Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Abnormal activation of the Wnt3a/ß-catenin signaling pathway promotes the expression of T-box transcription factor 3(TBX3) and the epithelial-mesenchymal transition pathway to mediate the occurrence of adenomyosis.

BACKGROUND: T-box transcription factor 3(TBX3) is a transcription factor that can regulate cell proliferation, apoptosis, invasion, and migration in different tumor cells; however, its role in adenomyosis (ADM) has not been previously studied. Some of ADM's pathophysiological characteristics are similar to those of malignant tumors (e.g., abnormal proliferation, migration, and invasion). METHODS AND RESULTS: We hypothesized that TBX3 might have a role in ADM. We used tamoxifen-induced Institute of Cancer research (ICR) mice to establish ADM disease model. The study procedure included western blotting and immunohistochemistry to analyze protein levels; additionally, we used intraperitoneal injection of Wnt/ß-catenin pathway inhibitor XAV-939 to study the relationship between TBX3 and Wnt/ß-catenin pathway as well as Anti-proliferation cell nuclear antigen( PCNA) and TUNEL to detect cell proliferation and apoptosis, respectively. TBX3 overexpression and epithelial-to-mesenchymal transition (EMT) in ADM mice was found to be associated with activation of the Wnt3a/ß-catenin pathway. Treatment with XAV-939 in ADM mice led to the inhibition of both TBX3 and EMT; moreover, abnormal cell proliferation was suppressed, the depth of invasion of endometrium cells was limited. Thus, the use of XAV-939 effectively inhibited further invasion of endometrial cells. CONCLUSION: These findings suggest that TBX3 may play an important role in the development of ADM. The expression of TBX3 in ADM was regulated by the Wnt3a/ß-catenin pathway. The activation of the Wnt3a/ß-catenin pathway in ADM promoted TBX3 expression and induced the occurrence of EMT, thus promoting cell proliferation and inhibiting apoptosis, ultimately accelerating the development of ADM. The study provides a reference for the diagnosis of ADM.

Inhibition of inflammation and infiltration of M2 macrophages in NSCLC through the ATF3/CSF1 axis: Role of miR-27a-3p.

Non-small cell lung cancer (NSCLC) imposes a significant economic burden on patients and society due to its low overall cure and survival rates. Tumour-associated macrophages (TAM) affect tumour development and may be a novel therapeutic target for cancer. We collected NSCLC and tumour-adjacent tissue samples. Compared with the tumour-adjacent tissues, the Activation Transcription Factor 3 (ATF3) and Colony Stimulating Factor 1 (CSF-1) were increased in NSCLC tissues. Levels of ATF3 and CSF-1 were identified in different cell lines (HBE, A549, SPC-A-1, NCI-H1299 and NCI-H1795). Overexpression of ATF3 in A549 cells increased the expression of CD68, CD206 and CSF-1. Moreover, levels of CD206, CD163, IL-10 and TGF-ß increased when A549 cells were co-cultured with M0 macrophages under the stimulation of CSF-1. Using the starbase online software prediction and dual-luciferase assays, we identified the targeting between miR-27a-3p and ATF3. Levels of ATF3, CSF-1, CD206, CD163, IL-10 and TGF-ß decreased in the miR-27a mimics, and the tumour growth was slowed in the miR-27a mimics compared with the mimics NC group. Overall, the study suggested that miR-27a-3p might inhibit the ATF3/CFS1 axis, regulate the M2 polarization of macrophages and ultimately hinder the progress of NSCLC. This research might provide a new therapeutic strategy for NSCLC.

Transcription factor 3 promotes migration and invasion potential and maintains cancer stemness by activating ID1 expression in esophageal squamous cell carcinoma.

Transcription factor 3 (TCF3) is a member of the basic Helix - Loop - Helix (bHLH) transcription factor (TF) family and is encoded by the TCF3 gene (also known as E2A). It has been shown that TCF3 functions as a key transcription factor in the pathogenesis of several human cancers and plays an important role in stem cell maintenance and carcinogenesis. However, the effect of TCF3 in the progression of esophageal squamous cell carcinoma (ESCC) is poorly known. In our study, TCF3 was found to express highly and correlated with cancer stage and prognosis. TCF3 was shown to promote ESCC invasion, migration, and drug resistance both from the results of in vivo and in vitro assays. Moreover, further studies suggested that TCF3 played these roles through transcriptionally regulating Inhibitor of DNA binding 1(ID1). Notably, we also found that TCF3 or ID1 was associated with ESCC stemness. Furthermore, TCF3 was correlated with the expression of cancer stemness markers CD44 and CD133. Therefore, maintaining cancer stemness might be the underlying mechanism that TCF3 transcriptionally regulated ID1 and further promoted ESCC progression and drug resistance.

Saikosaponin A triggers cell ferroptosis in hepatocellular carcinoma by inducing endoplasmic reticulum stress-stimulated ATF3 expression.

Ferroptosis is a type of nonapoptotic necrotic cell death characterized by iron-dependent lipid peroxidation. Saikosaponin A (SsA), a natural bioactive triterpenoid saponin extracted from Radix Bupleuri, has shown potent antitumor activity against various tumors. However, the underlying mechanism of the antitumor activity of SsA remains unclear. Here, we discovered that SsA induced HCC cell ferroptosis in vitro and in vivo. Using RNA-sequence analysis, we found that SsA mainly affected the glutathione metabolic pathway and inhibited the expression of cystine transporter solute carrier family 7 member 11 (SLC7A11). Indeed, SsA increased intracellular malondialdehyde (MDA) and iron accumulation, while it decreased the levels of reduced glutathione (GSH) in HCC. Deferoxamine (DFO), ferrostatin-1 (Fer-1) and GSH could rescue SsA-induced cell death, whereas Z-VAD-FMK was found ineffective in inhibiting SsA-induced cell death in HCC. Importantly, our result indicated that SsA induced the expression of activation transcription factor 3 (ATF3). SsA-induced cell ferroptosis and suppression of SLC7A11 are dependent on ATF3 in HCC. Moreover, we revealed that SsA induced ATF3 upregulation via activation of endoplasmic reticulum (ER) stress. Taken together, our findings support that ATF3-dependent cell ferroptosis mediated the antitumor effects of SsA, opening the possibility to explore SsA as a ferroptosis inducer in HCC.

Research progress on activation transcription factor 3: A promising cardioprotective molecule.

Activation transcription factor 3 (ATF3), a member of the ATF/cyclic adenosine monophosphate response element binding family, can be induced by a variety of stresses. Numerous studies have indicated that ATF3 plays multiple roles in the development and progression of cardiovascular diseases, including atherosclerosis, hypertrophy, fibrosis, myocardial ischemia-reperfusion, cardiomyopathy, and other cardiac dysfunctions. In past decades, ATF3 has been demonstrated to be detrimental to some cardiac diseases. Current studies have indicated that ATF3 can function as a cardioprotective molecule in antioxidative stress, lipid metabolic metabolism, energy metabolic regulation, and cell death modulation. To unveil the potential therapeutic role of ATF3 in cardiovascular diseases, we organized this review to explore the protective effects and mechanisms of ATF3 on cardiac dysfunction, which might provide rational evidence for the prevention and cure of cardiovascular diseases.

Multi-Omics Analysis of the Prognostic and Immunological Role of Runt-Related Transcription Factor 3 in Pan-Cancer.

Runt-related transcription factor 3 (RUNX3) plays a pivotal role in tumor microenvironment and immune infiltration. However, the prognostic and immunological roles of RUNX3 in pancancer remain unclear. In the current study, we explored the expression profiles, prognostic landscape, and immune infiltration of RUNX3 in pancancer through a variety of online platforms, including HPA, ONCOMINE, UALCAN, GEPIA, PrognoScan, TCGA, TIMER, R2, and Reactome databases. In general, RUNX3 was widely expressed in tonsil, gallbladder, skin, spleen, lymph node, and bone marrow, and RUNX3 was frequently higher expression in tumor tissues compared to normal tissues. In prognostic analysis, the RUNX3 expression level was significantly correlated with the clinical outcomes of bladder cancer, blood cancer, brain cancer, breast cancer, colorectal cancer, lung cancer, and ovarian cancer. In mutation analysis, a total 72 mutation sites were located within amino acids 1 to 415 of RUNX3, including 65 missense sites and seven truncating sites, whereas the mutation frequency of skin cutaneous melanoma and uterine corpus endometrial carcinoma (UCEC) is relatively high (> 3%). In immune infiltration analysis, the RUNX3 expression level was significantly related to recognized markers and the immune infiltration levels of various types of immune cells in colon adenocarcinoma (COAD) and brain lower grade glioma (LGG). After that, 453 RUNX3 co-expressed genes were recognized in COAD, lymphoid neoplasm diffuse large B-cell lymphoma, LGG, and ovarian serous cystadenocarcinoma (OV). Pathway enrichment analysis revealed that RUNX3 co-expressed genes were remarkably enriched in immune system and tumor progression pathways. RUNX3 expression is associated with clinical prognosis, immune infiltration, and identified RUNX3 related pathways in a variety of tumors, which may serve as targets of promising prognostic markers and novel therapeutic targets for various human cancers.

Macrophage microvesicle-derived circ_YTHDF2 in methamphetamine-induced chronic lung injury.

Long-term abuse of methamphetamine (MA) can cause lung toxicity. Intercellular communication between macrophages and alveolar epithelial cells (AECs) is critical for maintaining lung homeostasis. Microvesicles (MVs) are an important medium of intercellular communication. However, the mechanism of macrophage MVs (MMVs) in MA-induced chronic lung injury remains unclear. This study aimed to investigate if MA can augment the activity of MMVs and if circ_YTHDF2 is a key factor in MMV-mediated macrophage-AEC communication, and to explore the mechanism of MMV-derived circ_YTHDF2 in MA-induced chronic lung injury. MA elevated peak velocity of the pulmonary artery and pulmonary artery accelerate time, reduced the number of alveolar sacs, thickened the alveolar septum, and accelerated the release of MMVs and the uptake of MMVs by AECs. Circ_YTHDF2 was downregulated in lung and MMVs induced by MA. The immune factors in MMVs were increased by si-circ_YTHDF. Circ_YTHDF2 knockdown in MMVs induced inflammation and remodelling in the internalised AECs by MMVs, which was reversed by circ_YTHDF2 overexpression in MMVs. Circ_YTHDF2 bound specifically to and sponged miRNA-145-5p. Runt-related transcription factor 3 (RUNX3) was identified as potential target of miR-145-5p. RUNX3 targeted zinc finger E-box-binding homeobox 1 (ZEB1)-related inflammation and EMT of AECs. In vivo, circ_YTHDF2 overexpression-MMVs attenuated MA-induced lung inflammation and remodelling by the circ_YTHDF2-miRNA-145-5p-RUNX3 axis. Therefore, MA abuse can induce pulmonary dysfunction and alveolus injury. The immunoactivity of MMVs is regulated by circ_YTHDF2. Circ_YTHDF2 in MMVs is the key to communication between macrophages and AECs. Circ_YTHDF2 sponges miR-145-5p targeting RUNX3 to participate in ZEB1-related inflammation and remodelling of AECs. MMV-derived circ_YTHDF2 would be an important therapeutic target for MA-induced chronic lung injury. KEY POINTS: Methamphetamine (MA) abuse induces pulmonary dysfunction and alveoli injury. The immunoactivity of macrophage microvesicles (MMVs) is regulated by circ_YTHDF2. Circ_YTHDF2 in MMVs is the key to MMV-mediated intercellular communication between macrophages and alveolar epithelial cells. Circ_YTHDF2 sponges miR-145-5p targeting runt-related transcription factor 3 (RUNX3) to participate in zinc finger E-box-binding homeobox 1 (ZEB1)-related inflammation and remodelling. MMV-derived circ_YTHDF2 would be an important therapeutic target for MA-induced chronic lung injury.

Increased transcription of hsa_circ_0000644 upon RUNX family transcription factor 3 downregulation participates in the malignant development of bladder cancer.

BACKGROUND: Studies are ongoing to examine the versatile functions of circular RNAs (circRNAs) in human diseases. This research investigates the effects of hsa_circ_0000644 (circ_644) and its related molecules on the malignant behavior of bladder cancer (BCa) cells. METHODS: Abundant bioinformatics analyses were performed to screen the key circRNA and its related molecules in BCa. Tumor tissues and the para-tumorous tissues were collected from 58 patients with BCa. Expression of RUNX family transcription factor 3 (RUNX3), circ_644, microRNA-143-3p (miR-143-3p), and musashi RNA binding protein 2 (MSI2) in BCa tissues or cells was determined. Molecular interactions were confirmed by chromatin immunoprecipitation, RNA pull-down, and luciferase assays. Gain and loss-of function assays were performed using two BCa cell lines (T24 and HT1376). RESULTS: Circ_644 was highly expressed whereas RUNX3, which could suppress circ_644 transcription, was lowly expressed in BCa tissues and cells. Upregulation of RUNX3 suppressed proliferation, colony formation, migration and invasion, and tumorigenicity of BCa cells and induced cell cycle arrest. However, the tumor-suppressive effects of RUNX3 were blocked by circ_644 upregulation. Circ_644 served as a sponge for miR-143-3p, and miR-143-3p bound to MSI2 mRNA. The rescue experiments showed that miR-143-3p inhibition or MSI2 overexpression restored the malignant behaviors of BCa cells induced by circ_644 knockdown or RUNX3 overexpression. CONCLUSION: This study demonstrates that transcriptional activation of circ_644 upon RUNX3 downregulation drives the malignant development of BCa through the miR-143-3p/MSI2 axis. RUNX3 restoration or specific inhibition of circ_644 or MSI2 may help block BCa progression.

Knockdown of hsa_circ_0043691 restrains the progression of gastric cancer by decoying miR-1294 to target pre-leukemia transcription factor 3.

BACKGROUND: Circular RNAs (circRNAs) have been found to render pivotal effects in gastric cancer (GC). However, the effect of hsa_circ_0043691 in GC still needs to be further unveiled. METHODS: The contents of hsa_circ_0043691, miR-1294, and pre-leukemia transcription factor 3 (PBX3) were examined using qRT-PCR or Western blot assay. In vitro colony formation, transwell, wound healing, flow cytometry, tube formation assays, glutaminolysis corresponding kit and in vivo Xenograft mice model were utilized to evaluate cell functions. The relationship between miR-1294 and hsa_circ_0043691 or PBX3 was further verified. RESULTS: The levels of hsa_circ_0043691 and PBX3 were upregulated, whereas miR-1294 expression was diminished in GC tissues and cells. Hsa_circ_0043691 deficiency significantly inhibited GC cell progression and glutaminolysis metabolism. Mechanistically, hsa_circ_0043691 was directly bound to miR-1294 to modulate PBX3 expression. Besides, silencing of hsa_circ_0043691 impeded tumor growth in vivo. CONCLUSION: Hsa_circ_0043691 knockdown repressed GC malignant phenotypes by miR-1294/PBX3 axis, which exhibited a novel therapeutic target for GC treatment.

Clinicopathological Significance of STAT3 and p-STAT3 among 91 Patients with Adenocarcinoma of the Esophagogastric Junction.

Adenocarcinoma of the esophagogastric junction (AEG) has increased rapidly worldwide during the last few decades. The purpose of this study is to investigate the clinical and prognostic characteristics of signal transduction and activator of transcription factor 3(STAT3) and phosphorylated STAT3 (p-STAT3) expression in AEG patients. We retrospectively analyzed the immunohistochemical results of 61 AEG patients and followed up for 5 years, while Western blot was performed on tissues from another 30 AEG patients. The results showed that STAT3 and p-STAT3 were overexpressed in AEG tissues (P < 0.05, P < 0.01). The high expression of STAT3 was significantly associated with the pTNM stage (P < 0.05), and the increased expression of p-STAT3 was significantly associated with depth of invasion (pT), lymph node metastasis (pN), and pTNM stage (P < 0.05, P < 0.05, P < 0.05). The 5-year survival rate for AEG patients was 41.0% and was significantly associated with tumor differentiation, pN, pTNM, and p-STAT3 (P < 0.05, P < 0.01, P < 0.05, P < 0.01). Cox regression analysis confirmed that tumor differentiation, pN, and high expression of p-STAT3 were independent risk factors for the 5-year survival rate in patients with AEG (P < 0.05, P < 0.01, P < 0.05). Our study showed that STAT3 and p-STAT3 play a critical role in AEG development.

Methylation of RUNX3 Promoter 2 in the Whole Blood of Children with Ulcerative Colitis.

Ulcerative colitis (UC) results from a complex interplay between the environment, gut microbiota, host genetics, and immunity. Runt-related transcription factor 3 (RUNX3) regulates Th1/Th2 balance and, thus, the synthesis of cytokines and inflammation. We aimed to analyze the dependence of RUNX3 promoter 2 (P2) methylation level on: age, sex, body mass index (BMI), C-reactive protein (CRP), serum albumin, disease duration, Pediatric Ulcerative Colitis Activity Index (PUCAI), the Paris classification, and exposure to medications. This multicenter, cross-sectional study recruited hospitalized children with UC. Methylation of RUNX3 P2 was measured with methylation-sensitive restriction enzymes in the whole blood DNA. Sixty-four children were enrolled, with a mean age of 14.5 ± 2.8 years. Half of them were female (51.6%), and the average BMI Z-score was -0.44 ± 1.14. The mean methylation of RUNX3 P2 was 54.1 ± 13.3%. The methylation level of RUNX3 P2 did not correlate with age, sex, nutritional status, CRP, albumin, PUCAI, or the extent of colitis (Paris E1-E4). RUNX3 P2 methylation did not differ between patients recruited within two and a half months of diagnosis and children who had UC for at least a year. Current or past exposure to biologics, immunosuppressants, or steroids was not associated with RUNX3 P2 methylation. Methylation of RUNX3 promoter 2 in whole blood DNA does not seem to be associated with the characteristics of UC in children.

Long noncoding RNA Linc01612 represses hepatocellular carcinoma progression by regulating miR-494/ATF3/p53 axis and promoting ubiquitination of YBX1.

Long noncoding RNAs (lncRNAs) play an important role in the progression of hepatocellular carcinoma (HCC). Linc01612 is a novel lncRNA that function remains unknown in the progression of cancers, including HCC. In this study, we discovered that Linc01612 is significantly down-regulated in HCC tissues than in non-tumor tissues and correlated with poor prognosis. Linc01612 mainly localizes in the cytoplasm and functions as a tumor suppressor by repressing the growth and metastasis of hepatoma cells in vitro and in vivo. Mechanistically, in p53-expressing hepatoma cells, Linc01612 acts as a competitive endogenous RNA and promotes the expression of activation transcription factor 3 (ATF3) by sponging microRNA-494 (miR-494), which in turn inhibits MDM2-mediated ubiquitination of p53 and activates the p53 pathway. Furthermore, in p53-null hepatoma cells, Linc01612 exerts its biological functions by physically interacting with Y-box binding protein 1 protein (YBX1) and promoting the ubiquitin-mediated degradation of YBX1. Interestingly, the Linc01612-YBX1 signaling pathway is also present in p53-expressing hepatoma cells. In conclusion, our study indicated that Linc01612 is a functional lncRNA in HCC and Linc01612 may serve as a potential diagnostic biomarker and therapeutic target for HCC.

Transcriptional factor 3 binds to sirtuin 1 to activate the Wnt/ß-catenin signaling in cervical cancer.

Transcriptional factor 3 (TCF3, also termed E2A), first reported to exert crucial functions during lymphocyte development, has been revealed to participate in the pathogenesis of human cancers. The aim of this work was to investigate the function of TCF3 in cervical cancer (CC) and the molecular interactions. The bioinformatics prediction suggested that TCF3 was highly expressed in CC and linked to poor prognosis. Increased TCF3 expression was identified in CC cell lines, and its downregulation reduced proliferation and migration of CC cells in vitro as well as growth of xenograft tumors in vivo. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that the TCF-3-related genes and genes showed differential expression between CC and normal tissues were mainly enriched in the Wnt/ß-catenin pathway. TCF3 bound to sirtuin 1 (SIRT1) promoter for transcriptional activation, and SIRT1 promoted deacetylation and nuclear translocation of ß-catenin in CC. SIRT1 overexpression blocked the role of TCF3 silencing and restored cell proliferation in vitro and tumor growth in vivo. Treatment with XAV-939, a ß-catenin inhibitor, significantly suppressed the cell proliferation and tumor growth induced by SIRT1 overexpression. In conclusion, this study demonstrates that TCF3 augments progression of CC by activating SIRT1-mediated ß-catenin signaling.

Overexpression of transcription factor 3 drives hepatocarcinoma development by enhancing cell proliferation via activating Wnt signaling pathway.

BACKGROUND: Transcription factor 3 (TCF3) plays pivotal roles in embryonic development, stem cell maintenance and carcinogenesis. However, its role in hepatocellular carcinoma (HCC) remains largely unknown. This study aimed to analyze the correlation between TCF3 expression and clinicopathological features of HCC, and further explore the underlying mechanism in HCC progression. METHODS: The expression of TCF3 was collected from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) HCC datasets, and further confirmed by immunostaining and Western blotting assays. The correlation between TCF3 expression and the clinicopathological features was evaluated. Bioinformatical analysis and in vitro experiments were conducted to explore the potential role of TCF3 in HCC development. RESULTS: Both the mRNA and protein levels of TCF3 were significantly higher in HCC tumor tissues compared to tumor adjacent tissues (P < 0.001 and P < 0.01). Analysis based on TCGA datasets showed that TCF3 was positively correlated with tumor clinical stage and grade, and patients with high TCF3 expression had shorter overall survival (P = 0.012), disease-specific survival (P = 0.022) and progression-free survival (P = 0.013). Similarly, the immunostaining results revealed that the high expression of TCF3 was closely correlated with tumor size (P = 0.001) and TNM stage (P = 0.002), and TCF3 was an independent risk factor of HCC. In vitro study exhibited that TCF3 knockdown dramatically suppressed cancer cell proliferation, and the underlying mechanism might be that the silencing of TCF3 reduced the expression of critical regulating proteins towards cell cycle and proteins involved in Wnt signaling pathways. CONCLUSIONS: TCF3 expression is significantly elevated in HCC and positively associated with the tumor size and TNM stage, as well as poor prognosis of HCC patients. The mechanism might be that TCF3 promotes cancer cell proliferation via activating Wnt signaling pathway.

[Expression of Runt-related Transcription Factor 3 in Human Colon Cancer Cell Line HCT-116 Resistant to 5-Fluorouracil and the Mechanism of Drug Resistance].

Objective To establish a human colon cancer cell line HCT-116/5-FU resistant to 5-fluorouracil(5-FU)and explore the relationship between runt-related transcription factor 3(RUNX3)and drug resistance of colorectal cancer.Methods The human colon cancer cell line HCT-116/5-FU with resistance to 5-FU was established by low concentration gradient increment combined with high-dose intermittent shock.CCK-8 method was used to determine the half maximal inhibitory concentration(IC50)of 5-FU on the parent line HCT-116 and drug-resistant line HCT-116/5-FU.The cell growth curve was established for the calculation of population doubling time(TD).The mRNA levels and protein levels of RUNX3,P-glycoprotein(P-gp),multidrug resistance-associated protein 1(MRP1),and lung resistance-related protein(LRP)in HCT-116 and HCT-116/5-FU cells were determined by qRT-PCR and Western blotting,respectively.The RUNX3 expression in HCT-116 cells was knocked down by siRNA technique,and the cells were divided into RUNX3 knockdown groups(si-RUNX3-1 group and si-RUNX3-2 group)and negative control group(si-NC group).The knockdown efficiency was verified by qRT-PCR at the mRNA level and Western blotting at the protein level.The IC50 in si-RUNX3 groups and si-NC group was determined with CCK-8 method,and the expression of P-gp,MRP1,and LRP in the two groups was detected by Western blotting.Results A stable human colon cancer drug-resistant cell line HCT-116/5-FU was successfully constructed.HCT-116/5-FU showed the TD 1.38 times as long as that of HCT-116(P=0.002)and changed morphology.The mRNA level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116 cells(P=0.048),and those of P-gp(P=0.008),MRP1(P=0.001),and LRP(P=0.001)showed the opposite trend.The protein level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116(P<0.001),and those of P-gp,MRP1,and LRP presented the opposite trend(all P<0.001).The HCT-116 cell model with low expression of RUNX3 was successfully established.The mRNA level of RUNX3 had no significant difference between si-RUNX3-1 group and si-NC group(P=0.064),while the level in si-RUNX3-2 group was significantly lower than that in si-NC group(P=0.034).The protein levels of RUNX3 in si-RUNX3-1 group and si-RUNX3-2 group were lower than that in si-NC group(both P<0.001).The results demonstrated higher knocking efficiency in si-RUNX3-2 group,which was thus selected to complete the follow-up test.The IC50 of si-RUNX3 group was significantly higher than that of si-NC group(P<0.001),which indicated that the down-regulated expression of RUNX3 could reduce the sensitivity of HCT-116 cells to 5-FU.The relative protein levels of P-gp,MRP1,and LRP in si-RUNX3 group were significantly higher than those in si-NC group(all P<0.001).Conclusion The down-regulation of RUNX3 expression can reduce the sensitivity of HCT-116 cells to 5-FU,which is considered to be related to the up-regulated expression of P-gp,MRP1,and LRP.

Apatinib Inhibits Angiogenesis in Intrahepatic Cholangiocarcinoma by Regulating the Vascular Endothelial Growth Factor Receptor-2/Signal Transducer and Activator of Transcription Factor 3/Hypoxia Inducible Factor 1 Subunit Alpha Signaling Axis.

INTRODUCTION: Intrahepatic cholangiocarcinoma (ICC), which is difficult to diagnose and is usually fatal due to its late clinical presentation and a lack of effective treatment, has risen over the past decades but without much improvement in prognosis. OBJECTIVE: The study aimed to investigate the role of apatinib that targets vascular endothelial growth factor receptor-2 (VEGFR2) in ICC. METHODS: MTT assays, cell scratch assays, and tube formation assays were used to assess the effect of apatinib on human ICC cell line (HuCCT-1) and RBE cells proliferation, migration, and angiogenic capacity, respectively. Expression of vascular endothelial growth factor (VEGF), VEGFR2, signal transducer and activator of transcription factor 3 (STAT3), pSTAT3, and hypoxia inducible factor 1 subunit alpha (HIF-1α) pathway proteins was assessed using Western blotting and mRNA expression analysis in HuCCT-1 was performed using RT-qPCR assays. The pcDNA 3.1(-)-VEGFR2 and pcDNA 3.1(-)-HIF-1α were transfected into HuCCT-1 and RBE cells using Lipofectamine 2,000 to obtain overexpressed HuCCT-1 and RBE cells. RESULTS: We found that apatinib-inhibited proliferation, migration, and angiogenesis of HuCCT-1 and RBE cells in vitro in a dose-dependent manner. We also proved that apatinib effectively inhibits angiogenesis in tumor cells by blocking the expression of VEGF and VEGFR2 in these cells. In addition, we demonstrated that apatinib regulates the expression of STAT3 phosphorylation by inhibiting VEGFR2. Finally, we showed that apatinib regulates ICC angiogenesis and HIF-1α/VEGF expression via STAT3. CONCLUSIONS: Based on the above findings, we conclude that apatinib inhibits HuCCT-1 and RBE cell proliferation, migration, and tumor angiogenesis by inhibiting the VEGFR2/STAT3/HIF-1α axis signaling pathway. Apatinib can be a promising drug for ICC-targeted molecular therapy.

Rspo2 inhibits TCF3 phosphorylation to antagonize Wnt signaling during vertebrate anteroposterior axis specification.

The Wnt pathway activates target genes by controlling the ß-catenin-T-cell factor (TCF) transcriptional complex during embryonic development and cancer. This pathway can be potentiated by R-spondins, a family of proteins that bind RNF43/ZNRF3 E3 ubiquitin ligases and LGR4/5 receptors to prevent Frizzled degradation. Here we demonstrate that, during Xenopus anteroposterior axis specification, Rspo2 functions as a Wnt antagonist, both morphologically and at the level of gene targets and pathway mediators. Unexpectedly, the binding to RNF43/ZNRF3 and LGR4/5 was not required for the Wnt inhibitory activity. Moreover, Rspo2 did not influence Dishevelled phosphorylation in response to Wnt ligands, suggesting that Frizzled activity is not affected. Further analysis indicated that the Wnt antagonism is due to the inhibitory effect of Rspo2 on TCF3/TCF7L1 phosphorylation that normally leads to target gene activation. Consistent with this mechanism, Rspo2 anteriorizing activity has been rescued in TCF3-depleted embryos. These observations suggest that Rspo2 is a context-specific regulator of TCF3 phosphorylation and Wnt signaling.

Transcription Factor ß-Catenin Plays a Key Role in Fluid Flow Shear Stress-Mediated Glomerular Injury in Solitary Kidney.

Increased fluid flow shear stress (FFSS) in solitary kidney alters podocyte function in vivo. FFSS-treated cultured podocytes show upregulated AKT-GSK3ß-ß-catenin signaling. The present study was undertaken to confirm (i) the activation of ß-catenin signaling in podocytes in vivo using unilaterally nephrectomized (UNX) TOPGAL mice with the ß-galactosidase reporter gene for ß-catenin activation, (ii) ß-catenin translocation in FFSS-treated mouse podocytes, and (iii) ß-catenin signaling using publicly available data from UNX mice. The UNX of TOPGAL mice resulted in glomerular hypertrophy and increased the mesangial matrix consistent with hemodynamic adaptation. Uninephrectomized TOPGAL mice showed an increased ß-galactosidase expression at 4 weeks but not at 12 weeks, as assessed using immunofluorescence microscopy (p < 0.001 at 4 weeks; p = 0.16 at 12 weeks) and X-gal staining (p = 0.008 at 4 weeks; p = 0.65 at 12 weeks). Immunofluorescence microscopy showed a significant increase in phospho-ß-catenin (Ser552, p = 0.005) at 4 weeks but not at 12 weeks (p = 0.935) following UNX, and the levels of phospho-ß-catenin (Ser675) did not change. In vitro FFSS caused a sustained increase in the nuclear translocation of phospho-ß-catenin (Ser552) but not phospho-ß-catenin (Ser675) in podocytes. The bioinformatic analysis of the GEO dataset, #GSE53996, also identified ß-catenin as a key upstream regulator. We conclude that transcription factor ß-catenin mediates FFSS-induced podocyte (glomerular) injury in solitary kidney.

E47 upregulates ΔNp63α to promote growth of squamous cell carcinoma.

Targeted therapy has greatly improved both survival and prognosis of cancer patients. However, while therapeutic treatment of adenocarcinoma has been advanced greatly, progress in treatment of squamous cell carcinoma (SCC) has been slow and ineffective. Therefore, it is of great importance to decipher mechanisms and identify new drug targets involved in squamous cell carcinoma development. In this study, we demonstrate that E47 plays the distinctive and opposite roles on cell proliferation in adenocarcinoma and squamous cell carcinoma. While E47 suppresses cell proliferation in adenocarcinoma cells, it functions as a oncoprotein to promote cell proliferation and tumor growth of squamous cell carcinoma. Mechanistically, we show that E47 can directly bind to the promoter and transactivate ΔNp63 gene expression in squamous cell carcinoma cells, resulting in upregulation of cyclins D1/E1 and downregulation of p21, and thereby promoting cell proliferation and tumor growth. We further show that expression of E2A (E12/E47) is positively correlated with p63 and that high expression of E2A is associated with poor outcomes in clinical samples of squamous cell carcinoma. These results highlight that the E47-ΔNp63α axis may be potential therapeutic targets for treatment of squamous cell carcinoma.