Circular RNA circDLG1 contributes to HCC progression by regulating the miR-141-3p/WTAP axis.

This study aims to explore novel and reliable biomarkers for predicting hepatocellular carcinoma (HCC) prognosis. Circular RNAs (circRNAs) were determined by analysis of human circRNA arrays and quantitative reverse transcription polymerase reactions. To test for an interaction between circDLG1, we used luciferase reporter assays, RNA immunoprecipitation, and fluorescence in situ hybridization assays that were employed to test the interaction between circDLG1, miR-141-3p, and WTAP. q-RT-PCR and western blot were used to evaluate the target regulation of miR-141-3p and WTAP. shRNA-mediated knockdown of circDLG1, proliferation, migration, and invasion experiment of metastasis were used to evaluate the function of circDLG. CircDLG1 rather than lining DLG1 was upregulated in HCC tissues, from HCC patients as well as HCC cell lines compared to normal controls. circDLG1 high expression in HCC patients was correlated with shorter overall survival. Knockdown of circDLG1 and miR-141-3p mimic could inhibit the tumorigenesis of HCC cells in vivo and in vitro. Importantly, we demonstrated that circDLG1 could act as a sponge of miR-141-3p to regulate the expression of WTAP, and further suppress the tumorigenesis of HCC cells. Our study reveals that circDLG1 can serve as a novel potential circulating biomarker for the detection of HCC. circDLG1 participates in the progression of HCC cells by sponging miR-141-3p with WTAP, providing new insight into the treatment of HCC.

Plasma BNP level combined with surgical Apgar score to predict operative major cardiac adverse events in malignant obstructive jaundice patients.

OBJECTIVE: To investigate the predictive effect of major adverse cardiac events (MACE) in malignant obstructive jaundice (OJ) patients using plasma brain natriuretic peptide (BNP) level and surgical Apgar scoring (SAS) system. METHODS: Forty one malignant OJ patients undergoing surgical treatments were studied at a single center. Pre-and postoperative plasma BNP level, total bilirubin (TBil) and data of cardiac function (HR, CVP, CI, LVEF%) were detected, the SAS was calculated during the surgery, the relationship of both plasma BNP level and SAS with MACE after surgery was analyzed. RESULTS: Thirteen patients out of 41 (31.71%) experienced MACE without cardiac death. OJ patients had a higher plasma BNP level than baseline before operation (191.61±105.76 pg/ml VS 175 pg/ml, P<0.05), the cardiac function data was improved (CVP: t=4.761, p=0.000; CI: t=3.539, p=0.001; LVEF%: t=3.632, p=0.001) after the operation. Patients with lower SAS had increasing incidence of MACE after surgery. CONCLUSION: Malignant OJ patients with higher preoperative BNP level and lower surgical Apgar score were identified at high risk of MACE after surgery.

MEF2 transcription factors promotes EMT and invasiveness of hepatocellular carcinoma through TGF-ß1 autoregulation circuitry.

Invasion and metastasis is the main causes leading to the death of hepatocellular carcinoma (HCC) patients. However, the underlying mechanism is still to be explored. Transforming growth factor ß1 (TGF-ß1) is a stronger inducer of HCC cell invasion. However, the downstream effector of TGF-ß1 that promotes HCC invasion is still unknown. In this study, we found that PI3K/Akt activation takes place following the stimulation of TGF-ß1. The inhibition of PI3K/Akt activation abolished epithelial-mesenchymal transition (EMT) and invasion of HCC cells induced by TGF-ß1. Myocyte enhancer factors 2 (MEF2) family proteins were found to be overexpressed in HCC cells under the treatment of TGF-ß1 in a PI3K/Akt-dependent way. Silencing the expression of MEF2s was able to prevent the effect of TGF-ß1 on HCC EMT and invasion. Unexpectedly, MEF2 proteins were able to promote the expression of TGF-ß1 in HCC cells, suggesting the existence of regulatory circuitry consisting of TGF-ß1, PI3K/Akt, and MEF2. A natural compound, oleanolic acid, was demonstrated to suppress the invasion and EMT of HCC cells by downregulating MEF2, showing that targeting this pathway is an effective therapeutic strategy for HCC invasion. We believe that our findings can contribute to better understanding of the involved mechanism of HCC invasion and the development of preventive and therapeutic strategy.

CircANTXR1 Contributes to the Malignant Progression of Hepatocellular Carcinoma by Promoting Proliferation and Metastasis.

BACKGROUND: Circular RNA (circRNA) is a key regulator for the malignant progression of cancer. However, the role of circRNA anthrax toxin receptor 1 (circANTXR1) in hepatocellular carcinoma (HCC) is still unclear. METHODS: Quantitative real-time PCR was performed to detect RNA expression. Cell proliferation, migration and invasion were determined using MTT assay, EdU staining, colony formation assay, wound healing assay and transwell assay. The protein levels of metastasis markers, x-ray repair cross complementing 5 (XRCC5) and exosome markers were examined using Western blot analysis. Xenograft tumor models were built to investigate the role of circANTXR1 in HCC tumorigenesis. The relationship between microRNA (miR)-532-5p and circANTXR1 or XRCC5 was confirmed by dual-luciferase reporter assay and RNA pull-down assay. The identification of exosomes were performed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). RESULTS: CircANTXR1 was a stable and highly expressed circRNA in HCC. Silenced circANTXR1 inhibited the proliferation, migration and invasion of HCC cells in vitro, and suppressed HCC tumor growth in vivo. MiR-532-5p could be sponged by circANTXR1, and its inhibitor could reverse the inhibition of circANTXR1 silencing on HCC cells progression. In addition, we discovered that XRCC5 was a target of miR-532-5p. Furthermore, XRCC5 overexpression could reverse the suppressive effect of miR-532-5p overexpression on HCC cell proliferation, migration and invasion. Exosome was involved in the transport of circANTXR1 in HCC cells. Exosome circANTXR1 might be a potential serum biomarker for HCC patients. CONCLUSION: CircANTXR1 promotes the progression of HCC through the miR-532-5p/XRCC5 axis, which might be a potential serum biomarker and therapeutic target of HCC.

5-(2-Fur-yl)-3-methyl-1-(3-nitro-phen-yl)-4,5-dihydro-1H-pyrazole.

In the title compound, C(14)H(13)N(3)O(3), the pyrazoline ring assumes an envelope conformation with the furanyl-bearing C atom at the flap position. The dihedral angle between the furan and nitrobenzene rings is 84.40 (9)°. Weak inter-molecular C-H⋯O hydrogen bonding is present in the crystal structure.

3-Methyl-1-(3-nitro-phen-yl)-5-phenyl-4,5-dihydro-1H-pyrazole.

In the title compound, C(16)H(15)N(3)O(2), the planar [maximum deviation 0.156 (2) Å] pyrazoline ring is nearly coplanar with the 3-nitro-phenyl group and is approximately perpendicular to the phenyl ring, making dihedral angles of 3.80 (8) and 80.58 (10)°, respectively. Weak inter-molecular C-H⋯O hydrogen bonding is present in the crystal structure.

RUNX2 promotes hepatocellular carcinoma cell migration and invasion by upregulating MMP9 expression.

Runt-related transcription factor 2 (RUNX2) was first identified as a transcription factor to play an important role in different biological processes of osteoblast and chondrocyte, including differentiation and migration. Recently, RUNX2 has been implicated in promigratory/proinvasive behavior in different human malignancies. In the present study, we demonstrated that the RUNX2 mRNA and protein expression were both increased significantly in HCC tissues and cell lines. High RUNX2 expression was correlated obviously with poor clinicopathological characteristics including multiple tumor nodes, high histological grading, venous infiltration and advanced tumor-node-metastasis (TNM) stage. In addition, we demonstrated that RUNX2 was a prognostic indicator for predicting 5-year overall survival and disease-free survival of HCC patients. Our studies showed that RUXN2 overexpression promoted, while RUNX2 knockdown inhibited HCC cell migration and invasion in vitro. Notably, RUNX2 positively regulated matrix metalloproteinase 9 (MMP9) accumulation in HCC cells. Furthermore, we confirmed that RUNX2 was positively correlated with MMP9 expression in HCC tissues by Pearson correlation analysis. Mechanistically, we demonstrated that MMP9 overexpression increased HCC cell migration and invasion, while MMP9 knockdown reduced HCC cell migration and invasion in vitro. Alteration of MMP9 expression partially abrogated the effects of RUNX2 on HCC cell migration and invasion, which suggests that RUNX2 developed its pro-metastatic biological function by upregulating the expression of MMP9 in HCC cells. In conclusion, our results reveal that RUNX2 promotes HCC cell migration and invasion by MMP9-mediated pathway, and potentially serves as a new prognostic biomarker and in therapeutic strategies for HCC.

[Analysis of the treatment of unexpected gallbladder cancer].

OBJECTIVE: To investigate the secondary operation methods and the effects on the prognosis of unexpected gallbladder cancer (UGC). METHODS: A retrospective analysis on the clinical data was made for 41 patients who underwent extended radical excision from June 1995 to December 2002. Among the patients, 12 were male, 29 were female. The average age was 51 years old. The 41 patients had undergone gallbladder excision because of cholecystitis complicated lithiasis of gallbladder (32 cases), polypi of gallbladder or adenoma (9 cases). Postoperative pathology showed that 32 cases were adenocarcinoma of gallbladder, 6 cases were squamous carcinoma, 3 cases were squamous adenocarcinoma. Six cases were on the stage of Nevin I, 16 on Nevin II, 17 on Nevin III, 2 on Nevin IV. The second operation was performed after 6-30 d of the first operation. The second operation chose the improved method of Glenn excision of carcinoma of gallbladder. RESULTS: On the second operation, 14 cases were with lymphatic metastasis, 14 with gallbladder metastasis, 6 with bile duct metastasis, 2 with pancreas metastasis. Fourteen cases were on the stage of Nevin IV, 9 on Nevin V, none on Nevin I, II and III. After the second operation, 1 year survival rate was 100% (41 cases); The three-year survival rate was 53.8% (22 cases); The five-year survival rate was 17.5% (7 cases). CONCLUSION: Extended radical excision is one of the most important methods for the treatment of UGC.

Increased expression of miR-21 predicts poor prognosis in patients with hepatocellular carcinoma.

BACKGROUND: MicroRNAs (miRNAs) play critical roles in hepatocellular carcinoma (HCC) development and progression. Aberrant miR-21 expression has been reported in several cancers. However, the clinical significance of miR-21 in human HCC is still unclear. METHODS: A total of 112 patients with primary HCC who underwent a curative liver resection were included in this retrospective study. The differentially expressed amount of the miR-21 was validated by quantitative real-time PCR (qRT-PCR). Survival rate was analyzed by log-rank test, and survival curves were plotted according to Kaplan-Meier. Multivariate analysis of the prognostic factors was performed with Cox regression model. RESULTS: As revealed by qRT-PCR analysis, miR-21 expression was significantly upregulated in HCC tissues when compared with adjacent non-tumor tissues (P<0.05). High miR-21 expression level was observed to be closely correlated with tumor differentiation, TNM stage and vein invasion (P<0.05). Patients who had high miR-21 expression had a shorter overall survival than patients who had low miR-21 expression (P<0.05). Moreover, multivariate analysis of the prognosis factors with a Cox proportional hazards model showed that high miR-21 expression was a significant independent predictor of poor survival in HCC (P<0.05). CONCLUSION: Our results suggested that increased expression of miR-21 was significantly correlated with tumor progression and could be a novel potential biomarker for HCC prognosis.

P2X7 blockade attenuates mouse liver fibrosis.

P2X7 is important in inflammation and tissue injury. The aim of the present study was to investigate the effect of P2X7 inhibition, using a specific inhibitor (A438079) to prevent the development of liver injury and fibrosis in a mouse model of liver fibrosis. The mouse liver fibrosis model was induced by carbon tetrachloride (CCl4). Mice received subcutaneous administration of vehicle (saline/olive oil), CCl4 or subcutaneous CCl4 and A438079. The pro­inflammatory and pro­fibrotic factors were determined by western blot analysis. The biochemistry, histopathology, collagen deposition and nuclear factor­κB (NF­κB) activity were also analyzed. Chronic CCl4 treatment resulted in liver injury and collagen accumulation. The expression levels of P2X7, pro­inflammatory and pro­fibrotic mediators, and the activity of NF­κB were markedly increased. Treatment with A438079 significantly inhibited CCl4­induced P2X7 expression, and attenuated CCl4­induced liver injury and the inflammatory response. P2X7 blockade also significantly reduced the formation of collagen in the liver and the expression of α-smooth muscle actin and transforming growth factor­ß1. This study demonstrated that P2X7 inhibition attenuated liver injury and fibrosis in a mouse model. Thus, P2X7 is a potential novel therapeutic target for liver injury and fibrosis.

Expression of long non-coding RNA LOC285194 and its prognostic significance in human pancreatic ductal adenocarcinoma.

INTRODUCTION: Dysregulation of long non-coding RNAs (lncRNAs) plays critical roles in tumor progression. lncRNA LOC285194 was previously shown to be correlated with aggressive clinicopathological features and poor prognosis in several cancers. The aim of this study was to investigate relationship between LOC285194 expression and clinical outcomes in human pancreatic ductal adenocarcinoma (PDAC). METHODS: Quantitative real-time PCR (qRT-PCR) assay was performed to detect the expression of lncRNA LOC285194 in human PDAC cells and tissue samples. The association of LOC285194 expression with clinicopathologic features was analyzed. Kaplan-Meier analyses were used to assess survival of patients. Univariate and multivariate analyses were performed using the Cox proportional hazards model to analyze the prognostic significance of LOC285194 expression. RESULTS: Our data showed that the relative level of LOC285194 in PDAC cells was significantly lower than that in normal human pancreatic duct epithelial cell line. Also, the expression of LOC285194 in PDAC tissues was significantly lower than that in adjacent non-tumor tissues. By statistical analyses, low LOC285194 expression was observed to be closely correlated with clinical stage, lymphnode metastasis and liver metastasis. Kaplan-Meier survival analysis revealed that patients with low LOC285194 expression had a poor overall survival compared with the high LOC285194 group (P < 0.05). Univariate and multivariate analyses showed that low LOC285194 expression was an independent poor prognostic factor for PDAC patients. CONCLUSIONS: Our data provided the first evidence that reduced LOC285194 in PDAC tissues was correlated with tumor progression, and lncRNA LOC285194 might be a potential molecular biomarker for predicting the prognosis of patients.

Effects of AFP gene silencing on apoptosis and proliferation of a hepatocellular carcinoma cell line.

Alpha fetoprotein (AFP) is an oncoembryonal protein that is highly expressed in the majority of hepatocellular carcinomas. Previous studies have shown that AFP may be involved in multiple cell growth regulating, differentiating, and immunosuppressive activities. We investigated the effects of AFP gene silencing by siRNA on apoptosis and proliferation of hepatocellular carcinoma cell line EGHC-9901, which highly expresses AFP and may serve as an ideal model for investigation of AFP functions. siRNA expressing plasmid targeting the AFP gene was first established and subsequently transfected into hepatocellular carcinoma cell line EGHC-9901; cells were then divided into three groups: siRNA-afp, transfected with AFP-siRNA; siRNA-beta-actin, transfected with siRNA-beta-actin as the positive group; and vector control, transfected with empty vector as the blank control group. After G418 positive clone selection for a couple of weeks, Western blot and RT (reverse transcription)-PCR assay demonstrated that AFP expression was almost completely inhibited by siRNA-afp, which indicates that siRNA expressing plasmid targeting the AFP gene has been successfully established. Furthermore, MTT (methyl thiazolyl tetrazelium) assay showed that cells transfected with siRNA-afp proliferated at a significantly lower speed than the other two groups and flat plate clone formation assay also witnessed less clones with diameters of more than 75 µm in siRNA-afp immunofluorescence indicating that the apoptosis rate of cells transfected with siRNA-afp was significantly higher than the other two groups. Furthermore, flow cytometry manifested approximately 20% more cells of siRNA-afp within G1 phase than those of the negative group, indicating that inhibition of AFP expression may cause G1 phase arrest. Finally, Western blot and RT-PCR assay demonstrated that siRNA-afp induced a higher expression of caspase-3 than the other two groups whereas there was no difference in expression of caspase-8, caspase-9, and Bcl-2 between the three groups.