Investigating the Role of Transcription Factors TWIST1, TWIST2 and PPARγ in the Progression of Nonalcoholic Steatohepatitis

Abstract Background: To investigate the role that transcription factors TWIST1, TWIST2 and PPARγ play in nonalcoholic steatohepatitis progression. Methods: The protein levels of TWIST1, TWIST2 and PPARγ were determined in the serum of NAFLD patients and healthy controls by ELISA. An in vivo model for fatty liver was established by feeding C57BL/6J mice a high fat diet (HFD). An in vitro model of steatosis was established by treating LO-2 cells with oleic acid (OA). RNA sequencing was performed on the untreated and OA treated LO-2 cells followed by TWIST1, TWIST2 and PPARγ gene mRNA levels analysis, Gene Ontology (GO) enrichment and pathway analysis. Results: The TWIST2 serum protein levels decreased significantly in all fatty liver groups (P<0.05) while TWIST1 varied. TWIST2 tended to be lower in mice fed a HFD and was significantly lower at 3 months. Similarly, in our in vitro model, TWIST2 protein level was down significantly at 48 and 72 hours after OA treatment. RNA sequencing of the LO-2 cells showed an approximately 2.3-fold decrease in TWIST2, with TWIST1 and PPARγ no obvious change. The PPAR signaling pathway was enriched with 4 genes up regulated in OA treated cells (P=0.0018). IL-17 and TNF signaling pathways were enriched in OA treated cells. Conclusions: Our results provide evidence that TWIST2 and PPARγ are important in NAFLD and shed light on a potential mechanism of steatosis.

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TWIST2 and the PPAR signaling pathway are important in the progression of nonalcoholic steatohepatitis

Lipids in Health and Disease ◽

10.1186/s12944-021-01458-0 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Yanmei Zhang ◽

Xiaoxiao Ge ◽

Yongqing Li ◽

Bingyang Zhang ◽

Peijun Wang ◽

...

Keyword(s):

Transcription Factor ◽

Fatty Liver ◽

Rna Sequencing ◽

Nonalcoholic Steatohepatitis ◽

In Vitro Model ◽

Bhlh Transcription Factor ◽

Protein Levels ◽

Ppar Signaling ◽

Vitro Model

Abstract Background To investigate the roles of the transcription factors twist family bHLH transcription factor 1 (TWIST1), twist family bHLH transcription factor 2 (TWIST2), and peroxisome proliferator activated receptor gamma (PPARγ) in the progression of nonalcoholic steatohepatitis. Methods The protein levels of TWIST1, TWIST2 and PPARγ were determined in the serum of nonalcoholic fatty liver disease (NAFLD) patients and healthy controls by enzyme-linked immunosorbent assay (ELISA). An in vivo model for fatty liver was established by feeding C57BL/6 J mice a high-fat diet (HFD). An in vitro model of steatosis was established by treating LO-2 cells with oleic acid (OA). RNA sequencing was performed on untreated and OA-treated LO-2 cells followed by TWIST1, TWIST2 and PPARγ gene mRNA levels analysis, Gene Ontology (GO) enrichment and pathway analysis. Results The TWIST2 serum protein levels decreased significantly in all fatty liver groups (P < 0.05), while TWIST1 varied. TWIST2 tended to be lower in mice fed an HFD and was significantly lower at 3 months. Similarly, in the in vitro model, the TWIST2 protein level was downregulated significantly at 48 and 72 h after OA treatment. RNA sequencing of LO-2 cells showed an approximately 2.3-fold decrease in TWIST2, with no obvious change in TWIST1 and PPARγ. The PPAR signaling pathway was enriched, with 4 genes upregulated in OA-treated cells (P = 0.0018). The interleukin (IL)-17 and tumor necrosis factor (TNF) signaling pathways were enriched in OA-treated cells. Conclusions The results provide evidence that the TWIST2 and PPAR signaling pathways are important in NAFLD and shed light on a potential mechanism of steatosis.

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TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

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The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice

Reproduction ◽

10.1530/rep-09-0518 ◽

2010 ◽

Vol 139 (4) ◽

pp. 759-769 ◽

Cited By ~ 42

Author(s):

F P Yuan ◽

X Li ◽

J Lin ◽

C Schwabe ◽

E E Büllesbach ◽

...

Keyword(s):

Mesenchymal Cell ◽

Postnatal Period ◽

Testosterone Replacement Therapy ◽

Developmental Defect ◽

Mrna Levels ◽

Lh Receptor ◽

Protein Levels ◽

Testis Descent

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT).In vivoandin vitroexperiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development inLhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5α-dihydrotestosterone (DHT) upregulated the expression ofRxfp2which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase inRxfp2mRNA levels in a time-dependent fashion inLhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediatedRxfp2knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent inLhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.

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Effect of the proinflammatory cytokine TNF-α on intestinal riboflavin uptake: inhibition mediated via transcriptional mechanism(s)

AJP Cell Physiology ◽

10.1152/ajpcell.00295.2018 ◽

2018 ◽

Vol 315 (5) ◽

pp. C653-C663 ◽

Cited By ~ 5

Author(s):

Kasin Yadunandam Anandam ◽

Omar A. Alwan ◽

Veedamali S. Subramanian ◽

Padmanabhan Srinivasan ◽

Rubina Kapadia ◽

...

Keyword(s):

Ex Vivo ◽

Significant Inhibition ◽

Mrna Levels ◽

In Vivo Models ◽

Uptake Inhibition ◽

Tnf Α ◽

Vitro Model ◽

Factor Α

Riboflavin (RF), is essential for normal cellular metabolism/function. Intestinal RF absorption occurs via a specific carrier-mediated process that involves the apical transporter RFVT-3 ( SLC52A3) and the basolateral RFVT-1 (SLC52A1). Previously, we characterized different cellular/molecular aspects of the intestinal RF uptake process, but nothing is known about the effect of proinflammatory cytokines on the uptake event. We addressed this issue using in vitro, ex vivo, and in vivo models. First, we determined the level of mRNA expression of the human (h)RFVT-3 and hRFVT-1 in intestinal tissue of patients with inflammatory bowel disease (IBD) and observed a markedly lower level compared with controls. In the in vitro model, exposing Caco-2 cells to tumor necrosis factor-α (TNF-α) led to a significant inhibition in RF uptake, an effect that was abrogated upon knocking down TNF receptor 1 (TNFR1). The inhibition in RF uptake was associated with a significant reduction in the expression of hRFVT-3 and -1 protein and mRNA levels, as well as in the activity of the SLC52A3 and SLC52A1 promoters. The latter effects appear to involve Sp1 and NF-κB sites in these promoters. Similarly, exposure of mouse small intestinal enteroids and wild-type mice to TNF-α led to a significant inhibition in physiological and molecular parameters of intestinal RF uptake. Collectively, these findings demonstrate that exposure of intestinal epithelial cells to TNF-α leads to inhibition in RF uptake and that this effect is mediated, at least in part, via transcriptional mechanism(s). These findings may explain the significantly low RF levels observed in patients with IBD.

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Glucosamine Enhancement of BDNF Expression and Animal Cognitive Function

Molecules ◽

10.3390/molecules25163667 ◽

2020 ◽

Vol 25 (16) ◽

pp. 3667

Author(s):

Lien-Yu Chou ◽

Yu-Ming Chao ◽

Yen-Chun Peng ◽

Hui-Ching Lin ◽

Yuh-Lin Wu

Keyword(s):

Cognitive Function ◽

In Vivo Model ◽

Mrna Levels ◽

Bdnf Expression ◽

Object Recognition Test ◽

Creb Phosphorylation ◽

Downstream Signaling ◽

Vitro Model

Brain-derived neurotrophic factor (BDNF) is an important factor for memory consolidation and cognitive function. Protein kinase A (PKA) signaling interacts significantly with BDNF-provoked downstream signaling. Glucosamine (GLN), a common dietary supplement, has been demonstrated to perform a variety of beneficial physiological functions. In the current study, an in vivo model of 7-week-old C57BL/6 mice receiving daily intraperitoneal injection of GLN (0, 3, 10 and 30 mg/animal) was subjected to the novel object recognition test in order to determine cognitive performance. GLN significantly increased cognitive function. In the hippocampus GLN elevated tissue cAMP concentrations and CREB phosphorylation, and upregulated the expression of BDNF, CREB5 and the BDNF receptor TrkB, but it reduced PDE4B expression. With the in vitro model in the HT22 hippocampal cell line, GLN exposure significantly increased protein and mRNA levels of BDNF and CREB5 and induced cAMP responsive element (CRE) reporter activity; the GLN-mediated BDNF expression and CRE reporter induction were suppressed by PKA inhibitor H89. Our current findings suggest that GLN can exert a cognition-enhancing function and this may act at least in part by upregulating the BDNF levels via a cAMP/PKA/CREB-dependent pathway.

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Corilagin ameliorates schistosomiasis hepatic fibrosis through regulating IL-13 associated signal pathway in vitro and in vivo

Parasitology ◽

10.1017/s0031182016001128 ◽

2016 ◽

Vol 143 (12) ◽

pp. 1629-1638 ◽

Cited By ~ 13

Author(s):

HUA-RONG LI ◽

GANG LI ◽

MAN LI ◽

SHU-LING ZHANG ◽

HENG WANG ◽

...

Keyword(s):

Hepatic Fibrosis ◽

Quantitative Polymerase Chain Reaction ◽

Signal Pathway ◽

Signal Molecules ◽

Mrna Levels ◽

Protein Levels ◽

Set Up ◽

Inflammatory Changes

SUMMARYInterleukin (IL)-13-associated signal pathway plays an important role in schistosomiasis hepatic fibrosis. In this study we tried to investigate the effects of corilagin to ameliorate schistosomiasis hepatic fibrosis through regulating IL-13-associated signal pathway in vitro and in vivo. Cellular model was set up with hepatic stellate cells-T6 cells stimulated by rIL-13 and male Balb/c mice were infected with Schistosoma japonicum cercariaeas as animal model. Liver histological changes were observed with haematoxylin and eosin staining. Masson staining was employed to observe the change of egg granulomas. Expression of Col (collagen) and Col III were examined with Immunohistochemistry. Western bolt was employed to detect the JAK-1 and IL13Rα1 proteins. The mRNA expression of Col I, Col III, IL-13, JAK-1 and IL13Rα1 were tested by quantitative polymerase chain reaction. As a result, less inflammatory changes were found in all corilagin groups compared with model group and praziquantel group. The mRNA levels of Col I, Col III, IL-13, JAK-1 and IL13Rα1 were significantly decreased after corilagin intervention (P < 0·01). JAK-1 and IL-13Rα1 protein levels were also greatly decreased in the corilagin groups (P < 0·01). In conclusion, corilagin could ameliorate schistosomiasis hepatic fibrosis by down-regulating the expression of IL-13 and signal molecules in IL-13 pathway.

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The PPARγ ligand rosiglitazone attenuates hypoxia-induced endothelin signaling in vitro and in vivo

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00195.2011 ◽

2011 ◽

Vol 301 (6) ◽

pp. L881-L891 ◽

Cited By ~ 34

Author(s):

Bum-Yong Kang ◽

Jennifer M. Kleinhenz ◽

Tamara C. Murphy ◽

C. Michael Hart

Keyword(s):

Transcription Factors ◽

Hypoxia Inducible Factor ◽

Cell Counting ◽

Mouse Lung ◽

Therapeutic Effects ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Protein Levels

Peroxisome proliferator-activated receptor (PPAR) γ activation attenuates hypoxia-induced pulmonary hypertension (PH) in mice. The current study examined the hypothesis that PPARγ attenuates hypoxia-induced endothelin-1 (ET-1) signaling to mediate these therapeutic effects. To test this hypothesis, human pulmonary artery endothelial cells (HPAECs) were exposed to normoxia or hypoxia (1% O2) for 72 h and treated with or without the PPARγ ligand rosiglitazone (RSG, 10 μM) during the final 24 h of exposure. HPAEC proliferation was measured with MTT assays or cell counting, and mRNA and protein levels of ET-1 signaling components were determined. To explore the role of hypoxia-activated transcription factors, selected HPAECs were treated with inhibitors of hypoxia-inducible factor (HIF)-1α (chetomin) or nuclear factor (NF)-κB (caffeic acid phenethyl ester, CAPE). In parallel studies, male C57BL/6 mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 wk with or without gavage with RSG (10 mg·kg−1·day−1) for the final 10 days of exposure. Hypoxia increased ET-1, endothelin-converting enzyme-1, and endothelin receptor A and B levels in mouse lung and in HPAECs and increased HPAEC proliferation. Treatment with RSG attenuated hypoxia-induced activation of HIF-1α, NF-κB activation, and ET-1 signaling pathway components. Similarly, treatment with chetomin or CAPE prevented hypoxia-induced increases in HPAEC ET-1 mRNA and protein levels. These findings indicate that PPARγ activation attenuates a program of hypoxia-induced ET-1 signaling by inhibiting activation of hypoxia-responsive transcription factors. Targeting PPARγ represents a novel therapeutic strategy to inhibit enhanced ET-1 signaling in PH pathogenesis.

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Dissection of Mechanisms of Realgar Intervening Telomere Protein POT1, TRF1, TRF2 Expression to Regulate Telomere Dynamics in THP-1 Cells

Blood ◽

10.1182/blood.v128.22.5896.5896 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5896-5896

Author(s):

Rui-Rong Xu ◽

Nan-Xin Song ◽

Xiao-Long Wu ◽

Si-Yuan Cui ◽

Zhen-Zhen Wang ◽

...

Keyword(s):

Theoretical Basis ◽

Cell Cycle Distribution ◽

G1 Arrest ◽

Mrna Levels ◽

Protein Levels ◽

Qpcr Analysis ◽

Telomere Protein ◽

Telomere Dynamics

Abstract Objective: To observe the mechanisms of realgar intervening telomere protein POT1, TRF1, TRF2 expression to regulate telomere dynamics in THP-1 cells, and elucidate the experimental and theoretical basis for realgar targeted threapy of AML. Methods: 1.In vitro: (1)Cultured human acute myeloid leukemia THP-1 cells. (2), The cells were incubated in absence or presence of increasing concentration of realgar for 24/48 h, from which determined the IC50. Cell viability was tested by CCK-8 assay. (3)Apoptotic rate and cell cycle distribution were tested by FCM. (4)Each group of POT1, TRF1, TRF2 protein levels were tested by western-blot analysis. (5)Each group of POT1,TRF1,TRF2 mRNA levels were tested by RT-qPCR analysis. 2.In vivo: (1)Established THP-1 model in NOD/SCID mice, and the mouse were treated by realgar. (2)All the mouse were killed by institutionally approved method after 3 weeks. The apoptotic rate and cell cycle distribution of mice spleen cells were examined by FCM. (3)The changes of POT1, TRF1, TRF2 protein levels in organic tissue were detected by IHC. Results: 1.In vitro: (1)CCK-8 assay showed that after treated by realgar, THP-1 cells growth were inhibited and the cell viability decreased in a dose- and time- dependent manner, IC50 was 0.023μg/mL. Therefore, we choose a medium concentration was 0.015μg/mL (lower than IC50) at 48h. (2)Our study demonstrated that after treatment for 48h, the apoptotic rate and the G1 arrest of these THP-1 cell increased, compared with control group. (3)As shown by western blot analysis, compared with controls, realgar group POT1, TRF1 protein levels significantly increaesed and TRF2 decreased(P<0.01). (4) As shown by by RT-qPCR analysis, POT1, TRF1, TRF2 mRNA levels were consistent with their protein levels. 2.In vivo: (1) The THP-1/NOD-SCID model was established, and survival curve showed that compared with controls, realgar group mouse had longer life span(P<0.01). (2) The results of FCM showed that after treatment of realgar, the rate of THP-1 cell apoptosis and G1 arrest significantly increased(P<0.01). (3) The results of IHC showed that compared with controls, POT1, TRF1 protein levels significantly increased and TRF2 decreased in realgar group mouse(P<0.01). Conclusion: In vitro and in vivo studies results indicated that realgar could significantly prolong the life span of the THP-1/NOD-SCID mice, inhibit proliferation and induce apoptosis of THP-1 cell, and regulate telomere dynamics through intervening THP-1 cell telomere protein POT1, TRF1, TRF2 expression. Our studies results provided experimental and theoretical basis for realgar targeted threapy of AML. Disclosures No relevant conflicts of interest to declare.

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Epigenetic Regulator KDM4D Restricts Tumorigenesis via Modulating SYVN1/HMGB1 Ubiquitination Axis in Esophageal Squamous Cell Carcinoma

Frontiers in Oncology ◽

10.3389/fonc.2021.761346 ◽

2021 ◽

Vol 11 ◽

Author(s):

Wenjian Yao ◽

Jianjun Wang ◽

Li Zhu ◽

Xiangbo Jia ◽

Lei Xu ◽

...

Keyword(s):

Tumor Growth ◽

Bioinformatic Analysis ◽

Soft Agar ◽

Tumor Xenograft ◽

Mrna Levels ◽

Protein Levels ◽

Novel Biomarkers ◽

Underlying Mechanisms

BackgroundIncreasing researches have been reported that epigenetic alterations play critical roles in ESCC development. However, the role of the histone demethylase KDM4D in ESCC tumorigenesis is poorly investigated. This study aims to discover the underlying mechanisms between KDM4D and ESCC progression.MethodsCCK-8 assays, clone formation assay and soft-agar assays were performed to assess cell proliferation. Transwell assay was utilized to assess cell migration efficiency, while sphere formation assay was used to evaluate the cell self-renewal ability. Bioinformatic analysis was conducted to identify prognostic factors and predict the potential E3 ubiquitin ligases. In vitro ubiquitination assay was conducted to confirm the regulations between SYVN1 and HMGB1. The mRNA levels or protein levels of genes were detected by real-time PCR and western blot analysis. In vivo tumor xenograft models were used to determine whether the HMGB1 inhibition affected the malignant features of ESCC cells.ResultEpigenome screening and low-throughput validations highlighted that KDM4D is a tumor suppressor in ESCC. KDM4D expressed lowly in tumors that predicts poor prognosis. KDM4D deficiency significantly enhanced tumor growth, migration and stemness. Mechanistically, KDM4D transcriptionally activates SYVN1 expressions via H3K9me3 demethylation at the promoter region, thereby triggering the ubiquitin-dependent degradation of HMGB1. Low KDM4D depended on accumulated HMGB1 to drive ESCC progression and aggressiveness. Targeting HMGB1 (Glycyrrhizin) could remarkably suppress ESCC tumor growth in vitro and in vivo, especially in KDM4D-deficient cells.ConclusionsWe systematically identified KDM4D/SYVN1/HMGB1 axis in ESCC progression, proving novel biomarkers and potential therapeutic targets.

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Regulation of uridine diphosphate-glucuronosyltransferase 1A expression by miRNA-214-5p and miRNA-486-3p

Epigenomics ◽

10.2217/epi-2020-0244 ◽

2021 ◽

Author(s):

Stefan Paulusch ◽

Sandra Kalthoff ◽

Steffen Landerer ◽

Christian Jansen ◽

Robert Schierwagen ◽

...

Keyword(s):

Liver Fibrosis ◽

Potential Risk ◽

Mrna Levels ◽

Uridine Diphosphate ◽

Protein Levels ◽

Huh7 Cells ◽

Potential Risk Factors ◽

Cirrhotic Patients

Aim: This study aimed to identify novel miRNAs (miRs) as regulators of UGT1A gene expression and to evaluate them as potential risk factors for the development of liver fibrosis/cirrhosis. Materials & methods: miRNA target sites in UDP-glucuronosyltransferase 1A (UGT1A) 3′-UTR were predicted and confirmed by luciferase assays, quantitative real-time PCR and western blot using HEK293, HepG2 and Huh7 cells. UGT1A and miRNA expression were analyzed in cirrhotic patients and a mouse model of alcoholic liver fibrosis. Results: miR-214-5p and miR-486-3p overexpression reduced UGT1A mRNA, protein levels and enzyme activity in HepG2 and Huh7 cells. miR-486-3p was upregulated in cirrhotic patients and fibrotic mice livers, whereas UGT1A mRNA levels were reduced. Conclusion: In conclusion, we identified two novel miRNAs capable to repress UGT1A expression in vitro and in vivo. Furthermore, miR-486-3p may represent a potential risk factor for the development or progression of liver fibrosis/cirrhosis by means of a reduced UGT1A-mediated detoxification activity.

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