Wenmei Li

Combination of hsa-miR-375 and hsa-miR-142-5p as a predictor for recurrence risk in gastric cancer patients following surgical resection

BACKGROUND Recurrence is a major factor leading to treatment failure and death in gastric cancer (GC) patients following surgical resection. Importantly, the prediction of recurrence is critical in improving clinical outcomes. We isolated a group of microRNAs (miRNAs) and evaluated their usefulness as prognostic markers for the recurrence of GC. PATIENTS AND METHODS A total of 65 GC patients were selected for systematic analysis, 29 patients with recurrence and 36 patients without recurrence. Firstly, miRNAs microarray and bioinformatics methods were used to characterize classifiers from primary tumor samples (n = 8). Following, we validated these predictors both in frozen fresh and paraffin-embedded tissue samples (n = 57) using quantitative PCR. RESULTS We have identified 17 differential miRNAs including 10 up-regulated and 7 down-regulated miRNAs in recurrence group. Using k-top scoring pairs (k-TSP) method, we further ascertained hsa-miR-375 and hsa-miR-142-5p as a classifier to recognize recurrence and nonrecurrence cases both in the training and test samples. Moreover, we validated this classifier in 34 frozen fresh tissues and 38 paraffin-embedded tissues with consistent sensitivity and specificity with training set; among them, 15 cases were matched. A high frequency recurrence and poor survival were observed in GC cases with high level of hsa-miR-375 and low level of hsa-miR-142-5p (P < 0.001). In addition, we evaluated that hsa-miR-375 and hsa-miR-142-5p were involved in regulating target genes in several oncogenic signal pathways, such as TP53, MAPK, Wnt and vascular endothelial growth factor. CONCLUSION Our results indicate that the combination of hsa-miR-375 and hsa-miR-142-5p as a predictor of disease progression has the potential to predict recurrence risk for GC patients.

Differential expression of Rab27A/B correlates with clinical outcome in hepatocellular carcinoma

AIM To investigate the association of Rab27A and Rab27B expression with clinicopathological characteristics and prognosis of hepatocellular carcinoma (HCC). METHODS We used reverse transcription polymerase chain reaction (RT-PCR), real-time PCR, and Western blotting to detect Rab27A and Rab27B mRNA and protein expression in 5 human HCC lines and the immortalized hepatic HL-7702 cell line. We further examined 148 primary HCC samples matched with adjacent normal tissue and 80 non-HCC specimens by immunohistochemistry to evaluate the correlation of Rab27A and Rab27B expression with clinicopathological features and prognosis. RESULTS Our data showed that Rab27A and Rab27B were differentially expressed in cell lines and primary HCC tumors. Rab27A mRNA and protein were detected in 67% (4/6) of human cell lines and 80% (4/5) of HCC cell lines, while Rab27B was found in 50% (3/6) of human lines and 40% (2/5) of HCC lines. Rab27A expression was higher in primary HCC (46.2%, 66/143) than in matched adjacent tissue (24.3%, 33/136, P < 0.001), whereas immunopositivity for Rab27B was lower in primary HCC (57.4%, 81/141) than in matched adjacent tissue (87.5%, 119/136, P < 0.001). Analysis of clinicopathological characteristics of 148 HCC specimens revealed significant correlations between Rab27A and Rab27B expression and tumor tumor-node-metastasis (TNM) classification (P = 0.046 and P = 0.027, respectively), and between strong Rab27A expression and tumor differentiation grade (P = 0.008). Survival analyses revealed that patients with Rab27A(+) or Rab27B(+) tumors had significantly reduced overall survival compared with that of patients with Rab27A(-) or Rab27B(-) tumors (P = 0.015 and P = 0.005, respectively). Risk analyses revealed that Rab27B(+) and TNM III-IV were independent poor prognosis factors associated with a 3.36- and 3.37-fold higher relative risk of death, respectively. CONCLUSION Rab27A and Rab27B expression were closely correlated with tumor progression and can be valuable prognostic indicators for HCC patients.

miR-29b suppresses tumor growth and metastasis in colorectal cancer via downregulating Tiam1 expression and inhibiting epithelial-mesenchymal transition.

Recently, the role of miR-29b in colorectal carcinoma (CRC) development appears to be controversial. Until now, the expression and function of miR-29b in CRC have not been clarified clearly. We showed that decreased expression of miR-29b usually occurred in CRC cell lines and tissue samples. Loss- and gain-of-function assays in vitro revealed suppressive effects of miR-29b on cell proliferation and migration. Endogenous overexpression of miR-29b was sufficient to suppress aggressive behavioral phenotypes in mice. Proteomic analysis showed that miR-29b involved in integrate several key biological processes. In addition, miR-29b mediated the inhibition of epithelial–mesenchymal transition (EMT) and the inactivation of mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT signal transduction pathway. Further studies found that T lymphoma invasion and metastasis 1 (Tiam1) was identified as a direct target of miR-29b. In contrast to the phenotypes induced by miR-29b restoration, Tiam1-induced cell proliferation and migration partly rescued miR-29b-mediated biological behaviors. Our results illustrated that miR-29b as a suppressor has a critical role in CRC progression, which suggests its potential role in the molecular therapy of patients with advanced CRC.

MiR‐23a in amplified 19p13.13 loci targets metallothionein 2A and promotes growth in gastric cancer cells

Copy number variation (CNV) and abnormal expression of microRNAs (miRNAs) always lead to deregulation of genes in cancer, including gastric cancer (GC). However, little is known about how CNVs affect the expression of miRNAs. By integrating CNV and miRNA profiles in the same samples, we identified eight miRNAs (miR‐1274a, miR‐196b, miR‐4298, miR‐181c, miR‐181d, miR‐23a, miR‐27a and miR‐24‐2) that were located in the amplified regions and were upregulated in GC. In particular, amplification of miR‐23a‐27a‐24‐2 cluster and miR‐181c‐181d cluster frequently occurred at 19p13.13 and were confirmed by genomic real‐time PCR in another 25 paired GC samples. Moreover, in situ hybridization (ISH) experiments represented that mature miR‐23a was increased in GCs (75.5%, 40/53) compared with matched normal tissues (28.6%, 14/49, P = 0.001). Knocking down of miR‐23a expression inhibited BGC823 cell growth in vitro and in vivo. In addition, the potential target genes of miR‐23a were investigated by integration of mRNA profile and miRNA TargetScan predictions, we found that upregulation of miR‐23a and downregulation of metallothionein 2A (MT2A) were detected simultaneously in 70% (7/10) of the miRNA and mRNA profiles. Furthermore, an inverse correlation between miR‐23a and MT2A expression was detected in GCs and normal tissues. Through combining luciferase assay, we confirmed that MT2A is a potential target of miR‐23a. In conclusion, these results suggest that integration of CNV‐miRNA‐mRNA profiling is a powerful tool for identifying molecular signatures, and that miR‐23a might play a role in regulating MT2A expression in GC. J. Cell. Biochem. 114: 2160–2169, 2013.

Identification and characterization of a novel p42.3 gene as tumor-specific and mitosis phase-dependent expression in gastric cancer.

Multiple genetic alterations are attributed to gastric cancer (GC); however, only a few critical genes have been identified so far. In this study, we isolated and characterized a novel gene p42.3, represented as tumor-specific and mitosis phase-dependent expression protein in GC cell line BGC823. Our data showed that the expression of p42.3 was cell cycle-dependent in GC cell lines. Moreover, p42.3 was specifically expressed in primary GC tissues but not in the matched normal mucosa of stomach, and this gene was expressed in diverse embryonic tissues. Furthermore, significant suppression of cell proliferation and tumorigenicity were detected and G2/M phase arrest was observed in cell line BGC823 depleted of p42.3 expression by RNAi technique, and we confirmed the expression changes of cyclin B1 and Chk2 following the silence of p42.3. Taken together, we cloned and characterized p42.3 gene that was specifically expressed in GC tumors but not in normal gastric mucosa, and the gene was associated with M-phase regulation. Moreover, p42.3 might be involved in cell proliferation and tumorigenesis; therefore, this gene might have potential applications in the diagnosis or treatment of GC.

Evaluation of Hepatic-Metastasis Risk of Colorectal Cancer upon the Protein Signature of PI3K/AKT Pathway

Liver is the most common organ of colorectal cancer (CRC) metastasis, and hepatic metastasis (HM) is regulated by complex protein network. Hence, we initiated a proteomic survey to seek interrelated multiplex markers related with HM. A total of 34 unique differential proteins were identified in the primary tumor tissues from 14 CRC patients with/without HM. A differential protein cluster, consisting of 17 proteins throughout PI3K/AKT pathway, was deduced and validated by Western blot. A three-protein signature elicited from the protein cluster, phosphorylated IkappaBalpha, TNFalpha and MFAP3L, was detected by immunohistochemistry on 105 pairs of CRC and normal samples. The positive protein signature was specifically correlated with HM (P < 0.001), and classified the HM risk of CRC patients with high sensitivity (92.85 +/- 4.87%) and specificity (94.94 +/- 2.5%). The high-risk group had significantly decreased overall survival (P < 0.001). Furthermore, RKO and HT29, two colon cancer cells with different expression status of the protein signature, were used to construct the nude mouse model of HM. And the HM occurrence of RKO cell (4/5) was dramatically higher than that of HT29 cell (1/5). Therefore, the protein signature derived from PI3K/AKT pathway is likely a promising multiplex biomarker for HM of CRC.

A novel androstenedione derivative induces ROS-mediated autophagy and attenuates drug resistance in osteosarcoma by inhibiting macrophage migration inhibitory factor (MIF).

Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.

Decreased fructose-1,6-bisphosphatase-2 expression promotes glycolysis and growth in gastric cancer cells.

BackgroundIncreasing evidence suggests that cancer is a metabolic disease. Here, we investigated the potential role of fructose-1,6-bisphosphatase-2 (FBP2), the enzyme that catalyses the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate in glucose metabolism, in gastric cancer (GC) development.ResultsOur data indicated that FBP2 was downregulated in GC tissues (86.2%, 100/116), and absent or low FBP2 expression in GC tissues was correlated with poor survival of GC patients (P = 0.019). Conversely, ectopic expression of FBP2 in GC cells activated AMP-activated protein kinase (AMPK) signalling, inhibited the Akt-mTOR pathway, suppressed glucose metabolism, enhanced apoptosis, and reduced cell proliferation. Bisulphite genomic sequencing (BGS) in gastric cancer cell lines revealed that the FBP2 promoter region was densely methylated, and treatment of GC cells with the demethylation reagent, 5-aza-2-deoxycytidine (5-Aza), led to an increase in FBP2 expression. Importantly, forced expression of FBP2 abrogated tumour formation of these GC cells in nude mice.ConclusionOur results indicate that FBP2 does negatively regulate cell growth, and reduced expression of FBP2 may contribute to carcinogenesis for GC. These findings suggest that restoration of FBP2 expression can be a promising strategy for the target therapy of GC.

Dominant expression of 85-kDa form of cortactin in colorectal cancer

Purpose: Cortactin is commonly expressed in several human cancers, which may alter their invasive or metastatic properties. Eighty five kilodalton form (p85) and 80-kDa form (p80) of cortactin are two separate bands in SDS-PAGE representing different conformational states. The objective of this study was to investigate cortactin expression in colorectal cancer (CRC). Experimental Design: Cortactin expression was studied in an eight paired laser capture microdissection (LCM) CRC tissues and matched non-cancerous epithelia by immunoblotting. The expression in 58 CRC and two cell lines, HCT8 and HCT116, was studied respectively by immunohistochemistry and confocal laser scanning immunofluorescence. Results: Dominant expression of p85 was identified in LCM-procured CRC tissues compared with equal intensity of p85 and p80 forms in non-cancerous tissues, while the amount of total cortactin was approximate. Immunohistochemistry analysis demonstrated that cortactin located in the cytoplasm of tumor cells and adjacent non-cancerous cells, and its expression was negatively correlated with TNM staging and lymphatic invasion status. However, the invasion fronts in 3 of 58 primary tumors and 28 of 39 available lymph node metastases were intensively stained. Further, immunofluorescence analysis showed that cortactin was distributed in cytoplasm and enriched in the front of the extending lamellipodia at adhering side of cultured cancer cells. Conclusions: Our results demonstrated the dominant expression of p85 form of cortactin in CRC for the first time. The enrichment of cortactin in the invasion front of some tumor cells and in the extending lamellipodia of cultured cancer cells suggests that cortactin may help cancer cell movement.

Identification of Matrix Metalloproteinase 11 as a Predictive Tumor Marker in Serum Based on Gene Expression Profiling

Purpose: Prognostic markers discovery is a strategy for early diagnosis and individualization therapy for human cancer. In this study, we focus to integrate different methods to identify specific biomarker and elucidate its clinical significance. Experimental Design: A powerful tool named Digital Gene Expression Display online was applied to isolate differentially expressed genes correlated with gastric cancer. Matrix metalloproteinase 11 (MMP11) was selected and confirmed at both mRNA and protein level in 10 cell lines, 123 cases of tumor tissues, and 305 cases of gastric cancer serum specimen by semiquantitative PCR, immunohistochemistry staining, and ELISA techniques, respectively. Results: Our data showed that overexpression of MMP11 at mRNA and protein level was consistently detected in cell lines and primary tumors compared with matched normal tissues. Importantly, serum MMP11 levels were also significantly elevated in gastric cancer patients compared with those of the control subjects (P < 0.001), and the positive expression was well correlated with metastasis in gastric cancer patients (P = 0.009). Furthermore, we have shown that overexpression of MMP11 was associated with the malignant proliferation of AGS cells. Conclusions: Combination of gene expression profiling and specific clinical resource is a promising approach to validate gene expression patterns associated with malignant phenotype. As a secreted protein, MMP11 may play an important role in carcinogenesis and has potential implication as a biomarker for the diagnosis and prognosis of human cancers including gastric cancer.