Junwei Fan

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.

Multidrug-resistant gram-negative bacterial infections after liver transplantation – Spectrum and risk factors

OBJECTIVES Gram-negative bacilli infections, especially multidrug-resistant gram-negative bacilli infections, are the leading cause of high mortality after liver transplantation. This study sought to investigate the type of infection, infection rate, pathogenic spectrum, antibiotic-resistance profile, risk factors, and epidemiology of multidrug-resistant gram-negative bacterial infection. METHODS A retrospective cohort study was conducted and data of 217 liver transplant patients receiving cadaveric livers between January 2007 and April 2010 were analyzed. Antibiotic susceptibility was determined by minimum inhibitory concentration test. Extended-spectrum and metallo-β-lactamase assays were used to analyze β-lactamase-produced isolates, and repetitive-sequence polymerase chain reaction was used to differentiate bacterium subspecies. RESULTS Sixty-seven isolates of multidrug-resistant gram-negative bacteria were isolated from 66 infected liver transplant patients. Stenotrophomonas maltophilia (100%, 8/8), Klebsiella pneumoniae (61.5%, 8/13), Enterobacter cloacae (75%, 3/4) and Escherichia coli (81.3%, 13/16) were the most common extended-spectrum β-lactamase-producing bacilli. Metallo-β-lactamase expressing isolates were identified as S. maltophilia (100%, 8/8), Pseudomonas aeruginosa (83.3%, 5/6), Acinetobacter baumannii (95%, 19/20). Significant independent risk factors for multidrug-resistant gram-negative infection were extended use of pre-transplant broad-spectrum antibiotics (OR 9.027, P=0.001) and prolonged (≧72h) endotracheal intubation (OR 3.537, P=0.033). CONCLUSIONS To reduce the risk of acquiring MDR gram-negative bacillus infections after liver transplant, control measures are required to limit the use of prophylactic antibiotic in preventing infection during liver transplant and to shorten endotracheal intubation time.

FOXM1 expression predicts the prognosis in hepatocellular carcinoma patients after orthotopic liver transplantation combined with the Milan criteria.

Molecular biomarker has been proposed to improve patient selection and post-transplant prognostication, but rare achievement has been made. In the present study, Forkhead box M1 (FOXM1) expression and its prognostic role have been investigated in hepatocellular carcinoma (HCC) treated by orthotopic liver transplantation (OLT). We found that the notably higher level of FOXM1 in tumors was associated with malignant pathological features of HCC and unfavorable outcome after OLT. The status of FOXM1 expression combined with the Milan criteria could make the prognostication more accurate and may be of particular interest for expanding the criteria in selecting transplant candidates.

Overexpression of Forkhead box M1 protein associates with aggressive tumor features and poor prognosis of hepatocellular carcinoma.

The aim of this study was to detect the expression of the Forkhead box M1 (FOXM1) protein in human hepatocellular carcinoma (HCC) and to associate FOXM1 expression with clinicopathological features of the patients, and predict the prognosis of patients with FOXM1 expression. Surgical tissue specimens from 151 HCC patients were subjected to a tissue microarray construction and immunohistochemistry analysis of FOXM1 and the proliferation marker proliferating cell nuclear antigen (PCNA). The data showed that the FOXM1 protein was expressed in 59.3% of the HCC tissues, which was significantly higher compared to that of the surrounding non-tumorous tissues (23.8%; P<0.001). Moreover, FOXM1 expression was positively correlated with the labeling index of PCNA (P<0.001) in HCC and with aggressive tumor phenotypes, such as larger tumor size, multiple tumors, bilobar involvement, poor tumor cell differentiation, advanced stage and macrovascular invasion (P<0.05). In addition, HCC patients with FOXM1-positive tumors had a poorer recurrence-free and overall survival after hepatectomy than those with FOXM1-negative tumors. Multivariate Cox regression analysis demonstrated that FOXM1 expression was an independent predictor of unfavorable outcome (P<0.05). The data from the current study suggest that FOXM1 may play an important role in HCC progression and could be further evaluated as a prognostic biomarker and potential therapeutic target.

Phospholipase C epsilon plays a suppressive role in incidence of colorectal cancer

In our previous study, we have found that PLCE1 was down-regulated in sporadic colorectal cancer. But the role of PLCE1 in the incidence of colorectal cancer is still not definite. Therefore, in order to validate whether PLCE1 displays a suppressive role, in this study, we examined the expression of PLCE1 in sporadic colorectal cancer with a larger sample size and the effect of PLCE1 overexpression on cancer cell malignant degree. The expression level of PLCE1 in 50 colorectal cancers with their pair-matched normal tissues was measured by RT-PCR, Western blot, and immunohistochemistry. The effect of PLCE1 overexpression on cancer cell malignant degree was measured by MTT assay, plate colony formation assay, soft agar colony formation assay, cell cycle and apoptosis analysis, and xenograft assay. We found that PLCE1 was down-regulated in 42% (21/50) of colorectal cancer tissues compared with pair-matched normal tissues, more frequent in the poor differentiation tumor in patients under 60. Overexpression of PLCE1 significantly inhibited the proliferation of colon cancer cells and degraded its malignant degree. These results suggest that PLCE1 may be involved in the development of sporadic colorectal cancer through its inhibitory effect on cell proliferation. PLCE1 exhibits a suppressive role in incidence of colorectal cancer.

An epidemiologic study of irritable bowel syndrome in adolescents and children in South China: a school-based study.

BACKGROUND To explore the prevalence of irritable bowel syndrome (IBS) among school-aged children in South China, and identify distribution characteristics and contributing factors. METHODS Primary, middle and high school students (n= 2013) were recruited from schools in Shanghai. Students completed two questionnaires, one for IBS in adolescents and children and the Screen for Child Anxiety Related Emotional Disorders (SCARED). RESULTS (1) The prevalence of IBS among students was 20.72% and the incidence increased with age. (2) Several physical factors and overuse of analgesics were significantly associated with IBS. (3) The prevalence of anxiety disorder was higher in older students, and in females vs. males. Students with IBS tended towards anxiety-related emotional disorders (38.14% vs. 18.96%). (4) IBS students scored higher in all SCARED categories. Within IBS students, those who frequently sought medical care reported higher scores in the somatization/panic category. CONCLUSION (1) Irritable bowel syndrome is a common disorder among adolescents in South China and prevalence increases with age. (2) Gastrointestinal infection, abuse of analgesics and psychological factors might be related to the incidence of IBS. (3) The tendency towards anxiety-related emotional disorders also increases with age, suggesting a possible correlation with IBS and underlining the importance of positive family and school environments. Although the prevalence of anxiety-related emotional disorders was higher in females than males, this trend was not correlated with the occurrence of IBS.

Biglycan expression correlates with aggressiveness and poor prognosis of gastric cancer

Biglycan, a member of the small leucine-rich proteoglycan family, has been implicated in the development and progression of human cancers. However, the clinical significance of biglycan expression in gastric cancer has not been determined. In the present study, biglycan mRNA and protein concentrations were analyzed using quantitative realtime reverse transcription polymerase chain reaction and Western blot in 69 gastric cancer and adjacent non-tumorous tissues, respectively. Biglycan expression was further assessed using immunohistochemistry in tissue microarrays that contained 264 cases of gastric cancer, and others containing normal or metastasized lymph node tumor tissues. Biglycan was upregulated at the transcriptional and translational levels and there was a correlation between the expression of biglycan mRNA and protein (P = 0.000, κ = 0.769). Over-expression of biglycan was strongly associated with lymph node metastasis, tumor (T) classification, metastasis (M) classification, vascular invasion and Union for International Cancer Control (UICC) stage. Patients with biglycan-positive tumors had a significantly higher disease recurrence rate and poorer survival than patients with biglycan-negative tumors after the radical surgery. Multivariate analysis revealed that biglycan expression is an independent prognostic indicator for survival of patients with gastric cancer. The data from the current study demonstrate that elevated expression of biglycan may play an important role in the development and progression of gastric cancer, and could be further evaluated as a biomarker for predication of a poor clinical outcome.

Gene-expression profiling in Chinese patients with colon cancer by coupling experimental and bioinformatic genomewide gene-expression analyses: identification and validation of IFITM3 as a biomarker of early colon carcinogenesis.

Expression microarrays are widely used for investigating the nature and extent of global gene‐expression changes in human cancer. Accurate genomewide gene‐expression profiles have not been conducted in colon tumor and normal colon tissue specimens obtained from Chinese patients.

Downregulation of metallothionein 1F, a putative oncosuppressor, by loss of heterozygosity in colon cancer tissue.

PURPOSE Downregulation of metallothionein (MT) genes has been reported in several tumors with discrepant results. This study is to investigate molecular mechanism of MT gene regulation in colon cancer which is characterized by tumor suppressor gene alterations. EXPERIMENTAL DESIGN Integral analysis of microarray data with loss of heterozygosity (LOH) information was employed. Quantitative real-time PCR and immunohistochemistry were used to validate MT isoform expression in colon cancer tissues and cell lines. The effects of MT1F expression on RKO cell survival and tumorigenesis was analyzed. Bisulphite sequencing PCR (BSP) and methylation-specific PCR were employed to detect the methylation status of the MT1F gene in colon cancer tissues and cell lines. DNA sequencing was used to examine the LOH at the MT1F locus. RESULTS MT1F, MT1G, MT1X, and MT2A gene expression was significantly downregulated in colon cancer tissue (p<0.05). Exogenous MT1F expression increased RKO cell apoptosis and inhibited RKO cell migration, invasion and adhesion as well as in vivo tumorigenicity. Downregulation of MT1F gene in majority of human colon tumor tissues is mainly through mechanism by loss of heterozygosity (p=0.001) while CpG island methylation of MT1F gene promoter region was only observed in poorly differentiated, MSI-positive RKO and LoVo colon cancer cell lines. CONCLUSIONS MT1F is a putative tumor suppressor gene in colon carcinogenesis that is downregulated mainly by LOH in colon cancer tissue. Further studies are required to elucidate a possible role for MT1F downregulation in colon cancer initiation and/or progression.

STYK1 promotes epithelial-mesenchymal transition and tumor metastasis in human hepatocellular carcinoma through MEK/ERK and PI3K/AKT signaling.

Serine/threonine/tyrosine kinase 1 (STYK1) is known to be involved in tumor progression. However, its molecular role and mechanism in hepatocellular carcinoma (HCC) remains unknown. We evaluated the effect of STYK1 expression in HCC tissues and investigated the underlying mechanisms associated with progression. HCC tissues expressed greater levels of STYK1 than paired non-tumor tissues. Patients with HCC expressing low levels of STYK1 showed both, greater disease-free (p < 0.0001) and overall (p = 0.0004) survival than those expressing high levels of STYK1. Decreased expression of STYK1 was significantly associated with decreased cell proliferation, reduced migratory capability, and reduced invasive capability. Overexpression of STYK1 was significantly associated with increased cell proliferation, migratory capability, and invasive capability in vitro, as well as increased volume of tumor, weight of tumor, and number of pulmonary metastases in vivo. Furthermore, STYK1’s mechanism of promoting cancer cell mobility and epithelial-mesenchymal transition (EMT) was found to be via the MEK/ERK and PI3K/AKT pathways, resulting in increased expression of mesenchymal protein markers: snail, fibronectin, and vimentin, and decreased E-cadherin expression. Our results suggest that STYK1 acts as an oncogene by inducing cell invasion and EMT via the MEK/ERK and PI3K/AKT signaling pathways and it therefore may be a potential therapeutic target in HCC.