenovirus-mediated OTUB2 overexpression restrained experimental preelampsia-like symptoms in rats. Additionally, OTUB2 mediated deubiquitination of NR4A1 and delayed its degradation. The effects of OTUB2 were abolished by silencing of NR4A1, suggesting that OTUB2 function by regulating NR4A1 expression. These findings may provide novel insights for prevention and diagnosis of preeclampsia in clinic.

Materials and methods

Clinical specimens

Sixty-eight placentas were collected from participants (34 preeclamptic women and 34 healthy pregnant women) from Nov. 2022 to Jun. 2023 in Shenjing Hospital of China Medical University. According to the American College of Obstericians and Gynecologists (ACOG), preeclampsia was diagnosed as systolic blood pressure of ≥140 mmHg and diastolic blood pressure of ≥90 mmHg with proteinuria of 300 mg/day (or a protein/creatinine ratio of ≥0.3 mg/dl or proteinuria of ≥+1) or without proteinuria but with severe clinical features after 20 weeks of gestation in a woman with previously normal blood pressure56. Diagnosis criteria of severe preeclampsia were as follows: blood pressure of ≥160/110 mmHg with proteinuria of ≥300 mg/day. All participants had a singleton pregnancy. The cases with a history of hypertension, diabetes, kidney disease, immune disease, metabolic disease or genetic abnormality were excluded. Placenta samples were taken from a representative block of the central portion of tissue below one third of the placenta, near maternal side. This region was enriched for cytotrophoblasts, syncytiotrophoblasts and villous interstitium.

The placental tissue collection and detections were line with Declaration of Helsinki, and approved by the Ethics Committee of Shengjing Hospital of China Medical University (approval No. 2024PS568K).

Cell culture

The human primary trophoblasts were isolated from fresh placental tissue of healthy pregnant women after delivery in Shengjing Hospital of China Medical University according to the previous reports57,58. After washing, the chorinic villi was isolated immediately from placenta, cut into 5 × 5 × 5 cm cubes, and washed to remove the blood cells. Then the cubes were incubated with digestion solution containing 0.25% trypsase and 0.02% DNase for 20 min. After three repetitions, the solution was collected and filtered. The cell suspension was mixed with percoll, and underwent gradient centrifugation at 2000 × g for 20 min, and the cells in strata intermedium were collected as villous cytotrophoblasts for culture with DMEM supplemented with 15% fetal bovine serum (FBS) (Tianhang Biotechnology Co., Ltd, Zhenjiang, China) in a humid incubator with 37 °C, 8% O2 and 5% CO2. The trophoblasts were identified by immunofluorescence staining of cytokeratin 7 (CK7) and vimentin.

The human monocyte THP-1 was purchased from iCell Bioscience Inc (Shanghai, China), and cultured with RPMI-1640 medium added with 10% FBS. THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h to induce to macrophages (M0), which were continuedly induced by10 ng/ml interferon (IFN)-γ and 1 μg/ml LPS for 24 h to M1 polarization. For adenovirus infection, THP-1 cells were incubated with adenovirus suspension for 24 h, and the infection efficiency was confirmed with fluorescence photograph.

293 T cells were purchased from iCell Bioscience Inc, and cultured with DMEM and 10% FBS. 293 T cells were used to verify the interaction between OTUB2 and NR4A1.

Cycloheximide (CHX) (Aladdin Biochemical Technology Co., Ltd., Shanghai, China) was applied to intercept protein synthesis with 5 μM, and MG132 (10 μM, Macklin Inc., Shanghai, China) was applied to block proteasome degradation.

For cell co-culture, the THP-1 cells were cultured with medium of trophoblasts for 24 h, and the supernatant was collected, centrifuged at 1950 × g for 20 min, and filtered with a filter with 0.22 μm pores. Then the solution was used as conditional medium (CM) for incubation of trophoblasts.

RNA sequencing

Four placentas from preeclampsia patients and four placentas from healthy pregnant women were used for RNA sequencing in Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The total RNA was extracted, and reversely transcribed into cDNA with random hexamers, then the other strand was completed. The double-strand DNA was purified, terminal retrieved, and added with poly A. After screening, the DNA fragments with appropriate size were amplified to establish the library, which were applied for sequencing using NEBNext Ultra RNA Prep Kit for Illumina (NEB, USA). The original fluorescence image files obtained from Illumina platform were transformed to short reads (Raw data) by base calling and these short reads were recorded in FASTQ format, which contains sequence information and corresponding sequencing quality information. Quality control of the raw reads was performed using Fastp as follows: a, discard a paired reads if either one read contained adapter contamination; b, discard a paired reads if more than 10% of bases were uncertain in either one read; c, discard a paired reads if the proportion of low quality (phred quality <5) bases was over 50% in either one read.

Construction of vectors

To investigate the effects of OTUB2, the overexpression and silencing adenovirus vectors of OTUB2 were constructed. The human OTUB2 (NM_023112.3) or rat OTUB2 (NM_001108053.1) coding sequence was inserted into pAdTrack-CMV plasmid with SalI and XhoI sites, and short hairpin (sh) fragment targeting human OTUB2 was inserted into pAdTrack-CMV-H1 plasmid with BglII and SalI sites. The shRNA sequence targeting human NR4A1 (NM_001202234.1) was also inserted into pAdTrack-CMV-H1 plasmid. The adenovirus package was performed in 293 T cells, in the presence of pAdEasy-1 plasmid.

To study the interaction between OTUB2 and NR4A1, the human OTUB2 coding sequence or its C51S mutant sequence was inserted into pcDNA3.1-3×flag plasmid, human NR4A1 coding sequence into pcDNA3.1-HA plasmid, and human UBB (NM_018955.3) coding sequence into pcDNA3.1-myc plasmid.

The vectors were purchased from Fenghui Biotechnology Co., Ltd (Changsha, Hunan, China), and the shRNA targeting human OTUB2 or human NR4A1 was synthesized by General Biology Co., Ltd. (Chuzhou, Anhui, China). The shRNA sequence was shown below:

shOTUB2: 5′-CCGGGAGATCTTCAAGTTCAAAGTTCAAGAGACTTTGAACTTGAAGATCTCCCTTTTT-3′;

shNR4A1: 5′-CCGGCACCTTCATGGACGGCTACATTCAAGAGATGTAGCCGTCCATGAAGGTGCTTTTT-3′.

Animals

Healthy female Sprague-Dawley (SD) rats (10-12 weeks old, 200–250 g) were kept in a SPF environment with free access to food and water. The rats were randomly divided into four groups: control, LPS, LPS+Ad-NC, LPS+Ad-OTUB2. The male rats were kept with female rats in a cage overnight (male: female = 1: 2). The next morning, the present of vaginal suppository was used to confirm the successful pregnancy of female rats, and designated as gestation 0 day (GD0). The LPS rats were injected with LPS from E. coli via caudal vein using an infusion pump (Longerpump, Baoding, Hebei, China) (1 μg/kg, dissolved in saline solution, with a speed of 2 ml/h) at GD5. The rats in LPS+Ad-NC and LPS+Ad-OTUB2 groups were intravenously injected with adenovirus carrying rat OTUB2 coding sequence or control empty adenovirus at GD4 (5 × 108 PFU). The SP of pregnant rats was detected at GD0, GD3, GD6, GD9, GD12, GD15 and GD18, and urine protein in 24 h was assessed at GD3, GD10 and GD19. The euthanasia was performed at GD20, and blood and uterus were collected. The fetal rats were isolated, and the weight were measured. The placental tissues were isolated for subsequent detections.

The animal care and use were performed in accordance with Guide for the Care and Use of Laboratory Animals (NIH, 8th). The experiment procedures were approved by Ethic Committee of Shengjing Hospital of China Medical University (approval No. 2023PS1261K).

ELISA

The TNF-α and IL-1β levels in human specimens and cell supernatant were assessed using human TNF-α ELISA Kit or human IL-1β ELISA Kit (Multiscience Biotech, Co., Ltd., Shanghai, China), and serum TNF-α and IL-1β levels from rats were measured with rat TNF-α ELISA Kit or rat IL-1β ELISA Kit (Multiscience Biotech, Co., Ltd.) according to the manufacturer’s protocols. Briefly, the ELISA plates were incubated with sample and antibody at room temperature with slow shaking for 2 h. After washing, the plates were incubated with HRP-conjugated streptavidin at room temperature for 45 min. TMB reagent was added to react for 20 min in the dark, and the interaction was terminated by adding of termination reagent. Finally, the optical density (OD) at 450 nm and 570 nm was measured.

Real-time PCR

Total RNA was extracted with TRIpure RNA Extraction Kit (BioTeke Corporation Co., Ltd., Beijing, China), and the concentration was determined by NanoDrop 2000 ultraviolet spectrophotometer (Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA). Then the RNA was reversely transcribed into cDNA with BeyoRT II M-MLV reverse transcriptase (Beyotime Institute of Biotechnology, Shanghai, China), in the presence of oligo(dT)15 and random primers. Real-time PCR was performed to measure the expression of OTUB2 using SYBR GREEN (SolarBio, Beijing, China) and 2×Taq PCR MasterMix (SolarBio). The PCR procedure was set as below: 95 °C for 5 min 10 s, 60 °C for 10 s, 72 °C for 15 s, followed with 40 cycles of denaturation at 72 for 1 min 30 s, annealing at 40 °C for 1 min, melting at 60–94 °C every 1 °C for 1 s, and final incubation at 25 °C for several minutes. The PCR was carried out with ExicyclerTM 96 Real-Time Quantitative Thermal Block (Bioneer, Daejeon, Korea), and the data was calculated using 2−ΔΔCt (data in cell and animal experiments) or 2−ΔCt (data in clinical data). The primers were synthesized by General Biology Co., Ltd. (Chuzhou, Anhui, China), and the sequence was shown below:

human OTUB2 forward: 5′-GCCATCCGCAAGACCAAAGG-3′;

human OTUB2 reverse: 5′-CTCAAAGCCAGCAGCCAGAA-3′.

Western blot

Total protein was extracted with RIPA lysis supplemented with 1 mM phenylmethanesulfonyl fluoride (SolarBio) on the ice, and the concentration was assessed with a BCA Protein Assay Kit (SolarBio). Subsequently, the protein was separated with SDS-PAGE with 20 μg protein sample per lane. The concentration of polyacrylamide gel ranged from 5–14% according to protein size. After electrophoresis, the protein in the gel was transferred onto the polyvinylidene fluoride membrane Millar Instruments, Inc., Houston, TX, USA, which were blocked with Western Blocking Buffer (SolarBio), and incubated with primary antibodies at 4 °C overnight. After washing with TBST, the membrane was incubated with secondary antibody labeled with horse radish peroxidase (HRP) at 37 °C for 1 h, followed with reaction with ECL reagent and final signal explore in the dark. The information of antibodies was shown below: rabbit anti-OTUB2 (1:1000; Affinity Bioscience, Changzhou, Jiangsu, China), rabbit anti-iNOS (1:500; Affinity Bioscience), rabbit anti-CD86 (1:1000; ABclonal Technology Co., Ltd., Wuhan, Hubei, China), rabbit anti-NR4A1 (1:1000; Affinity Bioscience), mouse anti-GADPH (1:10000; ProteinTech, Rosemont, Illinois, USA), goat anti-rabbit IgG-HRP (1:3000; SolarBio), and goat anti-mouse IgG-HRP (1:3000; SolarBio).

MMP2/9 activity measurement

The enzymatic activity of MMP2 and MMP9 was determined by MMP2/9 Activity Fluorescence Quantitation Kits (Genmed Scientifics Inc., Shanghai, China). Briefly, the cells were lysed by reagent A, the lysate was centrifuged at 300 × g, and the supernatant was collected and incubated with reagent B on the ice. After centrifugation at 16,000 × g for 5 min, the supernatant was collected as sample. The protein concentration was determined by Bradford Protein Assay Kit (Beyotime Biotech Co., Ltd, Shanghai, China) according to the manufacturer’s protocol. The sample was incubated with the substrate (reagent D) in the buffer (reagent C), the relative fluorescence unit was determined with excitation wave of 330 nm and transmitting wave of 400 nm. The standard curve was drawn by herniarin of grading concentrations, and the enzymatic activity was calculated based on the standard curve.

Wound healing assay

Wound healing was performed to measure the migratory ability of trophoblasts. The cells were pre-treated by mitomycin C (MedChemExpres, Monmouth Junction, NJ, USA) in serum-free medium for 1 h. The wound was made by 200 μl pipette tip, and the cells continued to culture for 0 h or 24 h. The picture was acquired with a microscope with ×100 magnification, and the migration rate was calculated.

Transwell assay

The invasion of trophoblasts was assessed by transwell assay supplemented with Matrigel. The transwell chambers (Labselect, Hefei, Anhui, China) were pre-coated with Matrigel (Corning Incorporated, Corning, NY, USA) at 37 °C in 24-well plates. The cells were inoculated in the upper chamber with serum-free medium, and the lower chamber was added with medium supplemented with 10% FBS. After culture for 24 h, the cells on the reverse surface of the chamber were immobilized with 4% paraformaldehyde and stained with 0.5% cresol purple, and the cell number was counted under ×200 magnification.

TUNEL assay

The cells cultured on the glass slides were penetrated with 0.1% Triton X-100 at room temperature for 15 min. Then the cells were treated with In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instruction. The TUNEL reaction solution was prepared with enzyme solution and label solution with volume ratio 1:9. The cells were incubated with reaction solution at 37 °C in the dark for 60 min. After washing with PBS, the cells were counterstained with DAPI for 5 min. Finally, the slides were sealed with anti-fading reagent (SolarBio), and the pictures were acquired at ×400 magnification.

Immunohistochemistry staining

The tissues were immobilized with 10% formaldehyde overnight, washed with flowing water for 4 h, dehydrated with ethanol of grading concentrations (70% for 2 h, 80% overnight, 90% for 2 h, 100% for 1 h twice) and xylene for 30 min. Subsequently, the tissues were embedded by paraffin at 60 °C, and cut into 5-μm sections. After deparaffinization and antigen retrieval, the sections were blocked with 3% H2O2 to deactivate exogenous peroxidases, blocked with 1% bovine serum albumin (BSA) (Sangon Biotech Co., Ltd., Shanghai, China), and incubated with antibody against CK7 (1:1000; ProteinTech), OTUB2 (1:100; Affinity Bioscience) or NR4A1 (1:100; Affinity Bioscience) at 4 °C overnight. After incubation with secondary antibody labeled with HRP, the sections were incubated with DAB reagent, and stained with hematoxylin (SolarBio). Finally, the sections underwent re-dehydration with ethanol and xylene, and was sealed with gum. The images were acquired at ×400 magnification.

Immunofluorescence staining

The placental tissues were made into paraffin sections according to the description in immunohistochemistry staining section. After dehydration with xylene and ethanol, antigen retrieval was performed in boiling for 10 min. Subsequently, the sections were blocked with 1% BSA at room temperature for 15 min, and incubated with antibody against CD68 (1:50; Novus Biologicals, Littleton, CO, USA) or OTUB2 (1:100; Affinity Bioscience) at 4 °C overnight. Afterwards, goat anti-rabbit IgG-FITC or goat anti-mouse IgG-Cy3 (1:200; Abcam, Cambridge, MA, USA) were applied for incubation with sections. The nuclei were stained with DAPI, and the sections were sealed with anti-fading reagent (SolarBio).

For identification of primary trophoblasts, immunofluorescence staining with CK7 and vimentin was performed. The cells on the glass slides were immobilized with 4% paraformaldehyde (Sino Biological Inc., Beijing, China) for 15 min, permeated by 0.1% tritonX-100 for 30 min, blocked with 1% BSA for 15 min, and incubated with antibody against CK7 (1:200; ProteinTech) or vimentin (1:200; Affinity Bioscience) at 4 °C overnight. Then the sections were incubated with goat anti-rabbit IgG conjugated with Cy3 (1:200; Abcam) at room temperature for 60 min. After nucleus staining, the sections were sealed with an anti-fading reagent. The images were acquired at ×400 magnification.

Co-immunoprecipitation (co-IP)

The total protein was extracted on the ice, and used for co-IP with a PierceTM co-IP kit (Thermo Fisher Scientific Co., Ltd.) after concentration determination. The antibody was immobilized with resin in spin column, in the presence of sodium borohydride cyanide. The protein was incubated with the antibody in spin column with slow shaking at room temperature for 2 h. After washing, the antigen-antibody complex was eluted with elution buffer, and used for SDS-PAGE after denaturation in boiling. The details referred to those in western blot section. The information of IP antibody was shown below: rabbit anti-flag tag (ABclonal Technology Co., Ltd.), rabbit anti-HA tag (Affinity Bioscience), and rabbit anti-myc tag (ABclonal Technology Co., Ltd.).

Statistics and reproducibility

The data in this study were presented as mean ± standard deviation in three (cell experiments) or six independent individuals (animal experiments), and analyzed with GraphPad Prism software. The data in two independent groups were compared with student two-tailed t test, and data in multiple groups were analyzed by one-way ANOVA followed with Bonferroni’s multiple comparisons. A normality and lognormality test was performed before analysis. A p value less than 0.05 was considered as statistically significant.

Ethical statement

The placental tissue collection and detections were line with Declaration of Helsinki, and approved by the Ethics Committee of Shengjing Hospital of China Medical University (approval No. 2024PS568K). The informed consent was obtained from every placenta donor, and these placentae were used for RNA sequencing, cell isolation, or other detections.

Supplementary information

Supplementary Information (467.4KB, pdf)
Supplementary Data (205.9KB, pdf)
42003_2025_8755_MOESM3_ESM.docx (21.1KB, docx)

Description of Additional Supplementary Files

Acknowledgements

This work was supported by the Science and Technology Projects of Liaoning Province (2021JH2/10300093).

Author contributions

Yue W., Yang W., F.T.: conception and design, data collection and analysis, manuscript writing; Q.G., B.L.: experiment performance, data collection and analysis; Y.Z.: conception and design, final approval of manuscript, financial support. All authors have read and approved the final version of the manuscript.

Peer review

Peer review information

Communications Biology thanks Yi-Hui Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Dr Toshiro Moroishi and Dr Ophelia Bu.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. The numerical source data underlying all graphs were shown in Supplementary data. The original western blot images were shown in Supplementary Figs. The RNA sequencing data is shown in GEO databank (https://www.ncbi.nlm.nih.gov/geo/), and the accession number is GSE303543.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Yue Wang, Yang Wang, Fei Tong.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-025-08755-9.

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Supplementary Materials

Supplementary Information (467.4KB, pdf)
Supplementary Data (205.9KB, pdf)
42003_2025_8755_MOESM3_ESM.docx (21.1KB, docx)

Description of Additional Supplementary Files

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. The numerical source data underlying all graphs were shown in Supplementary data. The original western blot images were shown in Supplementary Figs. The RNA sequencing data is shown in GEO databank (https://www.ncbi.nlm.nih.gov/geo/), and the accession number is GSE303543.

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