Yugang Wen

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.

MicroRNA-20a-5p promotes colorectal cancer invasion and metastasis by downregulating Smad4

Background Tumor metastasis is one of the leading causes of poor prognosis for colorectal cancer (CRC) patients. Loss of Smad4 contributes to aggression process in many human cancers. However, the underlying precise mechanism of aberrant Smad4 expression in CRC development is still little known. Results miR-20a-5p negatively regulated Smad4 by directly targeting its 3′UTR in human colorectal cancer cells. miR-20a-5p not only promoted CRC cells aggression capacity in vitro and liver metastasis in vivo, but also promoted the epithelial-to-mesenchymal transition process by downregulating Smad4 expression. In addition, tissue microarray analysis obtained from 544 CRC patients’ clinical characters showed that miR-20a-5p was upregulated in human CRC tissues, especially in the tissues with metastasis. High level of miR-20a-5p predicted poor prognosis in CRC patients. Methods Five miRNA target prediction programs were applied to identify potential miRNA(s) that target(s) Smad4 in CRC. Luciferase reporter assay and transfection technique were used to validate the correlation between miR-20a-5p and Smad4 in CRC. Wound healing, transwell and tumorigenesis assays were used to explore the function of miR-20a-5p and Smad4 in CRC progression in vitro and in vivo. The association between miR-20a-5p expression and the prognosis of CRC patients was evaluated by Kaplan–Meier analysis and multivariate cox proportional hazard analyses based on tissue microarray data. Conclusions miR-20a-5p, as an onco-miRNA, promoted the invasion and metastasis ability by suppressing Smad4 expression in CRC cells, and high miR-20a-5p predicted poor prognosis for CRC patients, providing a novel and promising therapeutic target in human colorectal cancer.

Decreased Expression of RASSF6 Is a Novel Independent Prognostic Marker of a Worse Outcome in Gastric Cancer Patients after Curative Surgery

BackgroundOur previous study observed that the expression of RASSF6, a member of the Ras-association domain family, was down-regulated in gastric cancer cells. The present study further investigated the clinical significance of RASSF6 in gastric cancer.MethodsUsing real-time PCR, Western blot analysis, tissue microarray (TMA), and immunohistochemical staining, we evaluated RASSF6 mRNA and protein levels in tumor tissues and in the paired adjacent normal mucosa from patients with gastric cancers at different stages.ResultsRASSF6 mRNA and protein levels were decreased in gastric cancer tissues compared with the adjacent normal mucosa. Immunohistochemical detection of RASSF6 in a TMA that contained 264 paired specimens showed that a decreased cytoplasmic RASSF6 expression was significantly associated with the extent of cancer invasion, lymph node metastasis, distant metastasis, tumor histological grade, advanced clinical stage, and Ki-67 proliferative index. Moreover, RASSF6 expression in metastatic lymph nodes was lower than in the paired primary tumors. Patients with RASSF6-negative tumors had extremely higher disease recurrence rates and poorer survival than patients with RASSF6-positive tumors even after radical surgery. Stratification analysis revealed RASSF6 as an independent predictor for tumor recurrence in patients with gastric cancers irrespective of tumor stage.ConclusionsRASSF6 might contribute to the progression of gastric carcinogenesis and may function as a novel independent prognostic marker for the prediction of the recurrence of cancer in patients after curative operations.

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.

Identification and validation of Kallikrein-ralated peptidase 11 as a novel prognostic marker of gastric cancer based on immunohistochemistry †‡

It is important to identify and validate the differentially expressed genes in gastric cancer to screen diagnostic and/or prognostic tumor markers.

Overexpression of minichromosome maintenance 2 predicts poor prognosis in patients with gastric cancer

We examined the expression of minichromosome maintenance 2 (MCM2) in gastric cancer and adjacent normal tissues and estimated the possible value of MCM2 as a novel prognostic marker. Using real-time PCR, western blotting and immunohistochemistry, we examined the expression of MCM2 in gastric carcinoma and paired normal gastric mucosa. Statistical analysis of the expression of MCM2 mRNA and protein in gastric cancer and normal tissues was performed to evaluate the relationship between MCM2 expression and clinicopathological characteristics in gastric cancer. The expression of MCM2 mRNA and protein in gastric carcinomas was significantly higher compared to that in normal gastric mucosa (P=0.04). Immunohistochemistry analysis showed that MCM2 expression was significantly up-regulated in tumor and metastastic lymph node tissues compared with the corresponding non-cancerous mucosa (P<0.05). Positive expression of MCM2 was significantly associated with patient age, T category and the presence of lymph node metastasis (P<0.05). There were no differences between MCM2 expression and gender, tumor size, tumor location, M category, International Union Against Cancer (UICC) stage, vessel invasion and tumor differentiation. Patients with negative tumor MCM2 expression displayed a better survival time than those with positive MCM2 expression (P<0.05). Survival analysis showed that positive MCM2 expression (P<0.05), T stage (P<0.05) and N stage (P<0.05) were independent prognostic factors for disease-free survival (DFS) and overall survival (OS). Our data suggest that MCM2 could serve as a novel prognostic biomarker in gastric carcinoma.

miR-4775 promotes colorectal cancer invasion and metastasis via the Smad7/TGFβ-mediated epithelial to mesenchymal transition

BackgroundDespite advancements in the diagnosis and treatment of colorectal cancer (CRC), many patients die because of tumor metastasis or recurrence. Therefore, identifying new prognostic markers and elucidating the mechanisms of CRC metastasis and recurrence will help to improve the prognosis of the disease. As dysregulation of microRNAs is strongly related to cancer progression, the aim of this study was to identify the role of miR-4775 in the prognosis of CRC patients and the underling mechanisms involved in CRC progression.MethodsqPCR and in situ hybridization were used to evaluate the expression of miR-4775 in 544 pairs of paraffin-embedded normal and CRC tissues. Kaplan–Meier analysis with the log-rank test was used for survival analyses. Immunohistochemical staining was applied to investigate the expression of miR-4775-regulated Smad7/TGFβ pathway-associated markers. In vitro and in vivo invasion and metastasis assays were used to explore the function of miR-4775 in the progression of CRC.ResultsmiR-4775 was identified as a high-risk factor for CRC metastasis and recurrence, with high levels predicting poor survival among the 544 studied CRC patients. Furthermore, high miR-4775 expression promoted the invasion of CRC cells as well as metastasis and the epithelial to mesenchymal transition (EMT) via Smad7-mediated activation of TGFβ signaling both in vitro and in vivo. Downregulating miR-4775 or overexpressing Smad7 reversed the tumor-promoting roles of miR-4775/Smad7/TGFβ in vitro and in vivo.ConclusionmiR-4775 promotes CRC metastasis and recurrence in a Smad7/TGFβ signaling-dependent manner, providing a new therapeutic target for inhibiting the metastasis or recurrence of the disease.

Overexpression of HOXA10 promotes gastric cancer cells proliferation and HOXA10+/CD44+ is potential prognostic biomarker for gastric cancer

Gastric cancer (GC) is a malignant cancer with poor prognosis. This study aims to investigate the roles of homeobox A10 (HOXA10) in GC and the correlations between HOXA10/CD44 expression and GC prognosis. Based on qRT-PCR and Western Blot analyses in 50 pairs of fresh GC samples and adjacent normal samples, it is identified that HOXA10 was significantly up-regulated in GC tissues at mRNA and protein levels. Cell proliferation, migration, and invasion were enhanced in GC cells with overexpressed HOXA10, while inhibited in cells with silenced HOXA10. Through IPA software, HOXA10 was predicted to interact with CD44 via MSN, which was preliminarily confirmed by using Western Blot. Through immunohistochemistry and tissue microarray (N=264), it is found that HOXA10 expression was significantly correlated with tumor size (P=0.011) and CD44 expression (P<0.001), while CD44 expression was significantly correlated with tumor size (P<0.001), depth of tumor invasion (P<0.001), lymph node metastasis (P<0.001), distant metastasis (P=0.001), UICC stage (P<0.001), histological differentiation (P<0.001), and HOXA10 expression (P<0.001). Additionally, the over-all survival and disease-free survival of HOXA10(+)/CD44(+) patients were dramatically decreased in comparison with that of HOXA10(+)/CD44(-), HOXA10(-)/CD44(+), or HOXA10(-)/CD44(-) patients (P<0.001), suggesting that the combinatory expression of HOXA10 and CD44 was correlated with poor GC prognosis. In conclusion, HOXA10 and CD44 might play roles in GC tumorigenesis, metastasis, and invasion. HOXA10(+)/CD44(+) expression might serve as a prognostic biomarker for GC, which needs more studies to validate.

Downregulation of homeobox gene Barx2 increases gastric cancer proliferation and metastasis and predicts poor patient outcomes

Barx2 is a Bar family homeodomain transcription factor shown to play a critical role in cell adhesion and cytoskeleton remodeling, key processes in carcinogenesis and metastasis. Using quantitative real-time PCR, Western blotting, and immunohistochemistry, we found that Barx2 is expressed at lower levels in human gastric cancer (GC) tissues than in adjacent normal mucosa. In a multivariate analysis, Barx2 expression emerged as an independent prognostic factor for disease-free and overall survival. Kaplan-Meier survival analysis showed a trend toward even shorter overall survival in the patient group with Barx2-negative tumors, independent of advanced UICC stage and tumor relapse. Using in vitro and in vivo assays, we demonstrated that under normal conditions Barx2 inhibited GC cell proliferation and invasiveness through inhibition of the Wnt/β-catenin signaling pathway. These findings indicate that reduction or loss of Barx2 dis-inhibits GC cell proliferation and invasion, and that reduction in Barx2 could serve as an independent prognostic biomarker for poor outcome in GC patients.

Down-regulation of Barx2 predicts poor survival in colorectal cancer

Human BarH-like homeobox 2 (Barx2), a homeodomain factor of the Bar family, has an important role in controlling the expression of cell adhesion molecules and has been reported in an increasing array of tumor types except colorectal cancer (CRC). The purpose of the current study was to characterize the expression of Barx2 and assess the clinical significance of Barx2 in CRC. First, we analyzed the expression of Barx2 in two independent public datasets from Oncomine. Subsequently, we evaluated Barx2 mRNA and protein expression by quantitative real-time PCR and western blotting, respectively. It was determined that Barx2 expression was lower in tumor tissues than in adjacent non-tumorous colorectal tissues of CRC patients, consistent with results from the public datasets. Subsequently, a tissue microarray containing 196 CRC specimens was evaluated for Barx2 expression by immunohistochemical staining. It was found that low expression of Barx2 significantly correlated with TNM stage, AJCC stage, differentiation, and relapse in patients with CRC. Patients with lower levels of Barx2 expression showed reduced disease-free survival and overall survival. Furthermore, a trend toward shorter overall survival in the patient group with Barx2-negative tumors independent of advanced AJCC stage and poor differentiation was determined by Kaplan-Meier survival analysis. Based on univariate and multivariate analyses, Barx2 expression was an independent prognostic factor for determining CRC prognosis. Taken together, low Barx2 expression was associated with the progression of CRC and could serve as a potential independent prognostic biomarker for patients with CRC.