Transcription Factor 21 Research Articles

Tumor-intrinsic PRMT5 upregulates FGL1 via methylating TCF12 to inhibit CD8+ T-cell-mediated antitumor immunity in liver cancer

Protein arginine methyltransferase 5 (PRMT5) acts as an oncogene in liver cancer, yet its roles and in-depth molecular mechanisms within the liver cancer immune microenvironment remain mostly undefined. Here, we demonstrated that disruption of tumor-intrinsic PRMT5 enhances CD8+ T-cell-mediated antitumor immunity both in vivo and in vitro. Further experiments verified that this effect is achieved through downregulation of the inhibitory immune checkpoint molecule, fibrinogen-like protein 1 (FGL1). Mechanistically, PRMT5 catalyzed symmetric dimethylation of transcription factor 12 (TCF12) at arginine 554 (R554), prompting the binding of TCF12 to FGL1 promoter region, which transcriptionally activated FGL1 in tumor cells. Methylation deficiency at TCF12-R554 residue downregulated FGL1 expression, which promoted CD8+ T-cell-mediated antitumor immunity. Notably, combining the PRMT5 methyltransferase inhibitor GSK591 with PD-L1 blockade efficiently inhibited liver cancer growth and improved overall survival in mice. Collectively, our findings reveal the immunosuppressive role and mechanism of PRMT5 in liver cancer and highlight that targeting PRMT5 could boost checkpoint immunotherapy efficacy.

Research Note: Metabolomics revealed the causes of the formation of chicken structural blue earlobes

The earlobes of chickens exhibit a range of colors, but there has been relatively little research on the formation of structural blue earlobes. Previous results showed that the structural color earlobes were related to the interplay between melanin and collagen in light reflection. To investigate the metabolic differences in these earlobe colors, we conducted nontargeted liquid chromatograph mass spectrometer (LC-MS) for metabolomic sequencing on structural blue (Green and Blue groups) and nonstructural color (Black group) earlobes tissue of Jiangshan black-bone chickens. The content detection in earlobe tissues of different groups shows that there were significant differences in melanin and collagen content between the Black and Green group. The metabolome identified a total of 6,102 mass spectroscopic peaks and ultimately identified 919 annotated metabolites. Variable importance in the projection (VIP) analysis identified the common differential expressed metabolites (DMs) "Tyr Thr Ala Glu" among the 3 groups. By combining those DMs with differentially expressed genes (DEGs) in our previous transcriptome data from the same sample, and associated with KEGG pathway analysis, multiple pathways related to melanogenesis and collagen metabolism were enriched across the 3 groups. By analyzing the metabolites and genes in these pathways, as well as the interaction network diagram of DEGs, we identified some key genes, Wnt Family Member 6 (WNT6), Transcription Factor 7 (TCF7), Proopiomelanocortin (POMC) and Calcium/Calmodulin Dependent Protein Kinase II Alpha (CAMK2A), and some key DMs like DG (11M3/9M5/0:0) and gentisic acid. The differential gene expression and metabolic levels affect the production of melanin and collagen, leading to differences in the content in melanin and the thickness of the collagen layer between earlobe colors, while the thickness of the collagen layer could affect light scattering, ultimately resulting in different colored earlobes in Jiangshan black-bone chickens.

Inhibition of the TCF12/VSIG4 axis by palbociclib diminishes the proliferation and migration of glioma cells and decreases the M2 polarization of glioma-associated microglia.

The CDK4/CDK6 inhibitor palbociclib has shown the encouraging promise in the treatment of glioma. Here, we elucidated how palbociclib exerts suppressive functions in the M2 polarization of glioma-related microglia and the progression of glioma. Xenograft experiments were used to evaluate the function in vivo. The mRNA levels of transcription factor 12 (TCF12) and VSIG4 were detected by RT-qPCR, and their protein levels were assessed by immunoblotting. Cell migration was tested by wound-healing assay. Cell cycle distribution and M1/M2 microglia phenotype analysis were performed by flow cytometry. The levels of IFN-γ, TNF-α, IL-6，and TGF-β were measured by ELISA. The TCF12/VSIG4 association was verified by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. In U251 and LN229 glioma cells, TCF12 and VSIG4 were overexpressed, and palbociclib reduced their expression levels. TCF12 upregulation enhanced the proliferation and migration of glioma cells and the M2 polarization of glioma-associated microglia in vitro as well as the tumorigenicity of U251 glioma cells in vivo, which could be reversed by palbociclib. Mechanistically, TCF12 could enhance VSIG4 transcription and expression by binding to the VSIG4 promoter. TCF12 deficiency led to repression in glioma cell proliferation and migration as well as microglia M2 polarization, which could be abolished by increased VSIG4 expression. Our study reveals the novel TCF12/VSIG4 axis responsible for the efficacy of palbociclib in combating glioma, offering a rationale for the application of palbociclib in glioma treatment.

AB002. DNA methylation-regulated genes contribute to temozolomide (TMZ) resistance by scaffolding paraspeckle proteins.

Temozolomide (TMZ) resistance in glioblastoma (GBM) remains a challenge in clinical treatment and the mechanism is largely unknown. Emerging evidence shows that epigenetic modifications including DNA methylation and non-coding RNA were involved in diverse biological processes, including therapeutic resistance. However, the underlying mechanisms by which DNA methylation-mediated non-coding RNA regulates TMZ resistance remain poorly characterized. RNA microarray and DNA methylation chips of TMZ-resistant and parental GBM cells were performed for the gain of unreported long non-coding RNA HSD52. Quantitative reverse transcription polymerase chain reaction (PCR) and fluorescence in situ hybridization assays were used to detect HSD52 levels in GBM cells and tissues. The investigation into HSD52's impact on TMZ resistance was conducted utilizing both in vitro assays and intracranial xenograft mouse models. The mechanism of HSD52 expression and its relationships with paraspeckle proteins, non-POU domain-containing octamer-binding protein (NONO) and splicing factor proline/glutamine rich (SFPQ), as well as alpha-thalassemia mental retardation X-linked (ATRX) mRNA were determined by pyrosequencing assay, chromatin immunoprecipitation, chromatin isolation by RNA purification, RNA immunoprecipitation, RNA pulldown, immunofluorescence, and western blot assays. HSD52 was highly expressed in high-grade glioma and TMZ-resistant GBM cells. Phosphorylated p38 mitogen-activated protein kinase (p38 MAPK)/ubiquitin specific peptidase 7 (USP7) axis mediates H3 ubiquitination, impairs the interaction between H3K23ub and DNA methyltransferase 1 (DNMT1) and the recruitment of DNMT1 at the HSD52 promoter to attenuate DNA methylation, which makes the transcription factor 12 (TCF12) more accessible to the promoter region to regulate HSD52 expression. Further analysis showed that HSD52 can serve as a scaffold to promote the interaction between NONO and SFPQ, and then increase the paraspeckle assembly and activate the paraspeckle/ataxia telangiectasia mutated (ATM) kinase pathway in GBM cells. In addition, HSD52 forms an RNA-RNA duplex with ATRX mRNA, and facilitates the association of heteromer of SFPQ and NONO with RNA duplex, thus leading to the increase of ATRX mRNA stability and level. In clinical patients, HSD52 is required for TMZ resistance and GBM recurrence. Our results reveal that HSD52 in GBM could serve as a therapeutic target to overcome TMZ resistance, enhancing the clinical benefits of TMZ chemotherapy.

Astragaloside IV prevents enterovirus 71-induced pyroptosis through the TCF12-TXNIP-Keap1/Nrf2 axis

This study aimed to investigate the inhibitory effect of astragaloside IV (AS-IV) on enterovirus 71 (EV71)-induced pyroptosis. The results indicated that AST-IV inhibited EV71-induced pyroptosis in normal human gastric epithelial (GES-1) cells. Therefore, we investigated the mechanisms underlying pyroptosis inhibition by AST-IV. We found that EV71 inhibited the Nrf2-Keap1 pathway, whereas AST-IV treatment activated it. Overexpression of Nrf2 led to the inhibition of EV71-induced pyroptosis. EV71 infection and AST-IV treatment upregulated and downregulated thioredoxin interacting protein (TXNIP), respectively. Downregulation of TXNIP activated the Keap1/Nrf2 signaling pathway and inhibited EV71-induced pyroptosis. Furthermore, transcription factor 12 (TCF12) reduced the protein and mRNA levels of TXNIP, and AST-IV treatment upregulated TCF12. These results demonstrate that the TCF12-TXNIP-Keap1/Nrf2 axis participates in the inhibition of pyroptosis by AST-IV in EV71-infected GES-1 cells, suggesting that AST-IV could be used as a dietary supplement to alleviate EV71-induced pyroptosis.

Polymorphism rs2327430 in TCF21 predicts the risk and prognosis of gastric cancer by affecting the binding between TFAP2A and TCF21

BackgroundSingle nuclear polymorphisms (SNPs) have been published to be correlated with multiple diseases. Transcription Factor 21 (TCF21) is a critical transcription factor involved in various types of cancers. However, the association of TCF21 genetic polymorphisms with gastric cancer (GC) susceptibility and prognosis remains unclear.MethodsA case-control study comprising 890 patients diagnosed with GC and an equal number of cancer-free controls was conducted. After rigorous statistical analysis, molecular experiments were carried out to elucidate the functional significance of the SNPs in the context of GC.ResultsTCF21 rs2327430 (OR = 0.78, P = 0.026) provides protection against GC, while rs4896011 (OR = 1.39, P = 0.005) exhibit significant associations with GC risk. Furthermore, patients with the (TC + CC) genotype of rs2327430 demonstrate a relatively favorable prognosis (OR = 0.47, P = 0.012). Mechanistically, chromatin immunoprecipitation assay and luciferase reporter assay revealed that the C allele of rs2327430 disrupts the binding of Transcription Factor AP-2 Alpha (TFAP2A) to the promoter region of TCF21, resulting in increased expression of TCF21 and inhibition of malignant behaviors in GC cells.ConclusionOur findings highlight the significant role of TCF21 SNPs in both the risk and prognosis of GC and provide valuable insights into the underlying molecular mechanisms. Specifically, the disruptive effect of rs2327430 on TCF21 expression and its ability to modulate malignant cell behaviors suggest that rs2327430 may serve as a potential predictive marker for GC risk and prognosis.

TCF12 Transcriptionally Activates SPHK1 to Induce Osteosarcoma Angiogenesis by Promoting the S1P/S1PR4/STAT3 Axis.

Transcription factor 12 (TCF12) is a known oncogene in many cancers. However, whether TCF12 can regulate malignant phenotypes and angiogenesis in osteosarcoma is not elucidated. In this study, we demonstrated increased expression of TCF12 in osteosarcoma tissues and cell lines. High TCF12 expression was associated with metastasis and poor survival rate of osteosarcoma patients. Knockdown of TCF12 reduced the proliferation, migration, and invasion of osteosarcoma cells. TCF12 was found to bind to the promoter region of sphingosine kinase 1 (SPHK1) to induce transcriptional activation of SPHK1 expression and enhance the secretion of sphingosine-1-phosphate (S1P), which eventually resulted in the malignant phenotypes of osteosarcoma cells. In addition, S1P secreted by osteosarcoma cells promoted the angiogenesis of HUVECs by targeting S1PR4 on the cell membrane to activate the STAT3 signaling pathway. These findings suggest that TCF12 may induce transcriptional activation of SPHK1 to promote the synthesis and secretion of S1P. This process likely enhances the malignant phenotypes of osteosarcoma cells and induces angiogenesis via the S1PR4/STAT3 signaling pathway.

Transcription factor 12-mediated self-feedback regulatory mechanism is required in DUX4 fusion leukaemia.

IGH::DUX4 is frequently observed in 4% B-cell acute lymphoblastic leukaemia patients. Regarding the IGH::DUX4-driven transactivation and alternative splicing, which are the main reasons behind this acute leukaemia outbreak, it remains unclear how transcriptional cofactors contribute to this oncogenic process. Further investigation is required to elucidate their specific role in leukaemogenesis. In order to investigate the cofactors of IGH::DUX4, integrated mining of Chromatin immunoprecipitation (ChIP)-sequencing and RNA-sequencing of leukaemia cells and patient samples were conducted. Furthermore, to elucidate the synergistic interaction between transcription factor 12 (TCF12) and IGH::DUX4, knockdown and knockout experiment, mammalian two-hybridisation assay, co-immunoprecipitation and in situ proximity ligation assays were carried out. Additionally, to further investigate the direct interaction between TCF12 and IGH::DUX4, AI-based structural simulations were utilised. Finally, to validate the synergistic role of TCF12 in promoting IGH::DUX4 leukaemia, cell proliferation, apoptosis and drug sensitivity experiments were performed. In this study, we observed that the IGH::DUX4 target gene TCF12 might be an important cofactor/helper for this oncogenic driver. The co-expression of IGH::DUX4 and TCF12 resulted in enhanced DUX4-driven transactivation. Supportively, knockdown and knockout of TCF12 significantly reduced expression of IGH::DUX4-driven target genes in leukaemia REH (a precursor B-cell leukaemia cell line) and NALM-6 cells (a precursor B-cell leukaemia cell line). Consistently, in TCF12 knockout cells, the expression of structure-based TCF12 mutant, but not wild-type TCF12, failed to restore the TCF12-IGH::DUX4 crosstalk and the synergistic transactivation. More importantly, the breakdown in TCF12-IGH::DUX4 cooperation impaired IGH::DUX4-driven leukaemia cell survival, caused sensitivity to the chemotherapy. Altogether, these results helped to define a previously unrecognised TCF12-mediated positive self-feedback regulatory mechanism in IGH::DUX4 leukaemia, which holds the potential to function as a pivotal drug target for the management of this particular form of leukaemia. Transcription factor 12 (TCF12) is a new novel cofactor in IGH::DUX4 transcriptional complexes/machinery. TCF12 mediates a positive self-feedback regulatory mechanism in IGH::DUX4-driven oncogenic transaction. IGH::DUX4-TCF12 structure/cooperation might represent a potent target/direction in future drug design against B-cell acute lymphoblastic leukaemia.

Knockout of M-LP/Mpv17L, a newly identified atypical PDE, induces physiological afferent cardiac hypertrophy in mice

M-LP/Mpv17L (Mpv17-like protein) is an atypical cyclic nucleotide phosphodiesterase (PDE) without the molecular structure characteristic of the PDE family. Deficiency of M-LP/Mpv17L in mice has been found to result in development of β-cell hyperplasia and improved glucose tolerance. Here, we report another phenotype observed in M-LP/Mpv17L-knockout (KO) mice: afferent cardiac hypertrophy. Although the hearts of M-LP/Mpv17L-KO mice did not differ in size from those of wild-type mice, there was marked narrowing of the left ventricular lumen and thickening of the ventricular wall. The diameter and cross-sectional area of cardiomyocytes in 8-month-old M-LP/Mpv17L-KO mice were increased 1.16-fold and 1.35-fold, respectively, relative to control mice, but showed no obvious abnormalities of cell structure, fibrosis or impaired cardiac function. In 80-day-old KO mice, the expression of hypertrophic marker genes, brain natriuretic peptide (BNF), actin alpha cardiac muscle 1 (ACTC1) and actin alpha 1 skeletal muscle (ACTA1), as well as the Wnt/β-catenin pathway target genes, lymphoid enhancer-binding factor-1 (LEF1), axis inhibition protein 2 (AXIN2) and transcription factor 7 (TCF7), was significantly up-regulated relative to control mice, whereas fibrosis-related genes such as fibronectin 1 (FN1) and connective tissue growth factor (CTGF) were down-regulated. Western blot analysis revealed increased phosphorylation of molecules downstream of the cAMP/PKA signaling pathway, such as β-catenin, ryanodine receptor 2 (RyR2), phospholamban (PLN) and troponin I (cTnI), as well as members of the MEK1-ERK1/2 signaling pathway, which is strongly involved in afferent cardiac hypertrophy. Taken together, these findings indicate that M-LP/Mpv17L is one of the PDEs actively functioning in the heart and that deficiency of M-LP/Mpv17L in mice promotes physiological cardiac hypertrophy.

CircUSP10 promotes liver cancer progression by regulating miR-211-5p/TCF12/EMT signaling pathway

There is no precise diagnosis or prognosis for liver cancer (LC) using a single biomarker. Circular RNAs (circRNAs) contribute to the pathogenesis of different cancers, but their role in LC is not entirely understood. In this study, circUSP10, an aberrantly expressed circRNA in LC, was screened using the Gene Expression Omnibus database, and its tissue-specific expression was verified using qRT-PCR. In vitro, functional assays and nude mouse tumorigenesis models were used to investigate circUSP10 role in LC. RNA immunoprecipitation and dual-luciferase reporter assays were performed to study the mechanistic relationship between circUSP10, miR-211-5p, and transcription factor 12 (TCF12). We found that circUSP10 expression was upregulated in LC tissues and cells. CircUSP10 expression was linked to tumor size and tumor node metastasis stage and negatively correlated with LC prognosis. In vitro assays confirmed circUSP10-mediated proliferation, migration, and invasion of LC cells and their association with the epithelial-mesenchymal transition (EMT) pathway. Mechanistically, circUSP10 adsorbed miR-211-5p, which regulated TCF12 and promoted tumorigenesis via the EMT signaling pathway. Therefore, our results suggest that circUSP10 may promote LC progression by modulating the miR-211-5p/TCF12/EMT signaling cascade and may serve as a potential biomarker for LC diagnosis and prognosis.

Mechanism of TCF21 Downregulation Leading to Immunosuppression of Tumor-Associated Macrophages in Non-Small Cell Lung Cancer.

Lung cancer, as one of the high-mortality cancers, seriously affects the normal life of people. Non-small cell lung cancer (NSCLC) accounts for a high proportion of the overall incidence of lung cancer, and identifying therapeutic targets of NSCLC is of vital significance. This study attempted to elucidate the regulatory mechanism of transcription factor 21 (TCF21) on the immunosuppressive effect of tumor-associated macrophages (TAM) in NSCLC. The experimental results revealed that the expression of TCF21 was decreased in lung cancer cells and TAM. Macrophage polarization affected T cell viability and tumor-killing greatly, and M2-type polarization reduced the viability and tumor-killing of CD8+T cells. Meanwhile, overexpression of TCF21 promoted the polarization of TAM to M1 macrophages and the enhancement of macrophages to the viability of T cells. Furthermore, there appears to be a targeting relationship between TCF21 and Notch, suggesting that TCF21 exerts its influence via the Notch signaling pathway. This study demonstrated the polarization regulation of TAM to regulate the immunosuppressive effect, which provides novel targets for the treatment of lung cancer.

FOXO1 promotes the expression of canonical WNT target genes in examined basal-like breast and glioblastoma multiforme cancer cells.

Basal-like breast cancer (BBC) and glioblastoma multiforme (GBM) are aggressive cancers associated with poor prognosis. BBC and GBM have stem cell-like gene expression signatures, which are in part driven by forkhead box O (FOXO) transcription factors. To gain further insight into the impact of FOXO1 in BBC, we treated BT549 cells with AS1842856 and performed RNA sequencing. AS1842856 binds to unphosphorylated FOXO1 and inhibits its ability to directly bind to DNA. Gene Set Enrichment Analysis indicated that a set of WNT pathway target genes, including lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 7 (TCF7), were robustly induced after AS1842856 treatment. These same genes were also induced in GBM cell lines U87MG, LN18, LN229, A172, and DBTRG upon AS1842856 treatment. By contrast, follow-up RNA interference (RNAi) targeting of FOXO1 led to reduced LEF1 and TCF7 gene expression in BT549 and U87MG cells. In agreement with RNAi experiments, CRISPR Cas9-mediated FOXO1 disruption reduced the expression of canonical WNT genes LEF1 and TCF7 in U87MG cells. The loss of TCF7 gene expression in FOXO1 disruption mutants was restored by exogenous expression of the DNA-binding-deficient FOXO1-H215R. Therefore, FOXO1 induces TCF7 in a DNA-binding-independent manner, similar to other published FOXO1-activated genes such as TCF4 and hes family bHLH transcription factor 1. Our work demonstrates that FOXO1 promotes canonical WNT gene expression in examined BBC and GBM cells, similar to results found in Drosophila melanogaster, T-cell development, and murine acute myeloid leukemia models.

DNMT3a-mediated methylation of TCF21/hnRNPA1 aggravates hepatic fibrosis by regulating the NF-κB signaling pathway

Hepatic fibrosis is caused by liver damage as a consequence of wound healing response. Recent studies have shown that hepatic fibrosis could be effectively reversed, partly through regression of activated hepatic stellate cells (HSCs). Transcription factor 21 (TCF21), a member of the basic helix-loop-helix (bHLH) transcription factor, is involved in epithelial-mesenchymal transformation in various diseases. However, the mechanism by which TCF21 regulates epithelial-mesenchymal transformation in hepatic fibrosis has not been elucidated. In this research, we found that hnRNPA1, the downstream binding protein of TCF21, accelerates hepatic fibrosis reversal by inhibiting the NF-κB signaling pathway. Furthermore, the combination of DNMT3a with TCF21 promoter results in TCF21 hypermethylation. Our results suggest that DNMT3a regulation of TCF21 is a significant event in reversing hepatic fibrosis. In conclusion, this research identifies a novel signaling axis, DNMT3a-TCF21-hnRNPA1, that regulates HSCs activation and hepatic fibrosis reversal, providing a novel treatment strategy for hepatic fibrosis. The clinical trial was registered in the Research Registry (researchregistry9079).

Effect of thermal manipulation during embryogenesis on gene expression of myogenic upstream activation factors pre- and post-hatch in broilers

This study aimed to determine the optimum timing of embryonic thermal manipulation (TM) that may result in improvement of mRNA expression of myogenic upstream activation factors viz. Wnt family member-3 (Wnt-3), sonic hedgehog gene (Shh), proliferating cell nuclear antigen (PCNA) and paired-box transcription factor-7 (Pax-7) during development and histogenesis of broiler muscle. Fertile eggs (1440) were divided randomly and equally into 5 treatment groups including control (no TM) and four treatment groups (TM1, TM2 TM3 and TM4) that were daily subjected to 39 ̊C for 18 h with 65% relative humidity during embryonic days ED 7–11, ED 11–15, ED 15–18 and ED 7–18 respectively. Pectoral and thigh muscle mRNA expressions of myogenic upstream activation factors were evaluated by semi-quantitative real-time RT-PCR. Out of TM conditions that were investigated, TM1 resulted in a significant improvement of Wnt-3, Shh, PCNA and Pax-7 expressions in broiler pectoral and thigh muscles during embryonic and post-hatch life when compared to the control. Thus, thermal manipulation during early embryongenesis (embryonic days ED 7-11) enhance broiler skeletal muscle myoblast proliferation by triggering and inducing transcription factors that regulates myogenesis and subsequently may lead to improve cell number and size of skeletal muscle. The outcome of this study indicates that TM during ED7-11 improved muscle response to heat stress, was safe to the pectoral and thigh muscles and this method may enhance myogenesis and muscle growth in a positive manner.

Transcription factor 21 accelerates vascular calcification in mice by activating the IL-6/STAT3 signaling pathway and the interplay between VSMCs and ECs.

Vascular calcification is caused by the deposition of calcium salts in the intimal or tunica media layer of the aorta, which increases the risk of cardiovascular events and all-cause mortality. However, the mechanisms underlying vascular calcification are not fully clarified. Recently it has been shown that transcription factor 21 (TCF21) is highly expressed in human and mouse atherosclerotic plaques. In this study we investigated the role of TCF21 in vascular calcification and the underlying mechanisms. In carotid artery atherosclerotic plaques collected from 6 patients, we found that TCF21 expression was upregulatedin calcific areas. We further demonstrated TCF21 expression wasincreased in an in vitro vascular smooth muscle cell (VSMC) osteogenesis model. TCF21 overexpression promoted osteogenic differentiation of VSMC, whereas TCF21 knockdown in VSMC attenuated the calcification. Similar results were observed in ex vivo mouse thoracic aorta rings. Previous reports showed that TCF21 bound to myocardin (MYOCD) to inhibit the transcriptional activity of serum response factor (SRF)-MYOCD complex. We found that SRF overexpression significantly attenuated TCF21-induced VSMC and aortic ring calcification. Overexpression of SRF, but not MYOCD, reversed TCF21-inhibited expression of contractile genes SMA and SM22. More importantly, under high inorganic phosphate (3 mM) condition, SRF overexpression reduced TCF21-induced expression of calcification-related genes (BMP2 and RUNX2) as well as vascular calcification. Moreover, TCF21 overexpression enhanced IL-6 expression and downstream STAT3 activation to facilitate vascular calcification. Both LPS and STAT3 could induce TCF21 expression, suggesting that the inflammation and TCF21 might form a positive feedback loop to amplify the activation of IL-6/STAT3 signaling pathway. On the other hand, TCF21 induced production of inflammatory cytokines IL-1β and IL-6 in endothelial cells (ECs) to promote VSMC osteogenesis. In EC-specific TCF21 knockout (TCF21ECKO) mice, VD3 and nicotine-induced vascular calcification was significantly reduced. Our results suggest that TCF21 aggravates vascular calcification by activating IL-6/STAT3 signaling and interplay between VSMC and EC, which provides new insights into the pathogenesis of vascular calcification. TCF21 enhances vascular calcification by activating the IL-6-STAT3 signaling pathway. TCF21 inhibition may be a new potential therapeutic strategy for the prevention and treatment of vascular calcification.

Inhibition of ox-LDL-induced endothelial cell injury by LINC02381 knockdown through the microRNA-491-5p/transcription factor 7 axis.

Atherosclerosis (AS) is a complex multifactorial and chronic inflammatory vascular disease that contributes to the development of cardiovascular diseases. Abnormal cellular proliferation in human umbilical vein endothelial cells (HUVECs) is a crucial element in AS development. In this study, we investigated the potential role of the long noncoding RNA LINC02381/microRNA (miR)-491-5p/transcription factor 7 (TCF7) axis in regulating HUVEC injury in 30 participants suffering from AS and 30 healthy control participants. We established an in vitro model of AS in HUVECs using oxidized low-density lipoprotein (ox-LDL), and measured cellular mRNA and protein levels of LINC02381, miR-491-5p, and TCF7 in serum samples using reverse transcription-quantitative polymerase chain reaction and Western blotting assays. We evaluated cell viability, apoptosis, and inflammation using Cell Counting Kit-8, flow cytometry, and enzyme-linked immunosorbent assays, respectively. Moreover, we analyzed apoptosis-related protein expression using western blotting analysis and determined the association between miR-491-5p and LINC02381 or TCF7 using dual-luciferase reporter assay, RNA pull-down, and rescue experiments. We observed that LINC02381 was elevated, while miR-491-5p was downregulated in serum samples from participants with AS and in ox-LDL-treated HUVECs. LINC02381 knockdown was protective against HUVEC injury via miR-491-5p inhibition, which is its downstream target. Rescue experiments further demonstrated that miR-491-5p alleviated HUVEC injury by modulating TCF7. Thus, LINC02381 knockdown ameliorated HUVEC injury by regulating the miR-491-5p/TCF7 axis, which provides new insights into AS treatment strategies.

MicroRNA-92a-3p Inhibits Cell Proliferation and Invasion by Regulating the Transcription Factor 21/Steroidogenic Factor 1 Axis in Endometriosis

Endometriosis (EMS) is an estrogen-dependent disease. However, little is known about the regulation of estrogen, a potential therapeutic target, in EMS, which remains very poorly managed in the clinic. We hypothesized that microRNAs (miRNAs) can be exploited therapeutically to regulate transcription factor 21 (TCF21) and steroidogenic factor-1 (SF-1) gene expression. In our study, paired eutopic and ectopic endometrial samples were obtained from women with EMS and processed by a standard protocol to obtain human endometrial stromal cells (EMs) for in vitro studies. We found that miR-92a-3p levels were decreased in ectopic endometrium and ectopic stromal cells (ESCs) compared with paired eutopic lesions. miR-92a-3p overexpression significantly suppressed the proliferation and migration of ESCs, whereas a decreased level of miR-92a-3p generated the opposite results. Next, we identified TCF21 as a candidate target gene of miR-92a-3p. In vitro cell experiments showed that miR-92a-3p negatively regulated the expression of TCF21 and its downstream target gene SF-1. Moreover, cell proliferation and invasion ability decreased after the silencing of SF-1 and increased after SF-1 overexpression. We also observed that silencing SF-1 while inhibiting miR-92a-3p partially blocked the increase in cell proliferation and invasion ability caused by miR-92a-3p knockdown while overexpressing both SF-1 and miR-92a-3p mitigated the impairment in cell proliferation and invasion ability caused by miR-92a-3p overexpression. Our results may provide a novel potential therapeutic target for the treatment of EMS.

Circular RNA hsa_circ_0002938 (circCRIM1) promotes the progression of esophageal squamous cell carcinoma by upregulating transcription factor 12.

Growing evidence has indicated that circular RNAs (circRNAs) play crucial roles in the tumorigenesis and progression of diverse malignancies. However, the majority of circRNAs involved in esophageal squamous cell carcinoma (ESCC) remain undefined and the exact functions and underlying mechanisms of circRNAs in ESCC still need further exploration. In this study, we identified a novel onco-circRNA hsa_circ_0002938, derived from the exons of cysteine-rich transmembrane BMP regulator 1 (CRIM1) pre-mRNA, referred to as circCRIM1. We found that the expression of circCRIM1 was higher in ESCC tissues, compared to para-carcinoma tissues. Increased expression of circCRIM1 was positively correlated with clinical parameters of ESCC patients including tumor-node-metastasis (TNM) stage, tumor invasion range, and lymph node metastasis. Functionally, the results from the experiments in vitro showed that the knockdown of circCRIM1 suppressed proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in ESCC cells. By conducting bioinformatics algorithms analyses and microRNA (miRNA) rescue experiments, we found that circCRIM1 could act as a competing endogenous RNA (ceRNA) to sponge miR-342-3p in ESCC cells, and thereby upregulated the expression of transcription factor 12 (TCF12), a key regulator promoting the EMT process. Taken together, circCRIM1 facilitates the progression of ESCC by sponging miR-342-3p to regulate TCF12 and promote EMT, and the circCRIM1/miR-342-3p/TCF12 axis may be regarded as a potential predictive biomarker and therapeutic target for treating ESCC.

RNA-binding protein DHX9 promotes glioma growth and tumor-associated macrophages infiltration via TCF12.

Glioma is the most common malignant tumor of the central nervous system, with high heterogeneity, strong invasiveness, high therapeutic resistance, and poor prognosis, comprehending a serious challenge in neuro-oncology. Until now, the mechanisms underlying glioma progression have not been fully elucidated. The expression of DExH-box helicase 9 (DHX9) in tissues and cells was detected by qRT-PCR and western blot. EdU and transwell assays were conducted to assess the effect of DHX9 on proliferation, migration and invasion of glioma cells. Cocultured model was used to evaluate the role of DHX9 on macrophages recruitment and polarization. Animal study was performed to explore the role of DHX9 on macrophages recruitment and polarization in vivo. Bioinformatics analysis, dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP)-qPCR assay was used to explore the relation between DHX9 and TCF12/CSF1. DHX9 was elevated in gliomas, especially in glioblastoma multiforme (GBM). Besides promoting the proliferation, migration, and invasion of glioma cells, DHX9 facilitated the infiltration of macrophages into glioma tissues and polarization to M2-like macrophages, known as tumor-associated macrophages (TAMs). DHX9 silencing decreased the expression of colony-stimulating factor 1 (CSF1), which partially restored the inhibitory effect on malignant progress of glioma and infiltration of TAMs caused by DHX9 knockdown by targeting the transcription factor 12 (TCF12). Moreover, TCF12 could directly bind to the promoter region of CSF1. DHX9/TCF12/CSF1 axis regulated the increases in the infiltration of TAMs to promote glioma progression and might be a novel potential target for future immune therapies against gliomas.

TCF7/SNAI2/miR-4306 feedback loop promotes hypertrophy of ligamentum flavum

BackgroundHypertrophy of ligamentum flavum (HLF) is the mainly cause of lumbar spinal stenosis (LSS), but the precise mechanism of HLF formation has not been fully elucidated. Emerging evidence indicates that transcription factor 7 (TCF7) is the key downstream functional molecule of Wnt/β-catenin signaling, which participated in regulating multiple biological processes. However, the role and underlying mechanism of TCF7 in HLF is still unclear.MethodsWe used mRNAs sequencing analysis of human LF and subsequent confirmation with RT-qPCR, western blot and immunohistochemistry to identified the TCF7 in HLF tissues and cells. Then effect of TCF7 on HLF progression was investigated both in vitro and in vivo. Mechanically, chromatin immunoprecipitation, dual-luciferase reporter assays, and rescue experiments were used to validate the regulation of TCF7/SNAI2/miR-4306 feedback loop.ResultsOur results identified for first time that the TCF7 expression was obviously elevated in HLF tissues and cells compared with control, and also found that TCF7 expression had significant positive correlation with LF thickness and fibrosis score. Notably, TCF7 inhibition suppressed the hyper-proliferation and fibrosis phenotype of HLF cells in vitro and ameliorated progression of HLF in mice in vivo, whereas TCF7 overexpression promoted hyper-proliferation and fibrosis phenotype of HLF cells in vitro. Our data further revealed that TCF7 interacted with SNAI2 promoter to transactivated the SNAI2 expression, thereby promoting hyper-proliferation and fibrosis phenotype of HLF cells in vitro. Furthermore, miR-4036 negatively regulated by SNAI2 could negatively feedback regulate TCF7 expression by directly binding to TCF7 mRNA 3’-UTR, thus inhibiting the hyper-proliferation and fibrosis phenotype of HLF cells in vitro.ConclusionsOur study demonstrated that TCF7 inhibition could suppress HLF formation by modulating TCF7/SNAI2/miR-4306 feedback loop, which might be considered as a novel potential therapeutic target for HLF.