Six2 is negatively correlated with prognosis and facilitates epithelial-mesenchymal transition via TGF-β/Smad signal pathway in hepatocellular carcinoma

Background: Increasing evidence indicates that Six2 contributes to tumorigenesis in various tumor including hepatocellular carcinoma(HCC). This study aimed to determine the role of Six2 in HCC and to elucidate the association of Six2 with clinical pathological characteristics. Methods: The expressions of Six2 in HCC tumor, para-tumor tissue and portal vein tumor thrombus(PVTT) were detected by tissue microarray technique, immunohistochemistry, real-time RT-PCR and Western blotting. Chi-square and Kaplan-Meier analysis were used to analyze the correlation between Six2 expression and prognosis of HCC patients. Lentivirus mediated Six2 knockdown, spheroid formation assay, proliferation assay and subcutaneous tumor implantation were performed to determine the function of Six2. Results: In 274 HCC samples, Six2 was strongly expressed. Kaplan-Meier analysis revealed that high expression of Six2 was correlated with a shorter overall survival(OS) and disease-free survival(DFS). Moreover, Six2 expression was associated with sex, alpha-fetoprotein, tumor size and portal vein invasion. Six2 was highly expressed in PVTT. Six2 knockdown inhibited HCC cell lines proliferation, migration, and self-renewal in vitro and in vivo. In addition, low-expression of Six2 weakened TGF-β induced Smad4 activation and epithelial-mesenchymal transition in HCC cell lines. Conclusions: Elevated Six2 expression in HCC tumor patients was associated with negative prognosis. Upregulated Six2 promoted tumor growth and facilitated HCC metastasis via TGF-β/Smad signal pathway.     

Aberrant p16 methylation, a possible epigenetic risk factor in familial esophageal squamous cell carcinoma

Aim. Detection of methylation in the p16 gene, an inhibitor of cyclin D-dependent protein kinase, as a new tumor marker for early detection of esophageal squamous cell carcinoma (ESCC) in DNA derived from blood and serum. Method. A large family with clustering of ESCC was assessed in Khorasan province in northeastern Iran. The family had three histologically proven cases of ESCC in two consecutive generations and several other deceased cases with histories of ESCC. DNA from blood of 28 living family members in three consecutive generations, 30 sporadic ESCC cases (from serum, blood, and tumor tissues), and 30 healthy volunteers (from blood) were examined for the methylation status of p16 promoter using methylation-specific PCR (MSP). Results. Aberrant p16 promoter methylation was found in 64.3% (n=28) of ESCC family members and none (n=30) of our normal volunteers. Five of the 28 family members with esophageal cancer symptoms had negative endoscopy results for ESCC, while four of these members had p16 hypermethylation in their blood. The family members with negative endoscopy and positive p16 promoter methylation are being monitored closely for signs of ESCC development through regular check-ups and chromoendoscopies. In sporadic ESCC in northeastern Iran, 73.3% (n=30) of tumor tissue samples had p16 hypermethylation. Serum and blood samples from the same patients showed p16 hypermethylation in 26.6% and 43.3% of the samples, respectively. Conclusion. Aberrant p16 methylation may be a valuable diagnostic tool as a tumor marker for the early identification of individuals in high risk ESCC families.     

Toxicarioside A, isolated from tropical Antiaris toxicaria, blocks endoglin/TGF-β signaling in a bone marrow stromal cell line

ObjectiveTo investigate possible mechanism of toxicarioside A in HS-5 bone stromal cells.MethodsHS-5 bone stromal cells were cultured in media supplemented with various concentrations of toxicarioside A or control DMSO (not treatment). Endoglin and TGF-β were detected by Northern and Western blot analysis and quantified in a standard method. Downstream molecules of endoglin and TGF-β (Smad1, Smad2 and their active phosphorylated counterparts, pSmad1 and pSmad2) were also detected and quantified by Western blot analysis. In addition, cell proliferation assay and small interfering RNA (siRNA) against endoglin were used to certificate the function of endolgin in the HS-5 cells.ResultsCompared with the not treated (0 μg/mL) or DMSO treated control HS-5 cells, HS-5 cells treated with toxicarioside A were found significant attenuation of endolgin and TGF-β expression. Significant inhibition of cell proliferation was also found in the HS-5 cells treated with toxicarioside A. ALK1-related Smad1 and ALK5-related Smad2 were decreased in HS-5 cells treated with toxicarioside A. In addition, phosphorylated Smad1 (pSmad1) and Smad2 (pSmad2) were also found attenuation in toxicarioside A-treated HS-5 cells. RNA interference showed that blockage of endoglin by siRNA also decreased Smad1 and Smad2 expression in HS-5 cells.ConclusionsOur results indicate that toxicarioside A can influence bone marrow stromal HS-5's function and inhibit HS-5 cell proliferation by alteration of endoglin-related ALK1 (Smad1) and ALK5 (Smad2) signaling.     

Effects of miR-340 on hepatocellular carcinoma by targeting the DcR3 gene

In hepatocellular carcinoma (HCC), miR-340 plays a vital role in the regulation of tumor occurrence and deterioration, while DcR3 gene is involved in cancer cell proliferation and apoptosis. This study analyzed miR-340 in the serum of patients with HCC and healthy controls. Then, miR-340, DcR3, TGF-β1 and Smad2 expression were measured in HCC tissues and adjacent parts. Relationship between miR-340 and DcR3 was verified. Effects of miR-340 on human HepG2 cell proliferation and apoptosis were explored. miR-340, DcR3, TGF-β1, Smad2 mRNA and protein expression were also determined after miR-340 transfection. Compared with the control, miR-340 was significantly lower in the serum of the HCC patients (p < 0.01). miR-340 was lower in HCC tissues than in adjacent; however, DcR3, TGF-β1 and Smad2 were higher (p < 0.01). Furthermore, luciferase activity was significantly lower in the cells co-transfected with miR-340 mimics and DcR3-3′UTR-WT (p < 0.01), indicating that DcR3 was a target gene of miR-340. Moreover, decreased expression in DcR3, TGF-β1 and Smad2 was detected after miR-340 overexpression (p < 0.01), thus promoting apoptosis and blocking the proliferation of human HepG2 cells (p < 0.05). Furthermore, overexpression of DcR3 could activate the TGF-β1/Smad2 signal transduction pathway and increase the phosphorylation of Smad2. In conclusion, miR-340 plays a suppressive role in HCC development by targeting DcR3 and silencing the TGF-β1/Smad2 signaling pathway.     

Fibulin-1 Is Downregulated Through Promoter Hypermethylation in Colorectal Cancer: A Consort Study

Fibulin-1 (FBLN1) is involved in the progression of some types of cancer. However, the role of FBLN1 in colorectal cancer (CRC) has not been examined. The purpose of this study was to understand the molecular mechanisms and clinical significance of FBLN1 inactivation in CRC.The expression of FBLN1 in CRC tissues and adjacent normal tissues was analyzed by immunohistochemical analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing PCR (BSP) were performed to examine the methylation status of the FBLN1 gene promoter. Furthermore, the methylated level of FBLN1 was analyzed with the clinicopathological characteristics.Immunohistochemical analysis and qRT-PCR analysis showed that FBLN1 protein and messenger RNA (mRNA) levels in tumor tissues were both significantly decreased compared with that in adjacent nontumor tissues. The methylation rate of FBLN1 promoter was significantly higher in CRC tissues than that in adjacent nontumor tissues (P < 0.001). In addition, the correlation between FBLN1 hypermethylation, protein expression, and overall survival (OS) was statistically significant.Our results indicated that the FBLN1 gene may be a novel candidate of tumor suppressor gene in CRC, and that promoter hypermethylation of FBLN1 is an important reason for its downregulation and is also a good predictor of OS for CRC.     

Nintedanib inhibits epithelial-mesenchymal transition in A549 alveolar epithelial cells through regulation of the TGF-β/Smad pathway

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disorder. Recent studies have suggested that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells influences development of pulmonary fibrosis, which is mediated by transforming growth factor β (TGF-β). Tumor necrosis factor α (TNF-α), an important proinflammatory cytokine in IPF, has been shown to enhance TGF-β-induced EMT. Nintedanib, a multiple tyrosine kinase inhibitor that is currently used to treat IPF, has been shown to suppress EMT in various cancer cell lines. However, the mechanism of EMT inhibition by nintedanib and its effect on TGF-β and TNF-α signaling pathways in alveolar epithelial cells have not been fully elucidated.MethodsA549 alveolar epithelial cells were stimulated with TGF-β2 and TNF-α, and the effects of nintedanib on global gene expression were evaluated using microarray analysis. Furthermore, Smad2/3 phosphorylation was assessed using western blotting.ResultsWe found that in A549 cells, TGF-β2 and TNF-α treatment induces EMT, which was inhibited by nintedanib. Gene ontology analysis showed that nintedanib significantly attenuates the gene expression of EMT-related cellular pathways and the TGF-β signaling pathway, but not in the TNF-α-mediated signaling pathway. Furthermore, hierarchical cluster analysis revealed that EMT-related genes were attenuated in nintedanib-treated cells. Additionally, nintedanib was found to markedly suppress phosphorylation of Smad2/3.ConclusionNintedanib inhibits EMT by mediating EMT-related gene expression and the TGF-β/Smad pathway in A549 alveolar epithelial cells.     

Downregulation of transforming growth factor, beta receptor 2 and Notch signaling pathway in human abdominal aortic aneurysm

ObjectiveMutations in FBN1 and TGFBR2 genes are the main causative mutations identified in Marfan syndrome (MFS). The major vascular complication of MFS is aneurysm formation. Abdominal aortic aneurysm (AAA) is an acquired disease of later life of unknown etiology. The aim of this study was to examine if genetic aberrations in MFS-related genes FBN1 and TGFBR2 are present in patients with AAA.MethodsWe assessed the presence of copy number variation (CNV) in FBN1 and TGFBR2 genes in AAA biopsies from twelve patients. We also analyzed the expression of these genes in AAA biopsies compared to control biopsies from six organ donors. In addition we assessed the expression of two members of the Notch signaling pathway NOTCH3 and HEY2 as well as aortic smooth muscle cell (AoSMC) differentiation marker TAGLN in AAA and control biopsies.ResultsLoss of one copy (deletion) of the FBN1 exon 66 sequence and TGFBR2 exon 8 was identified in 7 (58%) and 11 (92%) of the 12 AAA biopsies. No copy number amplifications (duplications) were detected. Patients carrying TGFBR2 exon 8 deletion showed marked downregulation of this gene in AAA biopsies compared to control biopsies (0.699 vs. 1.765, p = 0.038). Notch signaling components NOTCH3 and HEY2 were markedly downregulated in AAA, while expression of the AoSMC differentiation marker TAGLN did not differ between AAA and control biopsies (0.468 vs. 0.486, p = 0.546).ConclusionThis study suggests an acquired impairment in TGF-β signaling that along with downregulation of the Notch signaling pathway may contribute to the pathogenesis of AAA.     

TGF-β1和Smad2在胃癌中的表达及意义

目的:检测TGF-β1和Smad2信号在胃癌中的表达,探讨TGF-β1和Smad2在胃癌发生、发展中的作用.方法:采用免疫蛋白印迹(Westernblot)和实时定量PCR检测45例胃癌组织及相应正常组织TGF-β1、Smad2蛋白及mRNA的表达,比较正常对照、癌组织中上述二者表达的差异,并进行统计学分析.结果:TGF-β1在胃癌组织中阳性表达率为77.8%(35/45),在正常组织中为33.3%(15/45),二者比较差异显著(P<0.05);Smd2蛋白在胃癌组织中阳性表达率为73.3%(33/45),在正常组织中为26.7%(12/45),二者比较有显著差异(P<0.05);TGF-β1、Smad2mRNA在胃组织中阳性表达率分别为88.9%(40/45)、84.4%(38/45),均显著高于正常组织(P<0.05).胃癌组织中TGF-β1、Smad2蛋白的表达与病理分级、浸润深度、淋巴结转移、脉管侵犯相关(P<0.05).结论:TGF-β1和Smad2可能在胃癌的发生、发展中发挥作用,并与胃癌的侵袭、转移相关.     

TGF-β1 signal pathway in the regulation of inflammation in patients with atrial fibrillation

ObjectiveTo observe the expression changes of inflammatory markers TGF-β1, Smad3 and IL-6 in patients with atrial fibrillation (AF), and to explore the significance of TGF-β1 signaling pathway in the structural remodeling of AF.MethodsThe expression of TGF-β1, Smad3 and IL-6 in 50 cases with AF and 30 normal cases were detected by RT-PCR and ELISA.ResultsThe TGF-β1, Smad3 and IL-6 mRNA and protein expression levels in patients with AF were significantly higher than that in the control group (P<0.05), but there was no significantly different between the paroxysmal AF group and the persistent AF group (P>0.05). The TGF-β1mRNA expression in the ? 50 years subgroup was significantly higher than that in the <50 years subgroups, and it was higher in the NYHA III subgroup than in the I/II grade subgroup. It was also higher in the left ventricular ejection fraction (LVEF) <50% subgroup than in LVEF ? 50% group, and it was significantly higher in the AF time ? 36 months subgroup than that in <36 months subgroup (P<0.05). The Smad3 and IL-6 expressions in the in the LVEF <50% subgroup were both high that than that in LVEF ? 50% group, and higher in the AF time ? 36 months subgroup than that in <36 months subgroup (P<0.05). There were a positive correlation between TGF-β1, Smad3 and IL-6 (r=0.687, r=0.547). There were also a positive correlation between Smad3 and IL-6 mRNA (r=0.823).ConclusionsAF is associated with inflammation, and the inflammation is also involved in the fibrillation and sustain of AF. The TGF-β1 signal pathway may be involved in the process of atrial structural remodeling in patients with AF, and iss related with the occurrence and maintenance of AF.     

KISS1 methylation and expression as predictors of disease progression in colorectal cancer patients

AIM:To examine the effect of aberrant methylation of the KISS1 promoter on the development of colorectal cancer(CRC)and to investigate reversing aberrant methylation of the KISS1 promoter as a potential therapeutic target.METHODS:KISS1 promoter methylation,mRNA expression and protein expression were detected by methylation-specific polymerase chain reaction(PCR),real-time quantitative PCR and Western blotting,respectively,in 126 CRC tissues and 142 normal colorectal tissues.Human CRC cells with KISS1 promoter hypermethylation and poor KISS1 expression were treated in vitro with 5-aza-2’-deoxycytidine(5-Aza-CdR).After treatment,KISS1 promoter methylation,KISS1 mRNA and protein expression and cell migration and invasion were evaluated.RESULTS:Hypermethylation of KISS1 occurred frequently in CRC samples(83.1%,105/126),but was infrequent in normal colorectal tissues(6.34%,9/142).Moreover,KISS1 methylation was associated with tumor differentiation,the depth of invasion,lymph node metastasis and distant metastasis(P<0.001).KISS1methylation was also associated with low KISS1 expression(P<0.001).Furthermore,we observed re-expression of the KISS1 gene and decreased cell migration after 5-Aza-CdR treatment in a CRC cell line.CONCLUSION:These data suggest that KISS1 is down-regulated in cancer tissues via promoter hypermethylation and therefore may represent a candidate target for treating metastatic CRC.     

Inhibition of PARP prevents angiotensin II-induced aortic fibrosis in rats

Background

Fibrosis is one of the major pathological features of hypertensive vascular disease. In this study, we aim to explore the possible protective effects of poly(ADP-ribose) polymerase (PARP) inhibitor on angiotensin II (AngII)-induced aortic fibrosis.

Methods

Sprague–Dawley rats were infused subcutaneously with AngII. PARP inhibitor was intraperitoneally injected once a day. Collagen deposition in thoracic aorta was assayed by Masson tricrome staining. The mRNA and protein expression of TGF-β target genes involved in extracellular matrix (ECM) remodeling in aorta was measured. Plasma level and aortic expression of TGF-β1 was assayed. Correlation of systolic blood pressure (SBP) with plasma level of TGF-β1 was analyzed. In cultured rat vascular smooth muscle cells (VSMCs), effects of PARP inhibition on TGF-β1 expression, Smad3 transactivity, and TGF-β/Smad3 target gene expression were investigated.

Results

Infusion of AngII promoted aortic PARP activation. Treatment with PARP inhibitor alleviated AngII-induced collagen deposition and expression of TGF-β target genes involved in ECM remodeling in aorta of rat. AngII increased plasma level and aortic expression of TGF-β1. A positive correlation between SBP and plasma level of TGF-β1 was revealed. Treatment with PARP inhibitor prevented AngII-induced elevation of SBP. Further experiments uncovered that AngII treatment increased TGF-β dependent gene expression through Smad3 pathway in cultured VSMCs. Inhibition of PARP prevented AngII-induced increases in TGF-β1 expression, Smad3 transactivity and its target gene expression.

Conclusions

These data indicate that inhibition of PARP prevents aortic fibrosis in AngII-induced hypertension in rats. This beneficial effect is mediated by inhibiting TGF-β/Smad3 pathway.     

Knockdown of DEAD-box 51 inhibits tumor growth of esophageal squamous cell carcinoma via the PI3K/AKT pathway

BACKGROUND Esophageal squamous cell carcinoma(ESCC)is one of the most prevalent malignancies that seriously threaten people’s health worldwide.DEAD-box helicase 51(DDX51)is a member of the DEAD-box(DDX)RNA helicase family,and drives or inhibits tumor progression in multiple cancer types.AIM To determine whether DDX51 affects the biological behavior of ESCC.METHODS The expression of DDX51 in ESCC tumor tissues and adjacent normal tissues was detected by Immunohistochemistry(IHC)analyses and quantitative PCR(qPCR).We knocked down DDX51 in ESCC cell lines by using a small interfering RNA(siRNA)transfection.The proliferation,apoptosis,and mobility of DDX51 siRNAtransfected cells were detected.The effect of DDX51 on the phosphoinositide 3-kinase(PI3K)/AKT pathway was investigated by western blot analysis.A mouse xenograft model was established to investigate the effects of DDX51 knockdown on ESCC tumor growth.RESULTS DDX51 exhibited high expression in ESCC tissues compared with normal tissues and represented a poor prognosis in patients with ESCC.Knockdown of DDX51 induced inhibition of ESCC cell proliferation and promoted apoptosis.Moreover,DDX51 siRNA-expressing cells also exhibited lower migration and invasion rates.Investigations into the underlying mechanisms suggested that DDX51 knock down induced inactivation of the PI3K/AKT pathway,including decreased phosphorylation levels of phosphate and tensin homolog,PI3K,AKT,and mammalian target of rapamycin.Rescue experiments demonstrated that the AKT activator insulin-like growth factor 1 could reverse the inhibitory effects of DDX51 on ESCC malignant development.Finally,we injected DDX51 siRNA-transfected TE-1 cells into an animal model,which resulted in slower tumor growth.CONCLUSION Our study suggests for the first time that DDX51 promotes cancer cell proliferation by regulating the PI3K/AKT pathway;thus,DDX51 might be a therapeutic target for ESCC.     

Effect of bone marrow mesenchymal stem cells on the Smad expression of hepatic fibrosis rats

Objective:To investigate the impact of bone marrow mesenchymal stem cells on Smad expression of hepatic fibrosis rats.Methods:A total of 48 adult female SD rats were randomly divided into three groups,normal control group(n=10),observation group(n=19)with liver fibrosis model rats injected with BMSCs cells:model group(n=19),with liver fibrosis model rats injected with physiological saline.Serum index,TGF-β1 and Smad expression were detected.Results:TypeⅢprocollagen,Ⅳcollagen,hyaluronic acid,laminin levels of observation group were significantly lower than those of model group(P0.05).The content and expression of TGF-β1in serum and liver tissue of observation group were significantly lower than those of model group(P0.05).Compared with normal control group,the Smad3,Smad4 mRNA and protein expression of model group were significantly increased,the Smad7 mRNA and protein expression were significantly reduced(P0.05).Compared with model group.Smad3,Smad4 mRNA and protein expression of observation group were significantly reduced,and Smad7 mRNA expression were significantly increased(P0.05).Conclusions:BMSCs can regulate Smad expression to some extent,and reduce the degree of liver fibrosis.     

头颈部鳞状细胞癌组织中 Smad7、Smad4蛋白的表达变化及意义

目的：观察Smad7及Smad4蛋白在头颈部鳞状细胞癌组织中的表达变化,并探讨其意义。方法采用免疫组化SP法分别对头颈部鳞状细胞癌组织、癌旁组织、活检留取的正常头颈部组织中的Smad7蛋白、Smad4蛋白、TGF-β1表达进行检测,分析三者间表达及其与肿瘤临床病理参数的关系。结果①TGF-β1和Smad7蛋白在肿瘤组织中的表达水平明显高于癌旁组织和正常组织（F分别为5．71、7．31）,而Smad4蛋白在肿瘤组织中的表达明显低于癌旁组织和正常组织（F为14．92）,P均＜0．05。②随肿瘤分化程度增高,TGF-β1及Smad7蛋白表达呈降低趋势（F分别为11．2、12．8）、Smad4蛋白呈升高趋势（F为7．91）,P均＜0．05;Smad4蛋白表达与肿瘤直径呈负相关、Smad7蛋白表达与肿瘤直径呈正相关（P均＜0．05）,TGF-β1表达与肿瘤直径无明显相关（P＞0．05）;Smad4蛋白表达在淋巴结转移者明显低于无转移者（P＜0．05）,有无淋巴结转移者Smad7蛋白及TGF-β1表达无显著差异（P＞0．05）。③Smad4蛋白表达与TGF-β1呈负相关（r＝-0．32）,与Smad7蛋白呈正相关（r＝0．39）,P均＜0．05。结论头颈部鳞状细胞癌组织中Smad7、Smad4蛋白表达异常,且可能通过调控上皮-间质转化参与肿瘤的侵袭、分化;检测两者（尤其是Smad4蛋白）可为临床分期、预后评估提供依据。