Low expression of c-Myc protein predicts poor outcomes in patients with hepatocellular carcinoma after resection.

BACKGROUND: Embryonic Liver Fodrin (ELF) is an adaptor protein of transforming growth factor (TGF-ß) signaling cascade. Disruption of ELF results in mislocalization of Smad3 and Smad4, leading to compromised TGF-ß signaling. c-Myc is an important oncogenic transcription factor, and the disruption of TGF-ß signaling promotes c-Myc-induced hepatocellular carcinoma (HCC) carcinogenesis. However, the prognostic significance of c-Myc in HCC is less understood METHODS: The expression of c-Myc protein and mRNA were measured by immunohistochemistry (IHC) and qRT- PCR, respectively. IHC was performed to detect TGF-ß1 and ELF expression in HCC tissues. Their relationship with clinicopathological factors and overall survival (OS) and disease free survival (DFS) were examined. RESULTS: The expression of c-Myc protein and mRNA in HCC tissues were significantly higher in HCC area than those in normal liver tissues. However, the expression were low compared with those adjacent to HCC area. c-Myc protein was independently predictive of DFS and OS, and it was negatively correlated with tumor size (P = 0.031), tumor number (P = 0.038), and recurrence (P = 0.001). Low c-Myc expression was associated with short-term recurrence and poor prognosis. The predictive value of c-Myc combined with TGF-ß1 or/and ELF was higher than that of any other single marker. Low c-Myc, high TGF-ß1 or/and low ELF expression was associated with the worst DFS and OS. CONCLUSIONS: Low expression of c-Myc protein predicts poor outcomes in patients with HCC with hepatectomy. The combination of the expression of c-Myc, TGF-ß1, and ELF can be used to accurately predict outcomes of patients with HCC.

MicroRNA-129-5p inhibits hepatocellular carcinoma cell metastasis and invasion via targeting ETS1.

MiR-129-5p is deregulated in various human cancers and has been associated with hepatocellular carcinoma (HCC) progression. However, the underlying mechanisms of miR-129-5p involvement in the development and progression of HCC and the effects of miR-129-5p deregulation on the clinical characteristics observed in HCC patients remain poorly understood. We therefore investigated the correlation between low miR-129-5p expression and vascular invasion, intrahepatic metastasis, and poor patient survival. Ectopic restoration of miR-129-5p expression in HCC cells suppressed cellular migration and invasion and the expression of v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS1), while inhibition of endogenous miR-129-5p caused an increase in these parameters. We identified the ETS1 gene as a novel direct target of miR-129-5p. SiRNA-mediated ETS1 knockdown rescued the effects of anti-miR-129-5p inhibitor in HCC cell lines, while the effects of miR-129-5p overexpression were partially phenocopied in the knockdown model. In addition, miR-129-5p levels inversely correlated with those of ETS1 in HCC cells and tissues. Taken together, our findings indicate an important role for miR-129-5p in the molecular etiology of invasive HCC and suggest that miR-129-5p could have potential therapeutic applications in HCC.

The prognostic value of combined TGF-ß1 and ELF in hepatocellular carcinoma.

BACKGROUND: Tumor suppression of Transforming Growth Factor (TGF-ß) signaling pathway requires an adaptor protein, Embryonic Liver Fodrin (ELF). Disruption of ELF expression resulted in miscolocalization of Smad3 and Smad4, then disruption of TGF-ß signaling. However, the prognostic significance of ELF for hepatocellular carcinoma (HCC) hasn't been clarified. This study aimed to investigate whether measuring both TGF-ß1 and ELF provides a more powerful predictor for HCC prognosis than either marker alone. METHODS: TGF-ß1 and ELF protein were detected by immunohistochemistry. The relationship between TGF-ß1/ELF expression and patients' clinicopathologic factors was analyzed. The association between TGF-ß1/ELF expression and disease-free survival and overall survival was analyzed by Kaplan-Meier curves, the log-rank test, and Multivariate Cox regression analyses. RESULTS: The expression of TGF-ß1 in HCC tissues was significantly higher than that in normal liver tissues. Conversely, the expression of ELF in HCC tissues declined markedly. ELF protein was correlated with HBsAg, tumor size, tumor number, TNM and recurrence. Data also indicated a significant negative correlation between ELF and TGF-ß1. Patients with high TGF-ß1 expression or/and low ELF expression appeared to have a poor postoperative disease-free survival and overall survival compared with those with low TGF-ß1 expression or/and high ELF expression. Furthermore, the predictive range of ELF combined with TGF-ß1 was more sensitive than that of either one alone. CONCLUSIONS: TGF-ß1 and ELF protein are potential and reliable biomarkers for predicting prognosis in HCC patients after hepatic resection. Our current study has demonstrated that the prognostic accuracy of testing can be enhanced by their combination.

Transducin ß-like 1 X-linked receptor 1 suppresses cisplatin sensitivity in nasopharyngeal carcinoma via activation of NF-κB pathway.

BACKGROUND: Transducin ß-like 1 X-linked receptor 1 (TBL1XR1) is an important transcriptional cofactor involved in the regulation of many signaling pathways, and is associated with carcinogenesis and tumor progression. However, the precise role of TBL1XR1 in these processes is not well understood. METHODS: We detected the expression of TBL1XR1 protein and mRNA in nasopharyngeal carcinoma (NPC) cell lines and biopsies by western blotting, real-time PCR and immunohistochemical staining (IHC). Overexpression of TBL1XR1 in NPC enhanced chemoresistance to cisplatin using two NPC cell lines in vitro and in vivo. RESULTS: TBL1XR1 was upregulated in NPC cell lines and clinical samples. The expression of TBL1XR1 was correlated with several clinicopathological factors including clinical stage, T classification, N classification and patient survival. Univariate and multivariate analysis revealed that TBL1XR1 was an independent prognostic factor for patient survival. In vitro and in vivo studies demonstrated that TBL1XR1 high expression induced resistance to cisplatin-induced apoptosis in NPC cells. Furthermore, we found that TBL1XR1 activated the NF-κB pathway and promoted transcription of genes downstream of NF-κB, especially anti-apoptotic genes. CONCLUSIONS: Upregulation of TBL1XR1 induces NPC cells resistance to cisplatin by activating the NF-κB pathway, and correlates with poor overall survival of NPC patients. TBL1XR1 has a pivotal role in NPC and could be a valuable prognostic factor as well as a novel biomarker for tailoring appropriate therapeutic regimes.

Down-regulation of Gli-1 inhibits hepatocellular carcinoma cell migration and invasion.

Glioma-associated oncogene homolog-1 (Gli-1) is considered a marker of Hedgehog pathway activation and is associated with the progression of several cancers. We have previously reported that Gli-1 was correlated with invasion and metastasis in hepatocellular carcinoma (HCC). However, the exact roles and mechanisms of Gli-1 in HCC invasion are unclear. In this study, we found that small interfering RNA mediated down-regulation of Gli-1 expression significantly suppressed adhesion, motility, migration, and invasion of both SMMC-7721 and SK-Hep1 cells. Furthermore, down-regulation of Gli-1 significantly reduced expressions and activities of both matrix metalloproteinase (MMP)-2 and MMP-9. In addition, we found that down-regulation of Gli-1 resulted in up-regulation of E-cadherin and concomitant down-regulation of Snail and Vimentin, consistent with inhibition of epithelial-mesenchymal transition (EMT). Taken together, our results suggest that down-regulation of Gli-1 suppresses HCC cell migration and invasion likely through inhibiting expressions and activations of MMP-2, 9 and blocking EMT.

miR-145 suppresses cell invasion in hepatocellular carcinoma cells: miR-145 targets ADAM17.

AIM: miR-145 is a candidate tumor suppressor miRNA. However, it is unknown whether miR-145 is involved in the invasion of hepatocellular carcinoma (HCC). Therefore, we aimed to explore the effect and mechanism of miR-145 in the control of HCC cell invasion. METHODS: HCC cell invasion was evaluated by transwell assays after transfection with pre-miR-145 or anti-miR-145. A luciferase reporter assay was used to determine whether a disintegrin and metalloprotease 17 (ADAM17) were a target of miR-145. The levels of miR-145 and ADAM17 mRNA were detected by a real-time polymerase chain reaction assay, and the level of ADAM17 protein was measured by western blot analysis. Pearson's correlation test was used to assess the correlation between ADAM17 mRNA expression and miR-145 expression in 20 HCC tissue samples. RESULTS: miR-145 was significantly downregulated in HCC tissues and cell lines. The loss of miR-145 expression was associated with the tumor-node-metastasis stage, vascular invasion and intrahepatic metastasis. The overexpression of miR-145 was able to suppress tumor MHCC-97H cell invasion, whereas the knockdown of miR-145 expression induced SMMC-7721 cell invasion. We demonstrated that miR-145 bound directly to the 3'-untranslated region of ADAM17 and inhibited the expression of ADAM17. The knockdown of ADAM17 in SMMC-7721 cells could partially reverse the effects of anti-miR-145. miR-145 expression was inversely associated with ADAM17 expression in 20 HCC tissue specimens. CONCLUSION: Our findings indicate that miR-145 could inhibit HCC cell invasion by regulating the expression of ADAM17.

Pathological characteristics of liver allografts from donation after brain death followed by cardiac death in pigs.

Donation after brain death followed by circulatory death (DBCD) is a unique practice in China. The aim of this study was to define the pathologic characteristics of DBCD liver allografts in a porcine model. Fifteen male pigs (25-30 kg) were allocated randomly into donation after brain death (DBD), donation after circulatory death (DCD) and DBCD groups. Brain death was induced by augmenting intracranial pressure. Circulatory death was induced by withdrawal of life support in DBCD group and by venous injection of 40 mL 10% potassium chloride in DCD group. The donor livers were perfused in situ and kept in cold storage for 4 h. Liver tissue and common bile duct samples were collected for hematoxylin and eosin staining, TUNEL testing and electron microscopic examination. Spot necrosis was found in hepatic parenchyma of DBD and DBCD groups, while a large area of necrosis was shown in DCD group. The apoptosis rate of hepatocytes in DBD [(0.56±0.30)%] and DBCD [(0.50 ± 0.11)%] groups was much lower than that in DCD group [(3.78±0.33)%] (P<0.05). And there was no significant difference between DBD group and DBCD group (P>0.05)). The structures of bile duct were intact in both DBD and DBCD groups, while the biliary epithelium was totally damaged in DCD group. Under electron microscope, the DBD hepatocytes were characterized by intact cell membrane, well-organized endoplasmic reticulum, mild mitochondria edema and abundant glycogens. Broken cell membrane, mild inflammatory cell infiltration and sinusoidal epithelium edema, as well as reduced glycogen volume, were found in the DBCD hepatocytes. The DCD hepatocytes had more profound cell organelle injury and much less glycogen storage. In conclusion, the preservation injury of DBCD liver allografts is much less severe than that of un-controlled DCD, but more severe than that of DBD liver allografts under electron microscope, which might reflect post-transplant liver function to some extent.

Sonic hedgehog signaling pathway induces cell migration and invasion through focal adhesion kinase/AKT signaling-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9 in liver cancer.

The aberrant activation of sonic hedgehog (SHH) pathway contributes to initiation and progression of various malignancies. However, the roles and underlying mechanisms of SHH signaling pathway in invasion and metastasis of liver cancer have not been well understood. In this study, we found that SHH signaling was activated and correlated with invasion and metastasis in hepatocellular carcinoma (HCC). Enhanced SHH signaling by recombinant human SHH N-terminal peptide (rSHH-N) promoted hepatoma cell adhesion, migration and invasion, whereas blockade of SHH signaling with SHH neutralizing antibody or cyclopamine suppressed hepatoma cell adhesion, migration and invasion. Furthermore, matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities were upregulated and downregulated by rSHH-N and SHH signaling inhibitor, respectively. The rSHH-N-mediated hepatoma cell migration and invasion was blocked by MMP-specific inhibitors or neutralizing antibodies to MMP-2 and MMP-9. In addition, phosphorylations of AKT and focal adhesion kinase (FAK) were increased and decreased by rSHH-N and SHH signaling inhibitor, respectively. Further investigations showed that activation of AKT and FAK were required for rSHH-N-mediated upregulation of MMP-2 and MMP-9, cell migration and invasion. Finally, we found that SHH protein expression was positively correlated with phosphorylatd FAK Tyr397, phosphorylatd AKT Ser473, MMP-2 and MMP-9 protein expressions in HCC samples. Taken together, our findings suggest that SHH pathway induces cell migration and invasion through FAK/AKT signaling-mediated MMP-2 and MMP-9 production and activation in liver cancer.

Analysis of the genome-wide DNA methylation profile of side population cells in hepatocellular carcinoma.

BACKGROUND: DNA methylation plays an important role in maintaining pluripotency and regulating the differentiation of stem cells, but the DNA methylation profile of stem cells in hepatocellular carcinoma (HCC) remains unclear. AIMS: To investigate the genome-wide DNA methylation profile of side population (SP) cells of HCC, a special subpopulation of cells enriched with cancer stem cells, by DNA methylation microarray analysis and to analyze the functions and signal pathways of the aberrantly methylated genes in SP cells. METHODS: Side population cells were isolated from HCC cell lines Huh7 and PLC/PRF/5 using flow cytometry, and the tumorigenicity of these SP cells was assessed in NOD/SCID mice. The genome-wide DNA methylation status of SP cells and non-SP (NSP) cells was detected and compared by DNA methylation microarray analysis. Genes with differential methylation between SP and NSP cells were further analyzed for their functions and roles in related signaling pathways. RESULTS: Subcutaneous inoculation of 1 × 10(3) SP cells yielded tumors in 60 % NOD/SCID mice, whereas no tumor was developed after the inoculation of 1 × 10(6) NSP cells. Genome-wide DNA methylation microarray analysis showed that 72 and 181 genes were hypermethylated and hypomethylated, respectively, in both Huh7 and PLC/PRF/5 SP cells as compared with their corresponding NSP cells. Analyses of signaling pathways revealed that hypermethylated and hypomethylated genes were related to four and eight pathways, respectively. CONCLUSIONS: Hepatocellular carcinoma SP cells possessed a differential DNA methylation status compared with NSP cells, and the differentially methylated genes in SP cells were involved in 12 signaling pathways. Our results provide valuable clues for further investigations in elucidating the importance of epigenetic regulation in sustaining HCC SP cells and tumorigenesis.

Reduced expression of Krüppel-like factor 17 is related to tumor growth and poor prognosis in lung adenocarcinoma.

Krüppel-like factor 17 (KLF17), a new member of the Krüppel-like factors (KLFs), has been reported to be a negative regulator of epithelial-mesenchymal transition (EMT) and metastasis in breast cancer. However, the biological role and clinical significance of KLF17 in lung adenocarcinoma has been less clear. In the present study, we showed that KLF17 expression was decreased in lung adenocarcinoma. Reduced expression of KLF17 was correlated significantly with a short survival time in patients with lung adenocarcinoma (P<0.0001). Moreover, KLF17 expression was an independent prognostic indicator for patients with lung adenocarcinoma. KLF17 expression level was correlated with the tumor stage (P=0.016) and tumor size (P=0.001) in lung adenocarcinoma. Overexpression of KLF17 inhibited cell growth in A549 and PC-9 cell lines. In conclusion, it is possible that KLF17 inhibits tumor growth in lung adenocarcinoma. The reduced expression of KLF17 is a valuable prognostic indicator for patients with lung adenocarcinoma, and KLF17 could be a novel target for treatment of lung adenocarcinoma.

Proteomics-based identification of haptoglobin as a favourable serum biomarker for predicting long-term response to splenectomy in patients with primary immune thrombocytopenia.

BACKGROUND: Splenectomy is the most effective treatment for patients with primary immune thrombocytopenia (ITP) who fail to respond to steroid therapy. Thus far, there is no effective means to predict the long-term haematological response of the procedure. The purpose of this study was to identify serum biomarkers as predictors of long-term response based on a proteomics approach. METHODS: The serum samples of ITP patients were collected before splenectomy and seven days after surgery. After depletion of the abundant serum proteins, pooled preoperative serum samples from four responders to splenectomy, four nonresponders and four healthy controls were subjected to two-dimensional gel electrophoresis (2-DE). Nine protein spots with at least a five-fold alteration in expression between responders and nonresponders were all identified as haptoglobin (Hp) by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometer (MS) analysis. The validation of serum Hp expression was performed using enzyme-linked immunosorbent assays (ELISA) in thirty-seven responders, thirteen nonresponders and twenty-one healthy controls. RESULTS: The preoperative serum levels of Hp in the nonresponders (925.9 ± 293.5 µg/ml) were significantly lower than those in the responders (1417.4 ± 315.0 µg/ml, p <0.001) and the healthy controls (1409.1 ± 354.2 µg/ml, p <0.001), while there was no significant difference between the latter two groups. The postoperative serum levels of Hp in responders and nonresponders were (1414.1 ± 225.0 µg/ml) and (952.9 ± 202.4 µg/ml), respectively. There were no significant differences between the serum Hp levels before and after surgery in both responders and nonresponders (p>0.05). The preoperative serum levels of Hp did not significantly correlate with preoperative platelet count of the same blood samples (r = 0.244, p = 0.087), while it positively correlated with postoperative peak platelet count (r = 0.622, p < 0.001). The optimal cutoff value of preoperative serum Hp levels (1173.80 µg/ml) derived from the receiver operating characteristic (ROC) curve led to 78.4% sensitivity and 84.6% specificity. CONCLUSIONS: These results suggest that serum Hp levels may serve as a favourable predictor for the long-term response to splenectomy in ITP and may help to understand the pathophysiological differences between responders and nonresponders.

Clinical significance of decreased nidogen-2 expression in the tumor tissue and serum of patients with hepatocellular carcinoma.

BACKGROUND AND OBJECTIVES: Nidogen-2 is a ubiquitous component of basement membrane (BM), which is modified by tumor cells to facilitate tumor invasion. However, the expression and function of nidogen-2 in hepatocellular carcinoma (HCC) remains unknown at present. In this study, we sought to investigate the potential role of nidogen-2 in HCC. METHODS: Nidogen-2 expression in HCC tissues, cell lines, and serum was evaluated by immunohistochemistry, immunoassay, and real-time PCR assays. The regulation of nidogen-2 expression was investigated using doxycycline induction and small interfering RNA analyses. RESULTS: Nidogen-2 was significantly decreased in both HCC tissues and serum (P < 0.001). The decreased expression of nidogen-2 in HCC tissues was significantly correlated with tumor progression factors (P < 0.05). Inhibition of matrix metalloproteinase (MMP)-9 led to significantly upregulate nidogen-2 expression in vitro assays. Moreover, patients with HCC had lowest serum nidogen-2 levels compared with patients with benign liver diseases and normal volunteers. Furthermore, the receiver operating characteristic curve analysis revealed a good diagnostic performance of nidogen-2 for HCC. CONCLUSIONS: These findings suggest that decreased expression of nidogen-2 may have a potential pathogenetic role in the development of HCC and may also have potential diagnostic value for HCC.

miR-338-3p suppresses invasion of liver cancer cell by targeting smoothened.

MicroRNAs are involved in human carcinogenesis and cancer progression. Our previous study has shown that loss of miR-338-3p expression is associated with clinical aggressiveness of hepatocellular carcinoma (HCC). However, the exact roles and mechanisms of miR-338-3p remain unknown in HCC. To determine whether and how miR-338-3p influences liver cancer cell invasion, we studied miR-338-3p in the liver cancer cell lines, and we found that miR-338-3p is down-regulated in treated cells. Forced expression of miR-338-3p in SK-HEP-1 cells suppressed cell migration and invasion, whereas inhibition of miR-338-3p in SMMC-7721 cells induced cell migration and invasion. Furthermore, smoothened (SMO) was identified as a direct target of miR-338-3p. Forced expression of miR-338-3p down-regulated SMO and matrix metalloproteinase (MMP)-9 expression, but inhibition of miR-338-3p up-regulated SMO and MMP9 expression. However, small interfering RNA targeted SMO reversed the effects induced by blockade of miR-338-3p. SMO and MMP9 were overexpressed and associated with invasion and metastasis in HCC tissues. These data indicate that miR-338-3p suppresses cell invasion by targeting the smoothened gene in liver cancer in vitro and miR-338-3p might be a novel potential strategy for liver cancer treatment.

Endoscopic stricturotomy in the treatment of anastomotic strictures in inflammatory bowel disease (IBD) and non-IBD patients.

BACKGROUNDS: Endoscopic stricturotomy (ESt) has been shown to be effective in treating inflammatory bowel disease (IBD)-associated anastomotic strictures. However, the outcome of ESt in benign, non-IBD conditions has not been described. The aim of this study was to evaluate the outcome of ESt in the management of IBD and non-IBD-associated strictures. METHODS: Data of all consecutive IBD and non-IBD patients with benign anastomotic strictures treated with ESt from 2009 to 2016 were extracted. The primary outcomes were surgery-free survival and procedure-related complications. RESULTS: A total of 49 IBD and 15 non-IBD patients were included in this study. The IBD group included 25 patients with Crohn's disease and 24 with ulcerative colitis and ileal pouches. Underlying diseases in the non-IBD group included colorectal cancer (n = 7), diverticulitis (n = 5), large bowel prolapse (n = 2), and constipation (n = 1). Immediate technical success was achieved in all patients in both groups. Bleeding complications occurred on five occasions (4.7% per procedure) in the IBD group, while no complication occurred in the non-IBD group (P = 0.20). Stricture improvement on follow-up endoscopy was found in 10 (20.4%) and 5 (33.3%) patients in the IBD and non-IBD groups, respectively (P = 0.32). Six (12.2%) patients in the IBD group and four (26.7%) patients in the non-IBD group eventually required stricture-related surgery (P = 0.23). IBD patients appeared to have a higher tendency for maintaining surgery-free after the procedure than non-IBD patients (P = 0.08). CONCLUSIONS: Endoscopic stricturotomy was shown to have comparable outcomes, though non-IBD patients seem to have a higher need for subsequent surgery but a lower complication rate than IBD patients.

Involvement of PI3K/PTEN/AKT/mTOR pathway in invasion and metastasis in hepatocellular carcinoma: Association with MMP-9.

AIM: To investigate the status of Phosphatidylinositol 3-kinase (PI3K)/PTEN/AKT/mammalian target of rapamycin (mTOR) pathway and its correlation with clinicopathological features and matrix metalloproteinase-2, -9 (MMP-2, 9) in human hepatocellular carcinoma (HCC). METHODS: PTEN, Phosphorylated AKT (p-AKT), Phosphorylated mTOR (p-mTOR), MMP-2, MMP-9 and Ki-67 expression levels were evaluated by immunohistochemistry on tissue microarrays containing 200 HCCs with paired adjacent non-cancerous liver tissues. PTEN, MMP-2 and MMP-9 mRNA levels were determined by real-time RT-PCR in 36 HCCs. The relationships between PI3K/PTEN/AKT/mTOR pathway and clinicopathological factors and MMP-2, 9 were analyzed in HCC. RESULTS: In HCC, PTEN loss and overexpression of p-AKT and p-mTOR were associated with tumor grade, intrahepatic metastasis, vascular invasion, TNM stage and high Ki-67 labeling index (P < 0.05). PTEN loss was correlated with p-AKT, p-mTOR and MMP-9 overexpression. Furthermore, PTEN and MMP-2, 9 mRNA levels were down-regulated and up-regulated in HCC compared with paired non-cancerous liver tissues, respectively (P < 0.01). PTEN, MMP-2 and MMP-9 mRNA levels were correlated with tumor stage and metastasis. There was an inverse correlation between PTEN and MMP-9 mRNA expression. However, PI3K/PTEN/AKT/mTOR pathway was not correlated with MMP-2. CONCLUSIONS: PI3K/PTEN/AKT/mTOR pathway, which is activated in HCC, is involved in invasion and metastasis through up-regulating MMP-9 in HCC.

Bead-based microarray analysis of microRNA expression in hepatocellular carcinoma: miR-338 is downregulated.

AIM: Recent studies have underlined causative links between microRNA (miRNA) deregulation and cancer development. However, the relevance of abnormally expressed miRNA to tumor biology has not been well understood in hepatocellular carcinoma (HCC). METHODS: A bead-based miRNA expression profiling method was performed on 20 pairs of surgically removed HCC and adjacent non-tumorous tissue (NT). Special miR-338 downregulations and miR-338 associated with clinical characteristics was validated in an extended samples set of 36 paired HCC and adjacent non-tumorous liver tissues by real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: Out of our bead-based microarray data, 12 upregulated and 19 downregulated miRNA were found to be associated with HCC. Further characterization of miRNA-338, in which 20 pairs of the samples were clustered clearly into two groups according to expression of miR-338, revealed that the level of miR-338 expression can be associated with clinical aggressiveness, such as, tumor size, tumor-node-metastasis stage, vascular invasion and intrahepatic metastasis. These results were validated by real-time RT-PCR analysis. CONCLUSION: Our study suggests that miRNA expression could have relevance to the clinical behavior of HCC and that the bead-based miRNA expression profiling method might be a suitable system to assay miRNA expression in large-scale diagnostic trails.

Bone marrow-derived CXCR4-overexpressing MSCs display increased homing to intestine and ameliorate colitis-associated tumorigenesis in mice.

BACKGROUND AND OBJECTIVE: Increasing interest has developed in the therapeutic potential of bone marrow-derived mesenchymal stem cells (MSCs) for the treatment of inflammatory bowel disease (IBD) and IBD-induced cancer. However, whether MSCs have the ability to suppress or promote tumor development remains controversial. The stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis is well known to play a critical role in the homing of MSCs. In this study, we aimed to evaluate the role of CXCR4-overexpressing MSCs on the tumorigenesis of IBD. METHODS: MSCs were transduced with lentiviral vector carrying either CXCR4 or green fluorescent protein (GFP). Chemotaxis and invasion assays were used to detect CXCR4 expression. A mouse model of colitis-associated tumorigenesis was established using azoxymethane and dextran sulfate sodium (DSS). The mice were divided into three groups and then injected with phosphate buffer saline (PBS), MSC-GFP or MSC-CXCR4. RESULTS: Compared with the mice injected with MSC-GFP, the mice injected with MSC-CXCR4 showed relieved weight loss, longer colons, lower tumor numbers and decreased tumor load; expression of pro-inflammatory cytokines decreased, and signal transducer and activator of transcription 3 (STAT3) phosphorylation level in colon tissue was down-regulated. CONCLUSION: CXCR4-overexpressing MSCs exhibited effective anti-tumor function, which may be associated with enhanced homing to inflamed intestinal tissues.

Engulfment and Cell Motility Protein 1 Protects Against DSS-induced Colonic Injury in Mice via Rac1 Activation.

BACKGROUND AND AIMS: Mucosal healing is an emerging therapeutic goal that could result in clinical remission of inflammatory bowel disease [IBD]. We sought to determine the role of engulfment and cell motility protein 1 [ELMO1] in wound healing in vitro and in vivo and to investigate the underlying pathways. METHODS: RNA transcriptome sequencing was performed to detect the expression profiles of mRNA between inflamed tissues and corresponding non-inflamed tissues of IBD patients, followed by Gene Expression Omnibus [GEO] datasets and western blot analysis. The effects of ELMO1 overexpression or knockdown on cell migration and proliferation were determined. The dependence of these effects on Rac1 was assessed using a Rac1 inhibitor [NSC23766] and a Rac1 pull-down assay. We identified the underlying pathways involved by Gene Ontology [GO] analysis. A dextran sulphate sodium [DSS]-induced colitis model was established to evaluate the role of ELMO1 in colonic mucosal healing. RESULTS: ELMO1 was upregulated in inflamed tissues compared with corresponding non-inflamed tissues. ELMO1 overexpression increased cell migration in a Rac1-dependent manner. Depletion of ELMO1, or NSC23766 administration, abolished this effect. GO analysis revealed that ELMO1 overexpression preferentially affected pathways involved in cytoskeletal regulation and wound healing, which was demonstrated by enhanced F-actin staining and increased numbers of extending lamellipodia in cells overexpressing ELMO1. In DSS-induced colitis, systemic delivery of pSin-EF2-ELMO1-Pur attenuated colonic inflammation and promoted recovery from colonic injury. The protective effect of ELMO1 was dependent on Rac1 activation. CONCLUSIONS: ELMO1 protects against DSS-induced colonic injury in mice through its effect on epithelial migration via Rac1 activation.

Expression and prognostic impact of PRL-3 in lymph node metastasis of gastric cancer: its molecular mechanism was investigated using artificial microRNA interference.

High PRL-3 expression had been reported to have close association with lymph node metastasis (LNM) of gastric cancer. However, the prognostic significance of highly expressing PRL-3 in LNM of human gastric cancer and the role in the metastasis remain unclear. Our study examined PRL-3 expression both in the LNM (n = 107) and in the primary lesion (n = 137) of gastric cancer, and compared the overall survival rates. RNA interference, induced by recombinant plasmid pcDNA.rPRL3-miR expressing artificial PRL-3 miRNA, was employed to knockdown PRL-3 expression in human SGC7901 gastric cancer cells. Invasion assay and migration assay in vitro were conducted to determine the role of PRL-3 in the metastasis. The role of PRL-3 in the proliferation of SGC7901 cells and tumor growth were also determined. We observed that high PRL-3 expression was more frequently detected in the LNM than in the matched primary lesion (72.9 vs. 47.7%, p < 0.001). Furthermore, the overall survival rate of the patients with high expression of PRL-3 in the LNM was significantly less than those with moderate/low expression (p = 0.003). Importantly, knockdown of PRL-3 can significantly reduce both invasion and migration potencies of SGC7901 cells (p < 0.001), and significantly suppressed the proliferation of SGC7901 cells and slowed down the tumor growth (p < 0.001). It was concluded that high expression of PRL-3 in the LNM had a negative impact on the prognosis of the patients, and plays important roles in LNM of gastric cancer and the tumor growth, which can be a potential therapeutic target and a prognostic factor.

Establishment and characteristics of two syngeneic human osteosarcoma cell lines from primary tumor and skip metastases.

AIM: To characterize and compare the different biological behaviors of 2 novel human osteosarcoma cell lines, Zos and Zos-M, established respectively from the primary tumor and the skip metastasis of an osteosarcoma patient. METHODS: In vitro studies included morphological observations, karyotype analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay, and cell sensitivity to chemotherapeutic drugs. Subcutaneous and intravenous inoculations into nude mice were carried out to study the tumorigenicity and the metastatic potential. RT-PCR was performed to assess the expression of the osteoblastic markers and some metastasis-related genes. RESULTS: Both cell lines remained stable for more than 100 passages in vitro without interruption. The RT-PCR examination indicated that they retained the molecular characteristics of an osteoblastic lineage. The karyotype analysis displayed aneuploidy and various structural abnormalities. Both cell lines are tumorigenic; Zos-M differs from Zos by the former's ability to develop lung metastasis after intravenous injection. The comparison of the expression patterns of some metastasis-related genes revealed that the decreased expression of cadherin-11 in Zos-M may correlate with a high potential of metastases. Moreover, both cell lines are less sensitive to the current chemotherapy protocols. CONCLUSION: The establishment of osteosarcoma cell lines, Zos and Zos-M, and related animal models provide a useful resource for studying the aggressive behavior of osteosarcoma and will be helpful for screening effective treatment strategies.