NEDD4L-induced β-catenin ubiquitination suppresses the formation and progression of interstitial pulmonary fibrosis via inhibiting the CTHRC1/HIF-1α axis

Interstitial pulmonary fibrosis (IPF) is a severe progressive lung disease with limited therapeutic options and poor prognosis. Initially, we found the downregulated level of neural precursor cell expressed developmentally down-regulated 4-like protein (NEDD4L) in IPF-related expression microarray dataset, and this study was thus performed to explore the molecular mechanism of NEDD4L in IPF. The expression of NEDD4L was subsequently validated in lung tissues of IPF patients and mouse models. Then, mouse primary lung fibroblasts (LFs) were collected for in vitro functional experiments, with CCK-8, Transwell, and immunofluorescence assays used to examine the viability, migration, and differentiation of LFs. The in vitro findings were further assessed using in vivo mouse models. The expression of NEDD4L was down-regulated in lung tissues of IPF patients and mouse models. Overexpression of NEDD4L restricted the formation and progression of IPF in mice and attenuated the proliferative, invasive and differentiative abilities of LFs. Further, NEDD4L halted LFs activity by enhancing β-catenin ubiquitination and down-regulating the CTHRC1/HIF-1α axis. Also, in vivo experiments then validated that NEDD4L silencing repressed β-catenin ubiquitination and activated the CTHRC1/HIF-1α axis, thereby aggravating IPF in mice. NEDD4L may suppress the formation and progression of IPF through augmenting β-catenin ubiquitination and inhibiting the CTHRC1/HIF-1α axis.     

Circular RNA VRK1 facilitates pre‐eclampsia progression via sponging miR‐221‐3P to regulate PTEN/Akt

Pre‐eclampsia (PE) is a worldwide pregnancy‐related disorder. It is mainly characterized by defect migration and invasion of trophoblast cells. Recently, circular RNAs (circRNAs) have been believed to play a vital role in PE. The expression patterns and the biological functions of circRNAs in PE remain elusive. Here, we performed a circRNA microarray to identify putative PE‐related circRNAs. Bioinformatics analyses were used to screen the circRNAs which have potential relationships with pre‐eclampsia, and we identified a novel circRNA (circVRK1) that was up‐regulated in PE placenta tissues. By using HTR‐8/SVneo cells, circVRK1 knockdown significantly enhanced cell migration and invasion abilities, as well as epithelial‐mesenchymal transition (EMT). Mechanistically, we found that circVRK1 and PTEN could function as the ceRNAs to miR‐221‐3p. Overexpression of miR‐221‐3p promoted cell migration, invasion and EMT via regulating PTEN. The cotransfection of miR‐221‐3p inhibitor or PTEN reversed the effect from circVRK1 knockdown. Moreover, the circVRK1/miR‐221‐3p/PTEN axis greatly regulated Akt phosphorylation. In general, circVRK1 suppresses trophoblast cell migration, invasion and EMT, by acting as a ceRNA to miR‐221‐3p to regulate PTEN, and further inhibit PI3K/Akt activation. The purpose of this paper is to open wide insights to investigate the onset of PE and provide new potential therapeutic targets in PE.     

Upregulated lncRNA UCA1 inhibits trophoblast cell invasion and proliferation by downregulating JAK2

Numerous studies have suggested that urothelial cancer-associated 1 (UCA1) acts as a suppressor gene affecting cell proliferation and migration. However, the biological role and the potential mechanism of UCA1 in the progression of pre-eclampsia (PE) remains unclear. The UCA1 level was markedly upregulated in PE pregnancies relative to non-PE ones in GSE75010 and tissues. A higher body mass index (BMI), maximum systolic blood pressure (BP), and maximum diastolic BP were observed in PE pregnancies, whereas the newborn weight z-score was lower compared with those of non-PE pregnancies. Knockdown of UCA1 accelerated the proliferative migratory abilities and cell cycle progression, but inhibited apoptosis of HTR-8/SVneo and JAR cells. Then, we found that Janus kinases 2 (JAK2) was negatively correlated with UCA1. In addition, JAK2 was downregulated in the placenta of PE pregnancies and was negatively regulated by UCA1. UCA1 was mainly enriched in the nucleus. Knockdown of UCA1 reduced the occupancies of the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and H3K27me3 on the Janus kinase 2 (JAK2) promoter regions. Finally, rescue experiments found that transfection of short-hairpin JAK2 attenuated proliferative and migratory abilities of trophoblasts, which were partially reversed after UCA1 knockdown. In short, UCA1 is upregulated in the trophocytes of PE pregnancies and accelerates trophoblast cell invasion and proliferation by downregulating JAK2.     

Placental villous mesenchymal cells trigger trophoblast invasion

The successful transformation of uterine spiral arteries by invasion trophoblasts is critical for the formation of the human hemochorial placenta. Placental trophoblast migration and invasion are well regulated by various autocrine/paracrine factors at maternal–fetal interface. Human placental multipotent mesenchymal stromal cells (hPMSCs) are a subpopulation of villous mesenchymal cells and have been shown to produce a wide array of soluble cytokines and growth factors including HGF (hepatocyte growth factor). The function of hPMSCs in placental villous microenvironment has not been explored. The interaction between hPMSCs and trophoblasts was proposed in vitro in a recent article. HGF produced by hPMSCs was able to engage c-Met receptor on trophoblast and induced the trophoblast cAMP expression. The cAMP activated PKA, which in turn, signaled to Rap1 and led to integrin β1 activation. The total integrin β1 protein expression by trophoblasts was not affected by HGF stimulation. Hypoxia downregulated HGF expression by hPMSCs. HGF and PKA activator 6-Bnz-cAMP increased trophoblast adhesion and migration that were inhibited by PKA inhibitor H89 or Rap1 siRNA. Thus, hPMSCs-derived paracrine HGF can regulate trophoblast migration during placentation. These findings provided insight revealing at least one mechanism by which hPMSCs implicated in the development of preeclampsia.     

Neuron specific enolase promotes tumor metastasis by activating the Wnt/β-catenin pathway in small cell lung cancer

Neuron-specific enolase (NSE) has been used as a specific biomarker for small cell lung cancer (SCLC) patients. Nevertheless, the biological function and mechanism of NSE in SCLC are still unclear. In this study, we clarified the role of NSE in the progression of SCLC and found that NSE expression was positively correlated with distant metastasis. Functional analysis showed that overexpression of NSE promoted migration and invasion of SCLC cells. Mechanism analysis showed that NSE overexpression induced epithelial-mesenchymal transition (EMT) of SCLC cells. Moreover, overexpression of NSE increased the protein expression of β-catenin and its downstream target genes, and silencing β-catenin eliminated NSE-mediated cell migration, invasion and EMT process. Furthermore, NSE interacted with β-catenin and inhibited the degradation of β-catenin. Besides, the animal experiments also indicated that NSE could promote the EMT process and distant metastasis of SCLC cells in vivo. In summary, our results revealed that NSE could promote the EMT process of SCLC cells by activating the Wnt/β-catenin signaling pathway, thereby promoting cell migration, invasion and distant metastasis, which might serve as a potential target for the therapy of SCLC patients.     

Overexpression of let-7d explains down-regulated KDM3A and ENO2 in the pathogenesis of preeclampsia

Pre-eclampsia (PE) is the leading cause of maternal death; however, the causative molecular basis remains largely unknown. Recent studies have revealed the important role microRNAs (miRNAs) play in PE. We aimed to explore the effects of let-7d on trophoblast proliferation, migration, invasion and apoptosis in PE and its underlying mechanism. Placental tissues were collected from PE patients and healthy pregnant women, and it was found that let-7d expression was increased, while KDM3A and ENO2 expression was decreased in PE tissues and cells. Bioinformatics analysis indicated the interaction among let-7d, KDM3A and ENO2, confirmed by dual luciferase reporter gene assay; ChIP experiment identified methylated modification to ENO2 by KDM3A. With gain- and loss-function method, silencing of let-7d increased KDM3A expression and enhanced the binding between KDM3A and ENO2. Furthermore, overexpression of let-7d suppressed cell proliferation, migration and invasion of trophoblasts, and induced apoptosis of trophoblasts, while these capacities were restored upon additional treatment of overexpressed ENO2. PE rat models were established to explore the effects of let-7d and ENO2 on PE in vivo. The results established that the silencing of let-7d alleviated the tissue injury and PE-related symptoms when reducing urine protein, TUNEL-positive cells and increasing ENO2, and KDM3A expression in rats. Cumulatively, let-7d suppressed cell progression of trophoblasts, and induced apoptosis through the down-regulation of KDM3A to promote ENO2 methylation, thereby promoting progression of PE. Such an epigenetic network of let-7d, KDM3A and ENO2 in the pathogenesis of PE might provide novel insight into targeted therapy against this disorder.     

miR-155-5p modulates malignant behaviors of hepatocellular carcinoma by directly targeting CTHRC1 and indirectly regulating GSK-3β-involved Wnt/β-catenin signaling

Background

Hepatocellular carcinoma (HCC) remains one of the most lethal cancers. MicroRNA-155 (miR-155) and collagen triple helix repeat containing 1 (CTHRC1) were found to be involved in hepatocarcinogenesis, but their detailed functions in HCC are unclear. Here, we aimed to investigate the underlying role of miR-155-5p and CTHRC1 in HCC.

Methods

miR-155-5p and CTHRC1 expression levels were detected by qRT-PCR, IHC and WB in HCC patients and cell lines. Dual-luciferase assay, qRT-PCR and WB were used to validate the target interaction between miR-155-5p and CTHRC1. Biological behaviors, including apoptosis, cell cycle progression, and cell proliferation, invasion and migration, were measured by flow cytometry, CCK-8 assay and Transwell tests. A xenograft model was established to examine the effects of miR-155-5p and CTHRC1 on tumor formation. WB was finally utilized to identify the role of GSK-3β-involved Wnt/β-catenin signaling in HCC growth and metastasis.

Results

Our results showed that miR-155-5p and CTHRC1 were down-regulated and up-regulated, respectively, in HCC patients and cell lines. Dual-luciferase assay verified that CTHRC1 was the direct target of miR-155-5p. Moreover, elevated miR-155-5p expression promoted apoptosis but suppressed cell cycle progression and cell proliferation, invasion and migration in vitro and facilitated tumor formation in vivo; elevated CTHRC1 expression abolished these biological effects. Additionally, miR-155-5p overexpression increased metastasis- and anti-apoptosis-related protein expression and decreased pro-apoptosis-related protein expression, while forced CTHRC1 expression conserved the expression of these proteins.

Conclusion

Altogether, our data suggested that miR-155-5p modulated the malignant behaviors of HCC by targeting CTHRC1 and regulating GSK-3β-involved Wnt/β-catenin signaling; thereby, miR-155-5p and CTHRC1 might be promising therapeutic targets for HCC patients.

     

MNSFβ regulates placental development by conjugating IGF2BP2 to enhance trophoblast cell invasiveness

ObjectivesSuccess in pregnancy in mammals predominantly depends on a well‐developed placenta. The differentiation of invasive trophoblasts is a fundamental process of placentation, the abnormalities of which are tightly associated with pregnancy disorders including preeclampsia (PE). Monoclonal nonspecific suppressor factor beta (MNSFβ) is an immunosuppressive factor. Its conventional knockout in mice induced embryonic lethality, whereas the underlying mechanism of MNSFβ in regulating placentation and pregnancy maintenance remains to be elucidated.MethodsTrophoblast‐specific knockout of MNSFβ was generated using Cyp19‐Cre mice. In situ hybridization (ISH), haematoxylin and eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) were performed to examine the distribution of MNSFβ and insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) at the foeto‐maternal interface. The interaction and expression of MNSFβ, IGF2BP2 and invasion‐related molecules were detected by immunoprecipitation (IP), immunoblotting and quantitative real‐time polymerase chain reaction (qRT‐PCR). The cell invasion ability was measured by the Transwell insert assay.ResultsWe found that deficiency of MNSFβ in trophoblasts led to embryonic growth retardation by mid‐gestation and subsequent foetal loss, primarily shown as apparently limited trophoblast invasion. In vitro experiments in human trophoblasts demonstrated that the conjugation of MNSFβ with IGF2BP2 and thus the stabilization of IGF2BP2 essentially mediated the invasion‐promoting effect of MNSFβ. In the placentas from MNSFβ‐deficient mice and severe preeclamptic (PE) patients, downregulation of MNSFβ was evidently associated with the repressed IGF2BP2 expression.ConclusionsThe findings reveal the crucial role of MNSFβ in governing the trophoblast invasion and therefore foetal development, and add novel hints to reveal the placental pathology of PE.     

Elevated Expression of KiSS-1 in Placenta of Chinese Women with Early-Onset Preeclampsia

Preeclampsia (PE) is a heterogeneous syndrome affecting 2% to 8% of all pregnancies and is the world’s leading cause of fetal and maternal morbidity and mortality. In many cases of PE, shallow trophoblast invasion results in inappropriate maternal spiral artery remodeling and impaired placental function. Multiple genes have been implicated in trophoblast invasion, among which are KiSS-1 and GPR54. The gene product of KiSS-1 is metastin, which is a ligand for the receptor GPR54. Both metastin and GPR54 are expressed in the placenta of normal pregnancy and have been implicated in modulating trophoblast invasion through inhibiting migration of trophoblast cells. We have previously reported that the expression level of KiSS-1 was higher in trophoblasts from women with preeclampsia as compared to normal controls. Here, using quantitative RT-PCR, Western blot analysis and immunohistochemistry, we extend our analysis to demonstrate that elevated KiSS-1 expression occurs only in early-onset preeclampsia (ePE) and not late-onset preeclampsia (lPE). However, no difference in the expression levels of GPR54 is observed between ePE, lPE, and normal controls. Further, we show that KiSS-1 expression is also increased in placenta of intrauterine death and birth asphyxia in comparison to normal newborns of ePE and lPE. Our findings suggest that aberrant upregulation of KiSS-1 expression may contribute to the underlying mechanism of ePE as well as birth asphyxia.     

Upregulated long noncoding RNA Linc00261 in pre-eclampsia and its effect on trophoblast invasion and migration via regulating miR-558/TIMP4 signaling pathway

Pre-eclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality but the exact underlying mechanisms of PE pathogenesis remain elusive. Accumulated data suggested that the long noncoding RNAs (lncRNAs) play important roles in the pathogenesis of PE. The present study identified the changes of lncRNA Linc00261 in PE and its effects on trophoblasts invasion and migration. Our results showed that the expression of Linc00261 was upregulated in placental tissues of PE women compared with those of healthy pregnant women. Overexpression of Linc00261 suppressed cell invasion and migration, induced cell apoptosis, and caused cell-cycle arrest at G₀/G₁ phase of HTR-8/SVneo cells; while knockdown of Linc00261 had the opposite effects on the HTR-8/SVneo cells. Mechanistic studies showed Linc00261 functioned as a competing endogenous RNA for miR-558 in HTR-8/SVneo cells, and miR-558 was negatively regulated by Linc00261. The expression level of miR-558 in the PE group was significantly lower than the control group, and the expression level of Linc00261 was negatively correlated with the expression level of miR-558 in the placental tissues of women with PE. Furthermore, miR-558 was found to negatively regulate the expression of TIMP metallopeptidase inhibitor 4 (TIMP4) via targeting the 3′ untranslated region in the HTR-8/SVneo cells. Overexpression of miR-558 increased HTR-8/SVneo cell invasion and migration, which was attenuated by TIMP4 overexpression. More importantly, both overexpression of miR-558 and knockdown of TIMP4 partially reversed the suppressive effects of Linc00261 overexpression on cell invasion and migration of HTR-8/SVneo cells. Collectively, our results for the first time showed the upregulation of Linc00261 in the placental tissues of severe PE patients. The mechanistic results indicated that Linc00261 exerted the suppressive effects on the trophoblast invasion and migration via targeting miR-558/TIMP4 axis, which may involve in the pathogenesis of PE.     

Extracellular vesicle-derived microRNA-18b ameliorates preeclampsia by enhancing trophoblast proliferation and migration via Notch2/TIM3/mTORC1 axis

Preeclampsia (PE), a common disorder of pregnancy, is characterized by insufficient trophoblast migration and inadequate vascular remodelling, such that promotion of trophoblast proliferation might ameliorate PE. In the current study, we sought to study the underlying mechanism of extracellular vesicle (EV)-derived microRNA-18 (miR-18b) in PE. Human umbilical cord mesenchymal stem cells (HUCMSCs) isolated from placental tissues were verified through osteogenic, adipogenic and chondrogenic differentiation assays. Bioinformatics analyses and dual-luciferase reporter gene assay were adopted to confirm the targeting relationship between miR-18b and Notch2. The functional roles of EV-derived miR-18b and Notch2 in trophoblasts were determined using loss- and gain-of-function experiments, and trophoblast proliferation and migration were assayed using CCK-8 and Transwell tests. In vivo experiments were conducted to determine the effect of EV-derived miR-18b, Notch2 and TIM3/mTORC1 in a rat model of PE, with monitoring of blood pressure and urine proteinuria. TUNEL staining was conducted to observe the cell apoptosis of placental tissues of PE rats. We found down-regulated miR-18b expression, and elevated Notch2, TIM3 and mTORC1 levels in the placental tissues of PE patients compared with normal placenta. miR-18b was delivered to trophoblasts and targeted Notch2 and negatively its expression, whereas Notch2 positively mediated the expression of TIM3/mTORC1. EV-derived miR-18b or Notch2 down-regulation enhanced trophoblast proliferation and migration in vitro and decreased blood pressure and 24 hours urinary protein in PE rats by deactivating the TIM3/mTORC1 axis in vivo. In summary, EV-derived miR-18b promoted trophoblast proliferation and migration via down-regulation of Notch2-dependent TIM3/mTORC1.     

MicroRNA-384 inhibits nasopharyngeal carcinoma growth and metastasis via binding to Smad5 and suppressing the Wnt/β-catenin axis

Nasopharyngeal carcinoma (NPC) is a major otorhinolaryngological disease with limited effective therapeutic options. This work focused on the function of microRNA-384 (miR-384) on the NPC pathogenesis and the molecules involved. miR-384 expression in cancer tissues and cells was detected. Gain- and loss-of-functions of miR-384 were performed to identify its role in NPC progression. The target mRNA of miR-384 was predicted on an online system and validated through a luciferase reporter assay. The activity of Wnt/β-catenin signaling was detected. Consequently, miR-384 was found to be poorly expressed in NPC tissues and cell lines and was linked to unfavorable survival rates in patients. Overexpression of miR-384 in 6-10B cells suppressed growth, migration, invasion and resistance to apoptosis of cells, but inverse trends were presented in C6661 cells where miR-384 was downregulated. miR-384 targeted Smad5 mRNA. Upregulation of Smad5 counteracted the roles of miR-384 mimic in cells. The NPC-inhibiting effects of miR-384 mimic were also blocked by Wnt/β-catenin activation. To conclude, miR-384 targets Smad5 and inactivates the Wnt/β-catenin pathway, which exerts a suppressing role in NPC cell behaviors as well as tumor growth in vivo. The findings may offer novel thoughts into NPC therapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10616-021-00458-3.     

Long non-coding RNA LINC01347 suppresses trophoblast cell migration,invasion and EMT by regulating miR-101–3p/PTEN/AKT axis

Recurrent miscarriage (RM) is one of the common complications of pregnancy, which is closely related to gene mutation. The profiling of non-coding RNAs showed that the expression level of long non-coding RNA LINC01347 (LINC01347) in the serum of patients with recurrent abortion was significantly increased, which could serve as a potential marker for early diagnosis. However, the biological functions of LINC01347 in the miscarriage remain to be elucidated. In this study, LINC01347 expression levels in HTR-8/SVneo cells and placenta samples were measured by RT-qPCR. The migration ability of HTR-8/SVneo cells was detected by wound-healing assay. Western blotting (WB) assay was conducted to measure E-cadherin, Vimentin, N-cadherin, PTEN, phospho-AKT(S473), phospho-AKT(T308) and AKT levels. Dual luciferase reporter assay and RNA pull-down analysis were performed to validate the molecular interactions. The results showed an upregulation of LINC01347 in the placenta samples of RM patients and HTR-8/SVneo cells. LINC01347 overexpression impaired the invasion and migration of trophoblast cells, while LINC01347 silencing promoted cell migration and invasion. LINC01347 level was also negatively correlated with the changes of epithelial-mesenchymal transition (EMT) markers in trophoblasts. We further demonstrated that miR-101–3p/PTEN/AKT axis played an important role in mediating the biological roles of LINC01347 in the invasion and migration of trophoblasts. In conclusion, our results revealed that LINC01347 suppresses the migratory ability and regulates the EMT processes in trophoblasts by regulating miR-101–3p/PTEN/AKT axis, suggesting that targeting LINC01347 may serve as a strategy to ameliorate RM.