[Expression and survival prediction of microRNA-155 in hepatocellular carcinoma after liver transplantation].

OBJECTIVE: To explore the expression of microRNA-155 in hepatocellular carcinoma (HCC) and its contribution to recurrence and prognosis of HCC after liver transplantation (LT). METHODS: The expression levels of microRNA-155 in 100 HCC samples were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Kaplan-Meier and Cox proportional regression analyses were utilized to determine the association of microRNA-155 expression with patient survivals. RESULTS: The expression levels of microRNA-155 were higher in primary HCC patients with post-LT recurrence (n = 45, mean relative level = 14.94) than those with non-recurrence (n = 55, mean relative level = 4.70) (P = 0.001) and correlated with micro-vascular invasion of HCC tissue samples (P = 0.001). The patients with a higher expression of microRNA-155 had significantly worse recurrence-free survival (RFS: (21.5 ± 3.2) months, log rank P < 0.001) and overall survival (OS: (29.3 ± 3.2) months, log rank P < 0.001) than those with a lower expression of microRNA-155 (RFS: (50.8 ± 3.2) months; OS: (54.6 ± 3.5) months). Multivariate analysis revealed that a high expression of miR-155 was an independent prognostic predictor. CONCLUSION: MicroRNA-155 is over-expressed in primary HCC with tumor recurrence and may serve as a novel biomarker for tumor recurrence and survival of HCC patients after LT. The detection of microRNA-155 is of clinical significance in HCC.

Identification of recurrence-related microRNAs in hepatocellular carcinoma following liver transplantation.

Tumor recurrence-related microRNAs (miRNAs) in hepatocellular carcinoma (HCC) following orthotopic liver transplantation (OLT) are not clear yet. This study was designed to determine whether altered miRNA expression is associated with HCC recurrence and prognosis following OLT. 18 miRNAs, including 6 up-regulated and 12 down-regulated miRNAs were identified by microarray in primary HCC samples of patients who had developed HCC recurrence (n = 5) compared to those with non-recurrence (n = 5) following OLT by using p < 0.05 as cutoff value. The six most significantly altered miRNAs (fold change ≥ 2: miR-19a, miR-886-5p, miR-126, miR-223, miR-24 and miR-147) were further confirmed by qRT-PCR in the remaining 105 HCC samples. In receiver-operating characteristic curve analysis, this six miRNAs were of high sensitivity and specificity in predicting HCC recurrence. Using Cox regression and risk score analysis, we built a six-miRNA signature based on their qRT-PCR readings for the prediction of outcome of HCC following OLT. Kaplan-Meier and Cox proportional regression revealed this six-miRNA signature was a significant independent predictor of overall survival (log-rank p = 0.020) and recurrence-free survival (log-rank p < 0.001). Finally, the data were further reconfirmed in an independent cohort of 50 patients from another transplant center. In addition, bioinformatics Gene Ontology and pathway analysis were also performed to better understand the critical roles of these miRNAs in HCC recurrence. Our study, in addition to suggesting a different miRNA expression pattern between HCC samples of patients with recurrence and those with non-recurrence, proposes that this six-miRNA signature may serve as biomarker for prognosis of HCC patients following OLT.

miR-203 expression predicts outcome after liver transplantation for hepatocellular carcinoma in cirrhotic liver.

Many recent studies have shown the utility of microRNAs (miRs) as cancer-related biomarkers. The aim of the present study was to evaluate the correlation between miR-203 expression and prognosis of patients with hepatocellular carcinoma (HCC) and cirrhosis after liver transplantation (LT). Sixty-six HCC samples from patients who had undergone LT were examined for miR-203 expression using quantitative reverse transcription-polymerase chain reaction. The data were correlated with clinicopathological parameters and prognosis. Patient survival was analyzed by the Kaplan-Meier method and log-rank test. Cox regression was used for multivariate analysis of prognostic factors. We found that miR-203 expression was low in tumor tissues of patients (n = 16) with post-LT HCC recurrence in comparison with those in patients with non-recurrence (n = 50) (P = 0.003). Patients with higher miR-203 expression had significantly better recurrence-free survival (RFS) and overall survival (OS) (P = 0.016 for RFS; P = 0.014 for OS). Multivariate analysis revealed that high-miR-203 expression was an independent predictor of good prognosis (HR 0.202, P = 0.006 for RFS; HR 0.332, P = 0.013 for OS). Our results suggest that miR-203 could be a novel prognostic marker in HCC patients who have undergone LT and might also be a potential therapeutic target.

Up-regulation of microRNA-155 promotes cancer cell invasion and predicts poor survival of hepatocellular carcinoma following liver transplantation.

PURPOSE: MicroRNAs play important roles in cancer development, progression, and metastasis. The aim of this study was to determine whether altered microRNA-155 expression is associated with hepatocellular carcinoma (HCC) recurrence and prognosis following orthotopic liver transplantation (OLT). METHODS: Tissue specimens from 100 HCC patients following OLT were recruited. MicroRNA-155 expression levels were detected using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Kaplan-Meier and Cox proportional regression analyses were utilized to determine the association of microRNA-155 expression with survival of patients. MicroRNA-155 expression levels of two HCC cell lines (HepG2 and SMMC-7721) and normal liver tissue were quantified using qRT-PCR. The potential function of miR-155 on invasiveness was evaluated in the above HCC cell lines. RESULTS: We found that microRNA-155 expression levels were high in tumor tissues in patients with post-OLT HCC recurrence (n = 45) compared with those in patients with non-recurrence (n = 55) (P = 0.001) and correlated with micro-vascular invasion of HCC tissue samples (P = 0.001). Patients with higher miR-155 expression had significantly poorer recurrence-free survival (RFS, log rank P < 0.001) and overall survival (OS, log rank P < 0.001). Multivariate analysis revealed that high miR-155 expression was an independent predictor of poor prognosis (HR 2.748, P = 0.001 for RFS; HR 5.752, P < 0.001 for OS). In addition, the invasiveness of HCC cells was significantly increased by higher microRNA-155 expression. CONCLUSIONS: MicroRNA-155 is a candidate oncogenic microRNA and plays an important role in promoting HCC cells invasion. Our findings suggest that microRNA-155 may serve as a novel biomarker for tumor recurrence and survival of HCC patients following OLT.

[Relationship of apoptotic receptor expression in normal human peripheral blood lymphocytes to cell apoptosis].

The aim of this study was to detect the expression and cell cycle specificity of Fas, TNFRI and TNFRII in human peripheral blood lymphocytes (PBL), and to study the potential role of Fas, TNFRIand TNFRII in cell cycle specific apoptosis. The improved double-parameter flow cytometry was used to detect the expressions of Fas, TNFRI and TNFRII and cell cycle specificity in PBL which were incubated for 24 hours in the presence or absence of phytohaematoagglutinin (PHA) respectively. Apoptosis induced by IgM type anti-Fas and TNF-alpha was detected by API method. The results showed that compared with PBL treated in the absence of PHA in G(0) phase, the ratio of Fas, TNFRI and TNFRII expressions in PHA-stimulated PBL entering cell cycle increased (35.55 +/- 6.63)%, (30.63 +/- 2.66)%, (26.62 +/- 5.14)% respectively (P < 0.01), and mainly appeared at G(1)-phase; no apoptosis was induced by anti-Fas and TNF-alpha in G(0)-phase PBL cultured in the absence of PHA. On the contrary, the apoptosis was induced by anti-Fas and TNF-alpha in PBL which entered cell cycle after stimulation with PHA and mainly initiated at G(1)-Phase. It is concluded that there is evident dose-effect relationship between apoptotic receptor and receptor-mediated apoptosis. Moreover, the cell cycle specificity of receptor-mediated apoptosis is correlated with the cell cycle specific expressions of apoptotic receptor. The induction of apoptosis by apoptotic factors (anti-Fas and TNF-alpha) depends on whether cell entering cell cycle or not.

[DNA damage and apoptosis of human airway epithelial cell lines caused by cigarette smoke extract].

BACKGROUND & OBJECTIVE: Cigarette smoke exposure has been reported to induce DNA damage in a variety of cell types. This study was to investigate DNA damage and apoptosis in normal human bronchial epithelial cell line NHBE and lung carcinoma cell line SPC-A1 caused by cigarette smoke extract. METHODS: NHBE and SPC-A1 cells were incubated with different concentrations of cigarette smoke extract. Cell viability was evaluated by MTT assay. Fluorescence-labeled anti-histone gamma-H2AX polyclonal antibody was used to detect DNA double-strand breaks (DSBS) in chromatin. DNA damage was analyzed by flow cytometry. The expression of gamma-H2AX was detected by Western blot. Cigarette smoke extract-induced cell apoptosis was detected by sub G1 peak method and annexin V-FITC/propidium iodide (PI) staining assay. Cell morphology of DNA damage and apoptosis was observed by confocal laser microscopy. RESULTS: MTT assay results showed that cigarette smoke extract decreased the viability of NHBE and SPC-A1 cells in time-and concentration-dependent manners. Cigarette smoke extract induced DSBS in NHBE cells and SPC-A1 cells, and led to H2AX phosphorylation (denoted gamma-H2AX) in time-and concentration-dependent manners. The maximal value of gamma-H2AX was seen about 4 h after the treatment, and the value decreased subsequently, but did not reduce to a normal level. Cell apoptosis appeared about 12 h after DNA damage. Cigarette smoke extract also initiated the accumulation of gamma-H2AX in these cells, and cell apoptosis morphology was observed 4 h later. CONCLUSION: Cigarette smoke extract can induce DSBS and apoptosis in NHBE and SPC-A1 cells in time-and concentration-dependent manners.

[Inducement of tumor cell autophagy and cell cycle analysis].

BACKGROUND & OBJECTIVE: Autophagy is the main phenomenon of type II programmed cell death which is also named as autophagic cell death, and autophagy has a close relationship with autophagic cell death. The relationship of apoptosis and cell cycle has been explored deeply, but little is known about the relationship of autophagic cell death and cell cycle. This study was to observe the correlation of autophagy induced by different methods to cell cycle. METHODS: Exponentially growing HeLa and SW480 cells, and peripheral blood lymphocytes (PBLs) from healthy donors, with or without 48 h stimulation of phytohemagglutinin (PHA), were treated with Hanks' solution (to produce starvation) or vincristine. Confocal laser microscope and transmission electron microscope (TEM) were used to detect autophagy; flow cytometry (FCM) was innovatively used to detect the cell cycle of autophagic cells with dipl-parameters of microtubule-associated protein 1 light chain 3 (MAP1-LC3-II)/PI. RESULTS: Autophagy of HeLa and SW480 cells induced by starvation or vincristine was observed in G1, S, and G2/M phases and increased along with the inducement time; no autophagy was observed in unstimulated PBLs. The positive rate of LC3-II, indicating the occurrence of autophagy, was lower than 2.62% when induced by starvation in Hanks' solution for 48 h, or 6.16% when induced by vincristine for 48 h. After PBLs were stimulated into cell cycle by PHA, autophagy was markedly detected 2 h after the indicated inducements. CONCLUSIONS: MAP1-LC3-II/DNA dipl-parameter analysis by FCM is a convenient and reliable method for simultaneously analyzing autophagy and cell cycle. Autophagy could be induced when cells are in cell cycle, while the cells in G0 phase are insensitive to the inducers.