Zhaofeng Chen

The clinical significance of downregulation of mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p in gastric cancer tumorigenesis

Dysregulated miRNAs in gastric cancer are usually screened by miRNA microarray from clinical samples, however, reports have indicated that results of each miRNA microarray screening are considerably different, and dysregulated miRNAs, especially downregulated miRNAs were contradictory. In view of this, the Human Cancer Pathway Finder miRNA PCR array was applied to compare 7 gastric cancer cell lines AGS, SGC-7901, MKN-45, MKN-28, MGC-803, BCG-823, and HGC-27 with an immortalized normal gastric cell line, GES-1 in cancer pathway-related miRNA expression profile, followed by qPCR verification, the clinical significance of downregulated miRNAs and the Enriched KEGG pathways and GO terms of their target genes were analyzed. Thirty-eight miRNAs were upregulated, and four miRNAs were downregulated in gastric cancer cell lines. Clinical significance of 4 miRNAs including mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p in gastric cancer tissue compared with adjacent non-tumor tissues of 58 patients indicated that the low-expression group of mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p showed more extensive lymph node metastasis, lymphatic invasion, venous invasion, high-stage Borrmann type, lymphatic invasion and poor differentiation than that of the high-expression groups, respectively (P<0.05; χ² test). Enriched KEGG pathway analyses showed that most of the targeted genes of the 4 miRNAs concentrated on 37 signaling pathways, and were involved in the same pathways related to cancer. Enriched GO terms showed that targeted genes of the 4 miRNAs concentrated on 339 terms, 24 of 339 terms are associated with cancer tumorigenesis. The Human Cancer Pathway Finder miRNA PCR array could be used to screen dysregulated miRNAs effectively, and 4 screened miRNAs, mir-124-3p, mir-146a-5p, mir-155-5p and mir-335-5p were found to be downregulated in gastric cancer. Clinical significance and bioinformatic analysis on the target genes of these 4 miRNAs indicated that they were deeply involved in tumorigenesis, suggesting roles such as miRNA tumor suppressors in gastric cancer tumorigenesis which could be applied in gastric cancer diagnosis and prognosis.

Celecoxib regulates apoptosis and autophagy via the PI3K/Akt signaling pathway in SGC-7901 gastric cancer cells

Gastric cancer, one of the most common malignancies worldwide, typically has a poor prognosis and poor survival rate. Previous studies have investigated the chemopreventive effect of celecoxib. In the present study, the SGC-7901 human gastric cancer cell line was utilized to examine the chemopreventive mechanisms of celecoxib. The inhibition of cell proliferation was determined using MTT assay, cell apoptosis was monitored by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and flow cytometry, and cell ultrastructural changes were assessed via transmission electron microscopy. The mRNA expression of Akt, caspase-8 and -9 was examined using quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) and p-Akt, procaspase-8 and -9 were analyzed via western blotting. The results showed that celecoxib inhibited the proliferation of SGC-7901 cells in a time- and dose-dependent manner. Additionally, celecoxib induced apoptosis as substantiated by typical apoptotic bodies, autophagosomes and an increased apoptotic rate. It was found that following celecoxib treatment, Akt mRNA expression was not significantly altered, and that p-Akt protein levels decreased in a time- and dose-dependent manner. Additionally, caspase-8 and -9 mRNA expression was significantly increased, while procaspase-8 and -9 protein expression decreased relative to the time- and dose-dependent effects. These results demonstrated that celecoxib induced apoptosis and autophagy of gastric cancer cells in vitro through the PI3K/Akt signaling pathway. Moreover, our findings suggested that celecoxib induces apoptosis in gastric cancer cells through the mitochondrial and death receptor pathways, providing additional understanding regarding the chemopreventive behaviors of celecoxib and its uses in cancer therapy.

COX-2 regulates E-cadherin expression through the NF-κB/Snail signaling pathway in gastric cancer

Cyclooxygenase-2 (COX-2) participates in cancer invasion and metastasis by decreasing the expression of E-cadherin. However, the molecular mechanisms through which COX-2 regulates E-cadherin expression and function have not yet been fully elucidated. The aim of this study was to investigate the possible molecular mechanisms through which COX-2 regulates E-cadherin expression in gastric cancer. The mRNA and protein expression of COX-2, nuclear factor-κB (NF-κB), Snail and E-cadherin was detected in gastric cancer cells by quantitative PCR and western blot analysis, respectively. The expression of these genes was also detected in healthy gastric mucosa and gastric cancer tissues by immunohistochemistry. We detected various levels of COX-2, nuclear factor-κB (NF-κB), Snail and E-cadherin expression in the normal gastric mucosa and cancer tissues; however, the expression patterns differed: the increased expression of COX-2, NF-κB and Snail was observed in the gastric cancer tissues, whereas there was a considerable reduction in E-cadherin expression in the cancer tissues compared to the normal gastric mucosa. The expression patterns of COX-2, NF-κB and Snail were similar. The increased expression of COX-2 in the gastric cancer tissues closely correlated with the increased expression of NF-κB and Snail, but inversely correlated with the expression of E-cadherin. Treatment of the SGC7901 cells (which express high levels of COX-2) with celecoxib, a COX-2 inhibitor, not only led to a marked dose- and time-dependent decrease in the expression of COX-2, NF-κB and Snail, but also led to a significant increase in the expression of E-cadherin, and this was associated with a reduction in cell invasion. By contrast, the same treatment did not alter the expression of these genes in another gastric cancer cell line, MGC803 (which barely expresses COX-2). These data suggest that COX-2 regulates the expression of E-cadherin through the NF-κB and Snail signaling pathway in gastric cancer.

Effects of the suppression of lactate dehydrogenase A on the growth and invasion of human gastric cancer cells

Lactate dehydrogenase A (LDH-A), which regulates glycolytic flux by catalyzing pyruvate to lactate in the cytoplasm, is believed to be one of the highly attractive therapeutic targets for cancers. Firstly, we detected the expression of LDH-A in gastric cancer (GC) cells. LDH-A inhibitor oxamate was then used to suppress the LDH-A activity in GC cells. Cell proliferation, lactic acid production, Transwell migration assay and apoptosis were assessed, respectively. The results showed that inhibition of LDH-A by oxamate decreased the lactate production. In the presence of glucose, oxamate inhibited cell proliferation in a dose-dependent manner. Flow cytometry assay further confirmed a pro-apoptotic effect of oxamate, and this was likely through increased expression of Bax, activated caspase-3, and decreased expression of Bcl-2. Therefore, we believe that oxamate inhibits cell growth, suppresses tumor invasion, and induces apoptosis in GC cells. LDH-A may be a potential therapeutic target for GC.

Clinical Significance of Upregulation of mir-196a-5p in Gastric Cancer and Enriched KEGG Pathway Analysis of Target Genes

BACKGROUND miRNAs are relatively recently discovered cancer biomarkers which have important implications for cancer early diagnosis, treatment and estimation of prognosis. Here we focussed on expression of mir-196a-5p in gastric cancer tissues and cell lines so as to analyse its significance for clinicopathologic characteristics and generate enriched KEGG pathways clustered by target genes for exploring its potential roles as a biomarker in gastric cancer. MATERIALS AND METHODS The expression of mir-196a-5p in poorly, moderate and well differentiated gastric cancer cell lines compared with GES-1 was detected by RT-qPCR, and the expression of mir-196a-5p in gastric cancer tissues comparing with adjacent non cancer tissues of 58 cases were also assessed by RT- qPCR. Subsequently, an analysis of clinical significance of mir-196a-5p in gastric cancer and enriched KEGG pathways was executed based on the miRWalk prediction database combined with bioinformatics tools DAVID 6.7 and Mirfocus 3.0. RESULTS RT-qPCR showed that mir-196a-5p was up-regulated in 6 poorly and moderate differentiated gastric cancer cell lines SGC-7901, MKN-45, MKN-28, MGC-803, BGC-823, HGC-27 compared with GES-1, but down-regulated in the highly differentiated gastric cancer cell line AGS. Clinical data indicated mir-196a-5p to beup-regulated in gastric cancer tissues (47/58). Overexpression of mir-196a-5p was associated with more extensive degree of lymph node metastasis and clinical stage (P <0.05; x2 test). Enriched KEGG pathway analyses of predicted and validated targets in miRWalk combined with DAVID 6.7 and Mirfocus 3.0 showed that the targeted genes regulated by mir-196a-5p were involved in malignancy associated biology. CONCLUSIONS Overexpression of mir-196a-5p is associated with lymph node metastasis and clinical stage, and enriched KEGG pathway analyses showed that targeted genes regulated by mir-196a-5p may contribute to tumorgenesis, suggesting roles as an oncogenic miRNA biomarker in gastric cancer.

Interaction between cyclooxygenase-2, Snail, and E-cadherin in gastric cancer cells.

AIM To investigate the mechanisms of how cyclooxygenase-2 (COX-2) regulates E-cadherin in gastric cancer cells. METHODS COX-2 expression in human gastric cancer cell lines SGC-7901, BGC-823, MGC-803 and AGS were measured at the mRNA and protein level. COX-2 rich cell line SGC-7901 was chosen for subsequent experiments. siRNA mediated gene knockdown was used to investigate the impact of COX-2 on nuclear factor-κB (NF-κB), Snail, and E-cadherin in gastric cancer cells. Gene expression was determined by Western blot and real-time polymerase chain reaction. To analyze whether NF-κB inhibition could interrupt the modulatory effect of COX-2 or prostaglandin E2 (PGE2) on E-cadherin, gastric cancer cells were treated with celecoxib or PGE2, in the presence of NF-κB specific siRNA. RESULTS Highest expression level of COX-2 was found in SGC-7901 cells, both at mRNA and protein levels. siRNA mediated down-regulation of COX-2 led to a reduced expression of NF-κB and Snail, but an increased expression of E-cadherin in SGC-7901 cells. siRNA mediated down-regulation of NF-κB also led to a reduced expression of E-cadherin and Snail in SGC-7901 cells. However, COX-2 expression did not alter after cells were treated with NF-κB specific siRNA in SGC-7901 cells. Treatment of SGC-7901 cells with celecoxib led to a reduced expression of Snail but an increased expression of E-cadherin. In contrast, treatment of SGC-7901 cells with PGE2 led to an increased Snail and a decreased E-cadherin. However, siRNA-mediated knockdown of NF-κB partially abolished the effect of celecoxib and PGE2 on the regulation of E-cadherin and Snail in SGC-7901 cells. CONCLUSION COX-2 likely functions upstream of NF-κB and regulates the expression of E-cadherin via NF-κB/Snail signaling pathway in gastric cancer cells.

Molecular mechanisms of lncRNA SMARCC2/miR-551b-3p/TMPRSS4 axis in gastric cancer

Decreased expression of miR-551b-3p has been identified in gastric cancer tissues but its biological role and underlying mechanism in this malignancy is poorly understood. In this study, we show that the expression of miR-551b-3p negatively correlates with the depth of tumour invasion and lymphatic metastasis, but it positively correlates with tumour differentiation and the patient survival. MiR-551b-3p negatively affects the proliferation, mobility and invasiveness of gastric cancer cells. LncRNA SMARCC2 inhibits the expression of miR-551b-3p through binding to its mRNA response elements in gastric cancer cells. Overexpression of LncRNA SMARCC2 enhances the proliferation and migration of gastric cancer cells, while inhibition of LncRNA SMARCC2 does the opposite. TMPRSS4 is a direct target gene of miR-551b-3p. We conclude that miR-551b-3p functions as a tumour suppressor gene in gastric cancer, and its function is regulated by LncRNA SMARCC2/miR-551b-3p/TMPRSS4 axis.