Jiantao Cui

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.

Gastrokine 1 induces senescence through p16/Rb pathway activation in gastric cancer cells

Background and aims Gastrokine 1 (GKN1) is a stomach-specific protein that is normally expressed in gastric mucosa but not in primary tumours and cell lines. Based on this evidence, it was presumed that GKN1 might play a role in gastric cancer development; however, its function and molecular mechanism are not clear. A systematic study was initiated that combined multiple approaches to define the molecular mechanism of GKN1 in gastric cancer cells. Method Proteomics, western blotting and immunohistochemistry were used to measure the expression level of GKN1. Western blotting combined with immunofluorescence was used to monitor the secretory process of this protein. Subsequently, the function and molecular mechanism of GKN1 was explored in vitro and in vivo. Results It was shown that GKN1 is an autocrine/paracrine protein and inhibits cell growth due to senescence, which resulted from activation of p16/Rb and p21waf pathways. Furthermore, sustained activation of Ras/Raf/MEK/ERK signalling was characterised in gastric cancer cells and a xenograft nude mouse model following GKN1 treatment. Conclusion These results provide comprehensive molecular evidence of GKN1 in inducing senescence of gastric cancer cells, and indicate that GKN1 might be a potential novel target for gastric cancer therapeutics.

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.

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.

Epigenetic Upregulation of Metallothionein 2A by Diallyl Trisulfide Enhances Chemosensitivity of Human Gastric Cancer Cells to Docetaxel Through Attenuating NF-κB Activation

Abstract Aims: Metallothionein 2A (MT2A) and nuclear factor-kappaB (NF-κB) are both involved in carcinogenesis and cancer chemosensitivity. We previously showed decreased expression of MT2A and IκB-α in human gastric cancer (GC) associated with poor prognosis of GC patients. The present study investigated the effect of diallyl trisulfide (DATS), a garlic-derived compound, and docetaxel (DOC) on regulation of MT2A in relation to NF-κB in GC cells. Results: DATS attenuated NF-κB signaling in GC cells, resulting in G2/M cell cycle arrest and apoptosis, culminating in the inhibition of cell proliferation and tumorigenesis in nude mice. The anti-GC effect of DATS was attributable to its capacity to epigenetically upregulate MT2A, which in turn enhanced transcription of IκB-α to suppress NF-κB activation in GC cells. The combination of DATS with DOC exhibited a synergistic anti-GC activity accompanied by MT2A upregulation and NF-κB inactivation. Histopathologic analysis of GC specimens from patients showed a significant increase in MT2A expression following DOC treatment. GC patients with high MT2A expression in tumor specimens showed significantly improved response to chemotherapy and prolonged survival compared with those with low MT2A expression in tumors. Innovation and Conclusion: We conclude that DATS exerts its anti-GC activity and enhances chemosensitivity of GC to DOC by epigenetic upregulation of MT2A to attenuate NF-κB signaling. Our findings delineate a mechanistic basis of MT2A/NF-κB signaling for DATS- and DOC-mediated anti-GC effects, suggesting that MT2A may be a chemosensitivity indicator in GC patients receiving DOC-based treatment and a promising target for more effective treatment of GC by combination of DATS and DOC. Antioxid. Redox Signal. 24, 839–854.

Cell cycle‐dependent expression of p42.3 promotes mitotic progression in malignant transformed cells

In an earlier study, we cloned the p42.3 gene and showed that its expression was specific to tumors in a number of tumor cell lines and primary tumor tissues. However, the biological role and function of this gene remains largely unknown. In this study, p42.3 expression was found to be cell cycle‐dependent at both the mRNA and protein levels in several human tumor cell lines. Typically, abundant expression was detected at G1 and M phases compared with S and G2 phases. Expression peaked at early G1 phase then decreased drastically at late G1, S, and G2. Furthermore, transfection of the p42.3 gene into NIH3T3 cells promoted malignant transformation, accompanied by accelerated mitotic progression and altered chromosome segregation. It was also observed that Cyclin B1 was upregulated and Cdc2‐Tyr15 was downregulated following p42.3 overexpression in NIH3T3 cells. Combined, these results indicate that p42.3 as a cell cycle‐regulated gene contributes to promoting cell cycle progression through disruption of mitotic regulation, and may play important roles in malignant transformation.

LncRNA16 is a potential biomarker for diagnosis of early-stage lung cancer that promotes cell proliferation by regulating the cell cycle

Early diagnosis of lung cancer greatly reduces mortality; however, the lack of suitable plasma biomarkers presents a major obstacle. Recent studies showed that long noncoding RNAs (lncRNAs) played important roles in cancer initiation and development. Here, we identified differentially expressed lncRNAs in 20 lung cancer samples by using custom designed microarray and evaluated their expression in 118 lung cancer samples by real-time PCR (qRT-PCR). lncRNA16 (ENST00000539303) expression was significantly higher in lung cancer tissues (80/118) than in adjacent matched normal tissues. Importantly, this increase was similar to that in plasma (53/84) of lung cancer patients, including early stage. The role of lncRNA16 in lung cancer was studied in vitro and in vivo by using the lung cancer cell lines and xenograft mouse models. The results reveal that knockdown of lncRNA16 inhibited proliferation of PC9 cells in vitro and also inhibited tumor growth in xenograft mouse models. Overexpression of lncRNA16 promoted proliferation of A549 cells in vitro and also promoted tumor growth in xenograft mouse models. Specifically, we showed that lncRNA16 promoted G2/M transition by regulating cyclin B1 transcription. Together, our findings suggest that lncRNA16 is a promising biomarker suitable for early diagnosis of lung cancer, and a potential target for lung cancer treatment.

Clinical Significance of a Point Mutation in DNA Polymerase Beta (POLB) Gene in Gastric Cancer.

Gastric cancer (GC) is a major cause of global cancer mortality. Genetic variations in DNA repair genes can modulate DNA repair capability and, consequently, have been associated with risk of developing cancer. We have previously identified a T to C point mutation at nucleotide 889 (T889C) in DNA polymerase beta (POLB) gene, a key enzyme involved in base excision repair in primary GCs. The purpose of this study was to evaluate the mutation and expression of POLB in a larger cohort and to identify possible prognostic roles of the POLB alterations in GC. Primary GC specimens and their matched normal adjacent tissues were collected at the time of surgery. DNA, RNA and protein samples were isolated from GC specimens and cell lines. Mutations were detected by PCR-RFLP/DHPLC and sequencing analysis. POLB gene expression was examined by RT-PCR, tissue microarray, Western blotting and immunofluorescence assays. The function of the mutation was evaluated by chemosensitivity, MTT, Transwell matrigel invasion and host cell reactivation assays. The T889C mutation was detected in 18 (10.17%) of 177 GC patients. And the T889C mutation was associated with POLB overexpression, lymph nodes metastases and poor tumor differentiation. In addition, patients with- the mutation had significantly shorter survival time than those without-, following postoperative chemotherapy. Furthermore, cell lines with T889C mutation in POLB gene were more resistant to the treatment of 5-fluorouracil, cisplatin and epirubicin than those with wild type POLB. Forced expression of POLB gene with T889C mutation resulted in enhanced cell proliferation, invasion and resistance to anticancer drugs, along with increased DNA repair capability. These results suggest that POLB gene with T889C mutation in surgically resected primary gastric tissues may be clinically useful for predicting responsiveness to chemotherapy in patients with GC. The POLB gene alteration may serve as a prognostic biomarker for GC.

STAT3 signaling drives EZH2 transcriptional activation and mediates poor prognosis in gastric cancer

BackgroundSTAT3 signaling plays the pivotal role in tumorigenesis through EZH2 epigenetic modification, which enhanced STAT3 activity by increased tyrosine phosphorylation of STAT3. Here, another possible feedback mechanism and clinical significance of EZH2 and STAT3 were investigated in gastric cancer (GC).MethodsSTAT3, p-STAT3 (Tyr 705) and EZH2 expression were examined in 63 GC specimens with matched normal tissues by IHC staining. EZH2 and STAT3 were also identified in five GC cell lines using RT-PCR and western blot analyses. p-STAT3 protein was detected by western blotting. In order to investigate whether EZH2 expression was directly regulated by STAT3, EZH2 expression was further detected using siRNA for STAT3 or IL-6 stimulation, with dual luciferase reporter analyses, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The clinical significance of STAT3, p-STAT3 and EZH2 expression was evaluated by multi-factor COX regression and Kaplan-Meier analyses.ResultsHyper-activation of STAT3, p-STAT3 and EZH2 expression were observed in GC cells and tissues. STAT3 signaling was correlated with EZH2 expression in GC (R = 0.373, P = 0.003), which was consistent with our data showing that STAT3 as the transcriptional factor enhanced EZH2 transcriptional activity by binding the relative promoter region (-214 ~ -206). STAT3 was an independent signature for poor survival (P = 0.002). Patients with STAT3+/EZH2+ or p-STAT3+/EZH2+ had a worse outcome than others (P < 0.001); Besides, high levels of STAT3 and EZH2 was associated with advanced TNM staging (P = 0.017). Moreover, treatment with a combination of siSTAT3 and EZH2-specific inhibitor, 3-deazaneplanocin A (DZNEP), increased the apoptotic ratio of cells. It is benefit for targeting STAT3-EZH2 interplay in GC treatment.ConclusionsOur results indicate that STAT3 status mediated EZH2 upregulation, associated with advanced TNM stage and poor prognosis, suggesting that combination with knockdown of STAT3 and EZH2 inhibitor might be a novel therapy in GC treatment. Collectively, STAT3, p-STAT3 and EZH2 expression were provided for the precision medicine in GC patients.