ALKBH5 suppresses autophagic flux via N6-methyladenosine demethylation of ZKSCAN3 mRNA in acute pancreatitis.

BACKGROUND: Increasing evidence has demonstrated that N6-methyladenosine (m6A) RNA modification plays an essential role in a wide range of pathological conditions. Impaired autophagy is a critical hallmark of acute pancreatitis (AP). AIM: To explore the role of the m6A modification of ZKSCAN3 in the regulation of autophagy in AP. METHODS: The AP mouse cell model was established by cerulein-treated mouse pancreatic acinar cells (MPC-83), and the results were confirmed by the levels of amylase and inflammatory factors. Autophagy activity was evaluated by specific identification of the autophagy-related microstructure and the expression of autophagy-related genes. ZKSCAN3 and ALKBH5 were knocked down to study the function in AP. A m6A RNA binding protein immunoprecipitation assay was used to study how the m6A modification of ZKSCAN3 mRNA is regulated by ALKBH. RESULTS: The increased expression of amylase and inflammatory factors in the supernatant and the accumulation of autophagic vacuoles verified that the AP mouse cell model was established. The downregulation of LAMP2 and upregulation of LC3-II/I and SQSTM1 demonstrated that autophagy was impaired in AP. The expression of ZKSCAN3 was upregulated in AP. Inhibition of ZKSCAN3 increased the expression of LAMP2 and decreased the expression of the inflammatory factors, LC3-II/I and SQSTM1. Furthermore, ALKBH5 was upregulated in AP. Knockdown of ALKBH5 downregulated ZKSCAN3 expression and restored decreased autophagic flux in AP. Notably, the bioinformatic analysis revealed 23 potential m6A modification sites on ZKSCAN3 mRNA. The m6A modification of ZKSCAN3 mRNA was significantly decreased in AP. Knockdown of ALKBH5 increased the modification of ZKSCAN3 mRNA, which confirmed that ALKBH5 upregulated ZKSCAN3 expression in a m6A-dependent manner. CONCLUSION: ALKBH5 inhibits autophagic flux through m6A demethylation of ZKSCAN3 mRNA in AP, thereby aggravating the severity of the disease.

Early laparotomy and timely reconstruction for patients with abdominal electrical injury: Five Case Reports and Literature Review.

INTRODUCTION: High-tension electricity can cause devastating injuries that may result in abdominal wall loss, visceral damage, and sometimes major threat to life. The visceral organ may be exposed after debridement and require flap cover, but the tensile strength of abdominal wall may be lack even if flap transplanted. METHODS: From April 2007 through May 2015, 5 patients with severe abdominal electrical injury were treated at our hospital. Exploratory laparotomy was performed based on their clinical manifestations and debridement findings of abdominal wall at early stage, and decision regarding technique for reconstruction of abdominal wall was based on an assessment of the location and extent of the defect. Medical records were reviewed for these data. RESULTS: Clinical evaluation and debridement findings of the abdomen revealed 4 patients with suspicious visceral damage. Laparotomy was performed in 4 cases, and revealed obvious lesion in 3 cases, including segmental necrosis of small intestine, partial necrosis of diaphragm, left liver and gastric wall, and greater omentum. Five patients underwent abdominal wall reconstruction using island retrograde latissimus dorsi myocutaneous flap or free/island composite anterolateral thigh myocutaneous flap. All flaps survived, abdominal bulging occurred in 3 cases after follow-up of 12 to 36 months. CONCLUSIONS: The clinical manifestations and wound features of abdomen collectively suggest a possible requirement of laparotomy for severe abdominal electrical burns. Retrograde latissimus dorsi myocutaneous flap or composite anterolateral thigh myocutaneous flap is an effective option for reconstruction of abdominal wall loss, the long-term complication of abdominal bulging, however, remains a significant clinical challenge.

Endocan expression correlated with poor survival in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death in China. We aimed to first present the expression of endocan in HCC tissue and its correlation with the clinicopathological features and overall survival of patients with HCC after curative hepatectomy. Immunohistochemical detection of endocan, CD34, and vascular endothelial growth factor (VEGF) were performed on samples from 100 patients with HCC. Endocan protein was expressed in endothelium of HCC tissue in all specimens, but was not expressed in endothelium of pericarcinomatous liver tissue and normal liver tissue. Microvessel density (MVD) denoted by endocan (endocan-MVD) in HCC was correlated with microscopic venous invasion and VEGF expression (P < 0.05). Survival analysis showed that overall survival of patients was inversely associated with endocan-MVD (P < 0.01). Multivariate analysis showed that endocan-MVD was an independent prognostic marker for overall survival of HCC (P < 0.01). In conclusion, endocan-MVD was a significant factor to predict the prognosis of HCC patients after curative hepatectomy.

Correlation between CD105 expression and postoperative recurrence and metastasis of hepatocellular carcinoma.

BACKGROUND: Angiogenesis is one of the mechanisms most critical to the postoperative recurrence and metastasis of hepatocellular carcinoma (HCC). Thus, finding the molecular markers associated with angiogenesis may help identify patients at increased risk for recurrence and metastasis of HCC. This study was designed to investigate whether CD105 or CD34 could serve as a valid prognostic marker in patients with HCC by determining if there is a correlation between CD105 or CD34 expression and postoperative recurrence or metastasis. METHODS: Immunohistochemical staining for the CD105, CD34 and vascular endothelial growth factor (VEGF) antibodies was performed in 113 HCC tissue specimens containing paracarcinomatous tissue and in 14 normal liver tissue specimens. The quantitation of microvessels identified by anti-CD105 and anti-CD34 monoclonal antibodies and the semiquantitation of VEGF expression identified by anti-VEGF monoclonal antibody were analyzed in conjunction with the clinicopathological characteristics of the HCC and any available follow-up information about the patients from whom the specimens were obtained. RESULTS: CD105 was not expressed in the vascular endothelial cells of any normal liver tissue or paracarcinomatous liver tissue but was expressed in the vascular endothelial cells of all HCC tissue. In contrast, CD34 was expressed in the vascular endothelial cells of normal liver tissue, paracarcinomatous tissue, and HCC tissue in the following proportions of specimens: 86.7%, 93.8%, and 100%, respectively. The microvascular densities (MVDs) of HCC determined by using an anti-CD105 mAb (CD105-MVD) and an anti-CD34 mAb (CD34-MVD), were 71.7 +/- 8.3 (SD) and 106.3 +/- 10.4 (SD), respectively. There was a significant correlation between CD105-MVD and CD34-MVD (r = 0.248, P = 0.021). Although CD34-MVD was significantly correlated with VEGF expression (r = 0.243, P = 0.024), CD105-MVD was more closely correlated (r = 0.300, P= 0.005). The correlation between microscopic venous invasion and CD105-MVD, but not CD34-MVD, was also statistically significant (r = 0.254, P = 0.018). Univariate analysis showed that CD105-MVD was significantly correlated with the 2-year overall survival rate (P = 0.014); CD34-MVD was not (P = 0.601). Multivariate analysis confirmed that CD105-MVD was an independent prognostic factor and that CD34-MVD was not. CONCLUSION: The anti-CD105 mAb is an ideal instrument to quantify new microvessels in HCC as compared with anti-CD34 mAb. CD105-MVD as compared with CD34-MVD is relevant a significant and independent prognostic indicator for recurrence and metastasis in HCC patients.

[Risk factors for metastasis and recurrence of hepatocellular carcinoma at different stages].

OBJECTIVE: To analyze the risk factors for metastasis and recurrence of hepatocellular carcinoma (HCC) postoperatively. METHODS: Data of 270 cases of postoperative HCC were analyzed by SPSS software retrospectively. RESULTS: Out of the 270 cases, 162 got follow-up study and 136 showed metastasis and recurrence. Lots of risk factors induced the recurrence of HCC, such as AFP, tumor form, venous blood invasion, HBV infection, resection dimension and perioperative transfusion. There were different risk factors at different stages. CONCLUSION: The early recurrence of HCC may be mediated by macro- or micro-vessel blood invasion and metastasis, the late recurrence by multicentric carcinogenesis or introhepatic cacinoma de novo.

Clinical analysis of the risk factors for recurrence of HCC and its relationship with HBV.

AIM: To comprehend the risk factors of recurrence of hepatocellular carcinoma (HCC) and its relationship with the infection patterns of hepatitis B virus (HBV). METHODS: All materials of 270 cases of postoperative HCC were statistically analyzed by SPSS software. Recurrence and metastasis were classified into early (< or =2 years) and late phase (>2 years). Risk factors for recurrence and metastasis after surgery in each group were analyzed. RESULTS: Out of 270 cases of HCC, 162 cases were followed up in which recurrence and metastasis occurred in 136 cases. There were a lot of risk factors related to recurrence and metastasis of HCC; risk factors contributing to early phase recurrence were serum AFP level, vascular invasion, incisal margin and operative transfusion, gross tumor classification and number of intrahepatic node to late phase recurrence. The HBV infective rate of recurrent HCC was 94.1%, in which "HBsAg, HBeAb, HbcAb" positive pattern reached 45.6%. The proportion of HBV infection in solitary large hepatocellular carcinoma (SLHCC) evidently decreased compared to nodular hepatocellular carcinoma (NHCC) (P<0.05). CONCLUSION: The early and late recurrence and metastasis after hepatectomy of HCC were associated with different risk factors. The early recurrence may be mediated by vascular invasion and remnant lesion, the late recurrence by tumor's clinical pathology propert, as multicentric carcinogenesis or intrahepatic carcinoma de novo. HBV replication takes a great role in this process. From this study, we found that SLHCC has more satisfactory neoplasm biological behavior than NHCC.

Role of AFP mRNA expression in peripheral blood as a predictor for postsurgical recurrence of hepatocellular carcinoma: a systematic review and meta-analysis.

AIM: To identify the role of alpha-fetoprotein (AFP) mRNA expression in peripheral blood one week after surgery as a predictor for recurrence of hepatocellular carcinoma (HCC). METHODS: Published studies fulfilling the selection criteria were identified by searching several databases online. After a methodology assessment using a quality scale designed by European Lung Cancer Working Party, data in each research were aggregated by means of meta-analysis. RESULTS: Altogether 368 cases were included in the 9 selected studies, which fulfilled the selection criteria. The quality scores ranged from 35% to 84% with a median score of 55%. The 'design' subscore had the lowest median value (38%). By aggregating the data, a high chi2 value (77.576) was presented. The fail-safe number was 136 and 64 for P = 0.05 and 0.01, respectively. CONCLUSION: AFP mRNA expression in peripheral blood 1 wk after surgery correlated with the recurrence of HCC and was a good predictor for tumor recurrence.

Expression of hypoxia-inducible factor 1alpha and vascular endothelial growth factor in hepatocellular carcinoma: Impact on neovascularization and survival.

AIM: To study the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC) and the impact on neovascularization and survival. METHODS: Expressions of HIF-1alpha, VEGF and microvessel density (MVD) are studied through immunohistochemistry in 36 cases of HCC and the corresponding paraneoplastic tissue and 6 cases of normal liver tissue. The relationship of the expressions of HIF-1alpha and VEGF with the clinicopathological data and survival are analyzed. RESULTS: The positive rate of VEGF in HCC was 32/36, which is significantly higher than that in paraneoplastic tissue and normal liver tissue (P<0.05). The expression of HIF-1alpha in HCC tissue is 24/36, also higher than that in paraneoplastic tissue and normal liver tissue (P<0.05). The expression of VEGF and HIF-1alpha in HCC with microscopic venous invasion is significantly higher than that in HCC without microscopic venous invasion (P<0.05). Spearman correlation analysis does not only show the expression of HIF-1alpha as correlated with the expression of VEGF (r(s) = 0.459, P<0.01), but it also shows the expression of HIF-1alpha and VEGF as correlated with MVD (r(s) = 0.412 and 0.336, respectively, P<0.05). The differences of the survival rates among VEGF positive group and VEGF negative group are significant (P<0.05), whereas the differences of the survival rates among the HIF-1alpha negative group and positive group are not significant (P>0.05). CONCLUSION: HIF-1alpha plays important roles in neovascularization in HCC possibly through regulation of VEGF transcription.

[Overexpression of the RhoC gene correlates with invasion and metastasis of hepatocellular carcinoma].

OBJECTIVE: Rho, a ras homologous gene, encodes a group of GTP-binding proteins. Our previous study suggested that one member of the Rho gene family, RhoC, was related to the progression of human hepatocellular carcinoma (HCC). This study is to elucidate correlation of Rho overexpression with invasion and metastasis of HCC. METHODS: The expression level of RhoC mRNA and protein in 25 cases of HCC and adjacent non-cancerous liver tissue was examined by RT-PCR and Western blot. Mutation of RhoC gene was examined by PCR-SSCP. RESULTS: The expression of RhoC mRNA and protein was found in all HCC and adjacent non-cancerous liver tissue. The expression level of RhoC mRNA and protein was significantly higher in tumor tissue than in adjacent non-cancerous liver tissue (1.8 +/- 1.1 vs 1.0 +/- 0.7; 33 992 +/- 10 384 vs 17 342 +/- 9998, P < 0.01). The degree of RhoC overexpression was even more marked in metastatic lesions than in primary tumors (P < 0.01). Overexpression of the rhoC gene was significantly correlated with such clinic-pathological findings as cell differentiation, portal vein invasion, number of primary tumor nodules and metastatic lesions (P < 0.05). Mutation of RhoC gene was found in none of the HCC specimens examined. CONCLUSION: Overexpression of RhoC gene may play an important role in carcinogenesis and progression of HCC.

Effects of PI3K and p42/p44 MAPK on overexpression of vascular endothelial growth factor in hepatocellular carcinoma.

AIM: To study the relationship between hypoxia or epidermal growth factor (EGF) and the overexpression of vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC) and the signal transduction pathway of the transcription of VEGF in hepatoma cells. METHODS: Cobalt chloride and recombinant human EGF were used to stimulate the hepatoma cell lines HepG(2). VEGF mRNA was detected by using of semi-quantitative polymerase chain reaction (RT-PCR). Specific inhibitors of phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen activated protein kinase (MAPK) were used to observe the effects of the two kinases on the regulation of the transcription of VEGF in hepatoma cells. RESULTS: The expression of VEGF mRNA in HepG(2) cells cultured in serum-free medium was 0.117. However, 100 mumol/L cobalt chloride for 24 h increased the expression of VEGF mRNA and VEGF mRNA increased gradually with the increase of the concentration and duration of cobalt chloride. Also, 25 ng/mL recombinant human EGF stimulated the expression of VEGF in HepG(2) cells and the expression increased with the increase of EGF concentration. 5 mumol/L LY294002 inhibited the expression of VEGF stimulated by cobalt chloride or recombinant human EGF and the inhibition decreased step by step with increase of the concentration of LY294002. But even 20 mumol/L LY294002 could not completely block the expression of VEGF. In contrast, PD98059 had no inhibitory effects on the transcription of VEGF stimulated by cobalt chloride or recombinant human EGF. CONCLUSION: The overexpression of VEGF in HCC could be promoted by hypoxia and EGF expression in HCC. The signal transduction pathway of VEGF transcription in HepG(2) cells may be through PI3K pathway, but not through p42/p44 MAPK pathway.

Metallothionein expression in hepatocellular carcinoma.

AIM: To investigate the expression of metallothioneins (MTs), which were recently thought to have close relationship with tumors, in human hepatocellular carcinoma. METHODS: Histological specimens of 35 cases of primary human hepatocellular carcinoma with para-neoplastic liver tissue and 5 cases of normal liver were stained for MTs with monoclonal mouse anti-MTs serum (E9) by the immunohistochemical ABC technique. RESULTS: MTs were stained in the 35 cases of HCC, including 6 cases negative (17.1 %), 23 weakly positive (65.7 %), and 6 strongly positive(17.1 %). But MTs were stained strongly positive in all the five cases of normal liver and 35 cases of para-neoplastic liver tissue. The differences of MTs expression between HCC and normal liver tissue or para-neoplastic liver tissue were highly significant (P<0.01). The rate of MTs expression in HCC grade I was 100 percent, higher than that in grade II(81 %) and grade III and IV (78 %). But the differences were not significant (P>0.05). No obvious correlations between MTs expression in HCC and tumor size, clinical stage or serum alpha fetoprotein concentration were found (P>0.05). CONCLUSION: Decrease of MTs expression in HCC may play a role in carcinogenesis of HCC. MTs are stained heterogenously in HCC. We can choose the anticancer agents according to the MTs concentration in HCC, which may improve the results of chemotherapy for HCC.