TET1 inhibits gastric cancer growth and metastasis by PTEN demethylation and re-expression.

Ten-Eleven Translocation 1 (TET1) is a member of ten eleven translocation enzymes, which convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). TET1 can promote CpG islands demethylation in specific genes and often absent in various cancers. Herein, we found that TET1 expression and 5-hmC content were low in gastric tumors compared to its adjacent non-tumor tissues. Cell proliferation, migration and invasion were enhanced upon TET1 knockdown in gastric cancer cells in vitro. This phenomenon was confirmed by an animal xeongraft model. We also found that TET1 directly binds to the promoter region of PTEN and activates its transcription through demethylation of CpG islands. TET1 knockdown activated AKT and FAK pathways, which were suppressed by PTEN. The activation of AKT and FAK facilitated tumor migration, invasion and accelerated cell growth. In conclusion, we found a novel mechanism that TET1 suppresses tumor cell growth, migration and invasion through demethylation of CpG island in PTEN promoter by increasing 5-hmC content. The re-expressed PTEN subsequently down regulates AKT and FAK activity.

Epigenetic silencing of microRNA-149 in cancer-associated fibroblasts mediates prostaglandin E2/interleukin-6 signaling in the tumor microenvironment.

Tumor initiation and growth depend on its microenvironment in which cancer-associated fibroblasts (CAFs) in tumor stroma play an important role. Prostaglandin E2 (PGE2) and interleukin (IL)-6 signal pathways are involved in the crosstalk between tumor and stromal cells. However, how PGE2-mediated signaling modulates this crosstalk remains unclear. Here, we show that microRNA (miR)-149 links PGE2 and IL-6 signaling in mediating the crosstalk between tumor cells and CAFs in gastric cancer (GC). miR-149 inhibited fibroblast activation by targeting IL-6 and miR-149 expression was substantially suppressed in the CAFs of GC. miR-149 negatively regulated CAFs and their effect on GC development both in vitro and in vivo. CAFs enhanced epithelial-to-mesenchymal transition (EMT) and the stem-like properties of GC cells in a miR-149-IL-6-dependent manner. In addition to IL-6, PGE2 receptor 2 (PTGER2/EP2) was revealed as another potential target of miR-149 in fibroblasts. Furthermore, H. pylori infection, a leading cause of human GC, was able to induce cyclooxygenase-2 (COX-2)/PGE2 signaling and to enhance PGE2 production, resulting in the hypermethylation of miR-149 in CAFs and increased IL-6 secretion. Our findings indicate that miR-149 mediates the crosstalk between tumor cells and CAFs in GC and highlight the potential of interfering miRNAs in stromal cells to improve cancer therapy.

Androgen receptor promotes gastric cancer cell migration and invasion via AKT-phosphorylation dependent upregulation of matrix metalloproteinase 9.

Androgen receptor (AR) plays an important role in many kinds of cancers. However, the molecular mechanisms of AR in gastric cancer (GC) are poorly characterized. Here, we investigated the role of AR in GC cell migration, invasion and metastatic potential. Our data showed that AR expression was positively correlated with lymph node metastasis and late TNM stages. These findings were accompanied by activation of AKT and upregulation of matrix metalloproteinase 9 (MMP9). AR overexpression induced increases in GC cell migration, invasion and proliferation in vitro and in vivo. These effects were attenuated by inhibition of AKT, AR and MMP9. AR overexpression upregulated MMP9 protein levels, whereas this effect was counteracted by AR siRNA. Inhibition of AKT by siRNA or an inhibitor (MK-2206 2HC) decreased AR protein expression in both stably transfected and parental SGC-7901 cells. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that AR bound to the AR-binding sites of the MMP9 promoter. In summary, AR overexpression induced by AKT phosphorylation upregulated MMP9 by binding to its promoter region to promote gastric carcinogenesis. The AKT/AR/MMP9 pathway plays an important role in GC metastasis and may be a novel therapeutic target for GC treatment.

RhoGDI2 confers resistance to 5-fluorouracil in human gastric cancer cells.

Resistance to 5-fluorouracil (5-FU) in patients with gastric cancer is a serious therapeutic problem and major efforts are underway to understand the underlying mechanisms. We have previously identified RhoGDI2 as a contributor to 5-FU resistance in colon cancer cells using 2D electrophoresis and mass spectrometry and the current study aimed to further investigate this role. The expression of RhoGDI2 in seven gastric cancer cell lines was positively correlated with resistance to 5-FU. Lower 5-FU sensitivity of isolated tumor cells from patients with gastric cancer was also associated with higher RhoGDI2 expression. Ectopic expression of RhoGDI2 in gastric cancer cells increased the resistance to 5-FU and reverted low dose 5-FU-induced G2/M phase arrest without affecting the population of sub-G1 cells. Overall, these findings suggest that RhoGDI2 is associated with 5-FU resistance and is a potential therapeutic target for enhancing chemotherapy efficacy in gastric cancer.

microrna expression signature of gastric cancer cells relative to normal gastric mucosa.

micrornas (miRNAs) play an important role in a wide range of physiological and developmental processes by negatively regulating the expression of target genes at the post-transcriptional level. In this study, we investigated the differential miRNA expression signature between gastric cancer cells and normal gastric mucosa to determine changes in miRNA expression during gastric cancer development. We analyzed the global miRNA expression profiles of 9 gastric cancer cell lines and 6 normal gastric mucosa lines using miRNA microarrays. In addition, we performed quantitative real-time PCR (Q-PCR) to validate the results. Correlations between the miRNA expression profile and tumor clinicopathological parameters were analyzed. We found that 17 miRNAs were upregulated in gastric cancer cell lines and 146 miRNAs were downregulated compared to normal gastric mucosa. Using microarray data and Q-PCR validation, 15 miRNAs were finally selected. These candidate miRNAs were associated with gastric cancer clinicopathology to various degrees. High expression levels of hsa-miR-93 were found to predict poor survival (median, 16 vs. 40 months; log-rank test p<0.05). These findings suggest that miRNAs play vital roles in human gastric cancer. The findings may also provide clues toward understanding the molecular functions of miRNAs in various biological processes.

[Screening and identification of genes associated with multi-drug resistance in colonic cancer].

OBJECTIVE: To identify novel multi-drug resistance-related genes, and to explore the mechanisms of multi-drug resistance. METHODS: Multi-drug resistant cell line Lovo/5-FU was established by incubation with increasing dose of 5-FU. The sensitivity to 5-FU and cis-diaminodichloroplatinum (CDDP) was measured by MTT assay. Two dimensional electrophoresis plus mass spectrum(2-DE/MS) was used to identify the differentially expressed protein between Lovo and Lovo/5-FU. The identified protein was then verified by Western blot analysis. RESULTS: The IC50 concentrations of Lovo/5-FU to 5-FU and CDDP were increased by 31 and 3 times, compared with Lovo (both P<0.01). 2DE-MS showed that CAP-G and RhoGDI2 were up-regulated, whereas 6-PGL, DCI, Prdx-6 and Maspin were down-regulated in Lovo/5-FU. Western blot analysis confirmed that the expression levels of RhoGDI2 and CAP-G in Lovo/5-FU were increased by 6.14 and 2.98 fold respectively (both P<0.01), whereas Maspin was decreased to 5.2% of Lovo(P<0.01). CONCLUSIONS: Multi-gene and multi-pathway are involved in the development of multi-drug resistance of colorectal cancer cells. CAP-G, RhoGDI2 and Maspin are potential multi-drug resistant genes.

[Interaction of SerpinB5 and MAFbx in gastric cancer cell and its action site].

OBJECTIVE: To explore the interaction between SerpinB5 and MAFbx in gastric cancer cell and to identify the interaction sites. METHODS: The interaction between SerpinB5 and MAFbx was screened and validated by yeast two-hybrid screening and co-immunoprecipitation. The expression of MAFbx was analyzed after SerpinB5 expression being modified by RNA interference and pGBKT7-SerpinB5 transfection. The impact of SerpinB5 on the expression of MAFbx was studied in gastric cancer cell line SUN-16. A model of MAFbx was constructed by homology modeling. The related residues for interaction were analyzed by Autodock4.0. RESULTS: The interaction between SerpinB5 and MAFbx was validated. The expression of MAFbx changed along with SerpinB5 expression. Amino acids including PRO261, ASN361, and LYS362 were key residue in the interaction of SerpinB5 and MAFbx. CONCLUSION: SerpinB5 interacts with MAFbx in gastric cancer cell. Amino acids including PRO261, ASN361, and LYS362 are potential binding sites.

microRNA-21 promotes tumor proliferation and invasion in gastric cancer by targeting PTEN.

Gastric cancer is one of the most common carcinomas in China. microRNAs, a type of non-coding RNA, are important specific regulators and are involved in numerous bioprocesses of an organism. microRNA-21 (miR-21) has been identified as the most suitable choice for further investigation because it is overexpressed in nearly all solid tumors; furthermore, it has been demonstrated that miR-21 is involved in the genesis and progression of human cancer. It has been reported that PTEN, an important tumour suppressor, is regulated by multiple miRNAs. Thus, in this study we focused on the expression and significance of miR-21 in gastric cancer tissues, and the role of miR-21 in the biological behaviour and the expression of PTEN in gastric cancer cells. Real-time PCR was used to detect miR-21 expression in gastric cancer tissues, the adjacent normal tissues, and the gastric cell lines. The gastric cancer cell line BGC-823 was transfected with pre-miR-21/miR-21 inhibitor to overexpress/downregulate miR-21. The influence of miR-21 on the biological behaviour of gastric cancer cells was evaluated using the CCK-8 kit, FCMs, the scratch healing assay and the transwell test. Western blotting and the Luciferase Reporter Assay were used to evaluate the change of PTEN expression after lowered expression of miR-21 in gastric cancer cell lines. Real-time PCR analysis indicated that miR-21 exhibited higher expression in gastric cancer tissues compared to the adjacent non-tumor tissues. miR-21 expression was significantly associated with the degree of differentiation of the tumour tissues (P=0.004), as well as local invasion and lymph node metastasis (P<0.01). After transfection, pre-miR21 BGC-823 cells grew faster than the negative and control groups (P<0.01). The reduction in miR-21 expression demonstrated a remarkable effect on the biological behaviour of gastric cancer cells (P<0.05); the pre-miR-21-transfected cells healed more quickly compared to the control cells in the scratch healing assay, whereas the transwell test indicated that cell migration in vitro was notably inhibited with the downregulation of miR-21 (P<0.05). The western blot results and Luciferase Reporter Assay demonstrated that PTEN expression was remarkably increased after miR-21 inhibition (P<0.05). microRNA-21 expression was upregulated in gastric carcinoma tissues and was significantly associated with the degree of differentiation of tumour tissues, local invasion and lymph node metastasis. Overexpression of miR-21 promoted BGC-823 cell growth, invasion and cell migration in vitro, whereas downregulation of miR-21 exhibited a stronger inhibitory effect on the biological behaviour of gastric cancer cells; additionally, miR-21 inhibition may upregulate the PTEN expression level, which indicates that PTEN may be a target gene for gastric cancer initiation and development.

SerpinB5 interacts with KHDRBS3 and FBXO32 in gastric cancer cells.

Mammary serine protease inhibitor B5 (SerpinB5) is a potential oncogene in gastric cancer (GC); however, the molecular mechanism by which SerpinB5 promotes oncogenesis remains elusive. In this study, SerpinB5-associated proteins were selected based on yeast two-hybrid screening and microarray analysis after RNA interference and were validated using co-immunoprecipitation (Co-IP) and RNA Co-IP. The expression profiles of the interacting proteins were analyzed by Western blotting and immunohistochemistry. The effects of SerpinB5 on KHDRBS3 and FBXO32 expression in GC cells were analyzed using real-time PCR and Western blotting after the expression of SerpinB5 was modified. By yeast two-hybrid screening and microarray analysis, FBXO32 and KHDRBS3 were found to be SerpinB5-interacting proteins. The interactions were confirmed by Co-IP. An RNA co-immunoprecipitation assay found that KHDRBS3 interacted with FBXO32 mRNA. The expression of SerpinB5 was much stronger in the nucleus of GC cells. FBXO32 was expressed at higher levels in the cytoplasm of GC cells. KHDRBS3 was primarily detected in the nucleus of normal mucosal cells. SerpinB5 expression was modified in GC cells, KHDRBS3 mRNA levels remained stable, however, FBXO32 mRNA levels changed 24 h after changes in KHDRBS3 protein levels were detected. In conclusion, SerpinB5 interacts with KHDRBS3 and FBXO32, and KHDRBS3 can interact with FBXO32 mRNA.

Cell cycle dependent genes from the gastric cancer cell MKN45 can affect tumorigenesis.

BACKGROUND/AIMS: To investigate the cell cycle dependent genes involved in gastric tumorigenesis, possibly determining the relationship between the cell cycle and tumorigenesis. METHODOLOGY: MKN45 cells were collected every hour from Oh to 12h after release from G2/M and G1/S blocks. Nine samples (a-i), chosen at key times of the cell cycle, were prepared for RNA isolation and cDNA microarray analysis. RESULTS: In 2001 viable clones, 959 genes showed periodic variations during the cell cycle. Among 2001 genes that were clustered, a series of up-regulated genes were assigned to different cell cycle phases. Many periodically dependent genes in the cell cycle were ubiquitously expressed and participated in various cell physiological functions, such as transcription, translation, ubiquitination and signal transduction. These cell cycle dependent genes could affect cancer cell proliferation, apoptosis, activation of oncogenes and inactivation of tumor suppressor genes. CONCLUSIONS: We provided a comprehensive understanding of the gene expression profile involved in gastric cancer cell cycles and laid a foundation for further research on mechanisms of gastric tumorigenesis.

Serum DJ-1 as a diagnostic marker and prognostic factor for pancreatic cancer.

OBJECTIVE: DJ-1 is an oncoprotein secreted by cancer cells. Therefore, it might be a diagnostic or prognostic biomarker for pancreatic cancer (PC). METHODS: The study involved 47 patients with PC, 43 with chronic pancreatitis, and 40 healthy subjects. We assayed the serum level of DJ-1 and the conventional tumor marker carbohydrate antigen 19-9 (CA 19-9) to define the diagnostic and prognostic value of DJ-1 for PC. RESULTS: Serum DJ-1 level was elevated in patients with PC compared with those with chronic pancreatitis and healthy individuals. The area under the curve (AUC) of serum DJ-1 was higher than CA 19-9 (DJ-1 vs. CA19-9, 0.8735 ± 0.0356 vs. 0.6647 ± 0.0572 ng/mL), and an 87.5% sensitivity was reached with a combination of serum DJ-1 and CA19-9. No association of serum DJ-1 level with tumor node metastasis (TNM) classification or tumor resectability was found. However, after resection, the median serum DJ-1 level was decreased from 2.00 to 0.78 ng/mL. In addition, higher serum DJ-1 was correlated with shorter overall survival as analyzed by both Kaplan-Meier test (P = 0.018) and COX regression analysis (P = 0.013). The median overall survival time of PC patients with serum DJ-1 level greater than or equal to 2.06 ng/mL was 7.00 ± 1.11 months, whereas that of patients with lower DJ-1 levels was 13.0 ± 2.5 months. CONCLUSIONS: These findings indicate the potential clinical significance for serum DJ-1 level to be used for the diagnosis and prognosis prediction of patients with PC.

Silencing invariant chains of dendritic cells enhances anti-tumor immunity using small-interfering RNA.

BACKGROUND: Genetic modification of dendritic cells (DCs) has been used as an effective approach to enhance anti-tumor immunity. RNA interference (RNAi), which can cause the degradation of any RNA in a sequence-specific manner, is a post-transcriptional gene silencing mechanism. In this study, small-interfering RNA (siRNA) specific for the Ii gene was transfected into DCs, and the anti-tumor immunity of Ii-silenced DCs was assessed. METHODS: The silencing effect of siRNA was evaluated by Western blotting and real-time PCR analyses. In vitro cytotoxic activity of T cells was evaluated using a Cytotox 96(®) non-radioactive cytotoxicity assay kit. The time to tumor onset and the tumor volumes were used as reliable indices to assess the anti-tumor immunity in vivo. To further examine the mechanisms underlying the anti-tumor immunity, flow cytometry analysis was used. RESULTS: The Ii expression of DCs was significantly reduced after Ii siRNA transfection. Significant in vitro anti-tumor ability was exhibited when DCs were co-transfected with Ii siRNA plus endogenous tumor antigen (P < 0.05). Furthermore, tumor growth was greatly inhibited when mice were immunized with DCs transfected with Ii siRNA plus tumor antigen prior to or subsequent to tumor implantation. Flow cytometry analysis in vitro and in vivo indicated that both CD4(+) and CD8(+) T cells were significantly activated in the Ii siRNA group (P < 0.05). CONCLUSION: Silencing of the Ii gene of DCs may offer a potential approach to enhance DC-based anti-tumor immunity.

[Demethylation of the gamma-synuclein gene CpG island in colorectal cancer and its clinical significance].

OBJECTIVE: To explore the relationship between gamma-synuclein gene expression and CpG island demethylation in colorectal cancer(CRC), and the relationship between the demethylation and clinicopathological factors of CRC. METHODS: The expression of gamma-synuclein mRNA was examined in 30 pairs of tumor tissues and tumor-matched non-neoplastic adjacent tissues(NNAT) by RT-PCR. CRC cell lines including COLO205, LoVo, and SW480 were used and treated with a demethylating agent, 5-aza-2'-deoxycytidine(5-aza-C). Before and after the treatment, the expression of gamma-synuclein mRNA in the cells was determined by RT-PCR, and bisulfite sequencing PCR was also used to analyze methylation status of CpG island. The methylation status of gamma-synuclein was then examined in 67 CRC samples and 30 NNAT samples by nested methylation-specific PCR (NMSP) and real time methylation-specific PCR(real-time MSP). The relationship between the demethylation of gamma-synuclein in CRC and clinicopathological factors was analyzed. RESULTS: The mean gamma-synuclein mRNA expression was 0.66+/-0.34 in CRC samples, which was much higher than 0.45+/-0.26 in NNAT samples(P=0.011). 5-aza-C could induce expression and demethylation of gamma-synuclein in COLO205, LoVo and SW480 cells. gamma-Synuclein gene was demethylated in 80.0%(24/30) of the CRC samples and 50.0%(15/30) of the NNAT samples. The demethylated status of gamma-synuclein was much higher in CRC samples than that in NNAT samples(P=0.030), and was significantly correlated with clinical stage, lymph node involvement, and distant metastasis of CRC(P<0.05). CONCLUSION: The upregulation of gamma-synuclein expression in CRC is primarily attributed to the demethylation of CpG island, which may be used as a marker for prognosis.

Expression of gamma-synuclein in colorectal cancer tissues and its role on colorectal cancer cell line HCT116.

AIM: To investigate the expression pattern of gamma-synuclein in colorectal cancer (CRC) tissues, and to study the effects of gamma-synuclein on CRC cell line HCT116 biological features in vitro. METHODS: The expression pattern of gamma-synuclein was determined in 54 CRC tissues and 30 tumor-matched nonneoplastic adjacent tissues (NNAT) 5 cm away from the tumor via real-time quantitative reverse transcription PCR (RT-PCR) and immunohistochemistry. The relationship between gamma-synuclein protein expression and clinicopathological factors of CRC tissues was analyzed. Three small interfering RNA (siRNA) targeting gamma-synuclein mRNA plasmids were constructed and transfected into the CRC cell line HCT116. The stable cell lines were selected with G-418 for 28 d, and the biological features of these cells were examined by cell growth curve, soft agar assay, and cell migration and invasion assays in vitro. RESULTS: The expression of gamma-synuclein mRNA and protein was much higher in CRC tissue samples than in NNAT samples (P = 0.02, P = 0.036). There was a significant correlation between the gamma-synuclein protein expression and clinical stage and lymph node involvement of CRC (P = 0.02, P = 0.033). In functional analysis we found that down-regulation of gamma-synuclein expression in HCT116 cells could inhibit the growth, colony formation rate, and migration and invasion ability of HCT116 cells. CONCLUSION: Increased expression of gamma-synuclein in CRC tissues and the biological effects of reduced gamma-synuclein expression on HCT116 cells suggest that gamma-synuclein may play a positive role in the progression of CRC.

Aberrant expression and demethylation of gamma-synuclein in colorectal cancer, correlated with progression of the disease.

Recent evidence suggests that gamma-synuclein is abnormally expressed in a high percentage of tumor tissues of diversified cancer types, but rarely expressed in tumor-matched non-neoplastic adjacent tissues (NNAT). The molecular mechanism of CpG island demethylation may underlie aberrant gamma-synuclein expression. To fully understand the roles of aberrant gamma-synuclein expression and demethylation in the development of colorectal cancer (CRC), we examined the expression and methylation status of gamma-synuclein in 67 CRC samples, 30 NNAT samples, and five CRC cell lines as well. By using reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry analyses, gamma-synuclein expression was detected in both HT-29 and HCT116 cells, and was much higher in CRC samples than in NNAT samples (P < 0.05). The demethylating agent, 5-aza-2 cent-deoxycytidine, can induce re-expression of gamma-synuclein in COLO205, LoVo, and SW480 cells. Unmethylated gamma-synuclein alleles were detected in HT-29, HCT116, and LoVo cells by nested methylation-specific PCR, and the demethylated status of gamma-synuclein was much higher in CRC samples than in NNAT samples by real-time quantitative methylation-specific PCR (P < 0.05). The results of genomic bisulfite DNA sequencing further confirmed that the aberrant gamma-synuclein expression in CRC was primarily attributed to the demethylation of CpG island. The protein expression and demethylation status of gamma-synuclein in 67 CRC samples correlated with clinical stage, lymph node involvement, and distant metastasis. These findings suggest an involvement of aberrant gamma-synuclein expression and demethylation in progression of CRC, especially in advanced stages.