Qinlong Gu

Overexpressed miR-301a promotes cell proliferation and invasion by targeting RUNX3 in gastric cancer

BackgroundMicroRNAs can promote or suppress the evolution of malignant behaviors by regulating multiple targets. We aimed to determine the expression of miR-301a recently screened in gastric cancer, to investigate the biological effects of miR-301a and to identify the specific miR-301a target gene.MethodsQuantitative real-time RT-PCR was used to test miR-301a expression. Functional effects were explored by a water-soluble tetrazolium salt assay, a colony formation assay in soft agar, a migration assay, an invasion assay and cytometry used to determine apoptosis and cell cycle. Nude mice were inoculated subcutaneously by retrovirus-mediated stably expressed SGC-7901 cells. The target gene was determined by bioinformatic algorithms, dual luciferase reporter assay and Western blot.ResultsFirstly, we found that miR-301a was significantly upregulated both in cells and tissues of gastric cancer. The expression level of miR-301a was inversely correlated with tumor differentiation of gastric cancer tissues. Secondly, miR-301a promoted cell growth, soft agar clonogenicity, migration, invasion, and decreased cell apoptosis induced by cisplatin in vitro, while blockage of miR-301a reduced the percentage of G2/M phase cells via flow cytometry in gastric cancer cells. Ectopic expression of miR-301a enhanced the subcutaneous tumorigenesis in vivo. Finally, miR-301a directly downregulated RUNX3 expression post-transcriptionally in gastric cancer.ConclusionOur results demonstrate that miR-301a plays important roles in the development of gastric cancer.

MicroRNA-409-3p regulates cell proliferation and apoptosis by targeting PHF10 in gastric cancer.

Emerging evidence has indicated microRNAs are involved in tumor development and progression, acting as tumor suppressors or oncogenes. Here we report that miR-409-3p was significantly downregulated in gastric cancer (GC) cell lines and tissues. Overexpression of miR-409-3p in SGC-7901 gastric cancer cells dramatically suppressed cell proliferation and induced cell apoptosis both in vitro and in vivo. Furthermore, we demonstrate that the transcriptional regulator PHF10 was a target of miR-409-3p. Taken together, these findings suggest that miR-409-3p may function as a novel tumor suppressor in GC and its anti-oncogenic activity may involve the direct targeting and inhibition of PHF10.

Down‐regulated miR‐625 suppresses invasion and metastasis of gastric cancer by targeting ILK

Accumulating evidence has shown that microRNAs are involved in multiple processes in cancer development and progression. Here, we report that expression of miR‐625 is significantly down‐regulated and negatively correlated with lymph node metastasis in gastric cancer. miR‐625 significantly inhibits invasion and metastasis of gastric cancer cells both in vitro and in vivo. Moreover, we identify that ILK is a direct target gene for miR‐625 and knockdown of ILK has a phenocopy of overexpression of miR‐625. Taken together, our findings suggest that miR‐625 plays an important role in the mechanism of tumor metastasis.

In vitro and in vivo evidence of metallopanstimulin-1 in gastric cancer progression and tumorigenicity.

Purpose: The metallopanstimulin-1 (MPS-1) gene is a growth factor–inducible gene, which is highly expressed in many human cancers and may be involved in the progression towards tumor malignancy. However, it is unclear whether MPS-1 plays any role in gastric cancer development or progression. Our studies were designed to clarify the MPS-1 expression pattern and to explore its potential role in gastric cancer. Experimental Design: The expression pattern of MPS-1 was determined in primary gastric cancer specimens and gastric cancer cell lines via immunohistochemistry and Western blotting. To investigate the functional significance of MPS-1 expression, three small interfering RNA (siRNA) expression plasmids were constructed and transfected into gastric cancer cell line SGC7901. The stable cell lines transfected with the siRNA targeting MPS-1 mRNA plasmids were selected and the biological features of these cells were examined. Results: MPS-1 was overexpressed in 86% of the gastric cancer tissues and all gastric cancer cells. In addition, MPS-1 expression was significantly increased and corresponded with the tumor-node-metastasis clinical stage, and was significantly higher in the late stage (P < 0.01). The MPS-1 expression level was significantly decreased in the transfected cells with MPS-1-specific siRNA expression plasmid pRNAT-133. Furthermore, the stable transfected cancer cells exhibited an increase in the incidence of spontaneous apoptosis and a decrease in growth ability and tumorigenicity in nude mice. Conclusions: These results provide strong evidence that MPS-1 plays an important role in gastric cancer cell proliferation and development, and suggests that MPS-1 is a promising target for gastric cancer treatment.

Hypermethylated FAM5C and MYLK in Serum as Diagnosis and Pre-Warning Markers for Gastric Cancer

Most cases of gastric cancer (GC) are not diagnosed at early stage which can be curable, so it is necessary to identify effective biomarkers for its diagnosis and pre-warning. We have used methylated DNA immunoprecipitation (MeDIP) to identify genes that are frequently methylated in gastric cancer cell lines. Promoter regions hypermethylation of candidate genes were tested by methylation-specific polymerase chain reaction (MSP) in serum samples, including GC (n = 58), gastric precancerous lesions (GPL, n = 46), and normal controls (NC, n = 30). Eighty two hypermethylated genes were acquired by array analysis and 5 genes (BCAS4, CHRM2, FAM5C, PRAC and MYLK) were selected as the candidate genes. Three genes (CHRM2, FAM5C and MYLK) were further confirmed to show methylation rates increased with progression from NC to GPL, then to GC. There was obvious decrease in detection of FAM5C and MYLK hypermethylation, but not CHRM2, from preoperative to postoperative evaluation (P < 0.001). Combined detection of FAM5C and MYLK hypermethylation had a higher sensitivity in GC diagnosis (77.6%,45/58) and pre-warning (30.4%,14/46) than one single gene detection and also had a high specificity of 90%. The combined hypermethylated status of FAM5C and MYLK correlated with tumor size (P < 0.001), tumor invasion depth (P = 0.001) and tumor-node-metastasis (TNM) stage (P = 0.003). Hypermethylated FAM5C and MYLK can be used as potential biomarkers for diagnosis and pre-warning of GC.

Stromal fibroblasts in the microenvironment of gastric carcinomas promote tumor metastasis via upregulating TAGLN expression

BackgroundFibroblasts play a critical role in tumorigenesis, tumor progression and metastasis. However, their detailed molecular characteristics and clinical significance are still elusive. TAGLN is an actin-binding protein that plays an important role in tumorigenesis.ResultsWe investigated the interaction between cancer cells and the tumor microenvironment to determine how the fibroblasts from human gastric carcinoma facilitate tumorigenesis through TAGLN. QRT-PCR and Western blot indicated that TAGLN expression was upregulated in gastric carcinoma-associated fibroblasts (CAFs) that promote gastric cancer cell migration and invasion. Using small interfering RNA (siRNA), we found that CAFs enhanced tumor metastasis through upregulated TAGLN in vitro and in vivo. The expression of matrix metalloproteinase-2 (MMP-2) was significantly lower after TAGLN knock-down by siRNA. TAGLN levels were elevated in human gastric cancer stroma than normal gastric stroma and associated with differentiation and lymph node metastasis of gastric cancer.ConclusionCAFs may promote gastric cancer cell migration and invasion via upregulating TAGLN and TAGLN induced MMP-2 production.

CEACAM6 Promotes Gastric Cancer Invasion and Metastasis by Inducing Epithelial-Mesenchymal Transition via PI3K/AKT Signaling Pathway

Overexpressed CEACAM6 in tumor tissues plays important roles in invasion, metastasis and anoikis resistance in a variety of human cancers. We recently reported that CEACAM6 expression is upregulated in Gastric cancer (GC) tissues and promoted GC metastasis. Here, we report that CEACAM6 promotes peritoneal metastases in vivo and is negatively correlated with E-cadherin expression in GC tissues. Overexpressed CEACAM6 induced epithelial-mesenchymal transition (EMT) in GC, as measured by increases in the EMT markers N-cadherin, Vimentin and Slug while E-cadherin expression was decreased in CEACAM6-overexpressing GC cells; opposing results were observed in CEACAM6-silenced cells. Furthermore, E-cadherin expression was negatively correlated with depth of tumor invasion, lymph node metastasis and TNM stage in GC tissues. Additionally, CEACAM6 elevated matrix metalloproteinase-9 (MMP-9) activity in GC, and anti-MMP-9 antibody could reverse the increasing invasion and migration induced by CEACAM6. CEACAM6 also increased the levels of phosphorylated AKT, which is involved in the progression of a variety of human tumors. We further observed that LY294002, a PI3K inhibitor, could reverse CEACAM6-induced EMT via mesenchymal-epithelial transition. These findings suggest that CEACAM6 enhances invasion and metastasis in GC by promoting EMT via the PI3K/AKT signaling pathway.

CEACAM6 promotes tumor angiogenesis and vasculogenic mimicry in gastric cancer via FAK signaling.

CEACAM6 is a member of glycosylphosphatidylinositol-linked immunoglobulin superfamily that is implicated in a variety of human cancers. In our previous study, we reported that CEACAM6 was overexpressed in gastric cancer tissues and promoted cancer metastasis. The purpose of this study is to determine the role of CEACAM6 in tumor angiogenesis and mimicry formation. We found that overexpressed CEACAM6 promoted tubule formation dependent on HUVEC cells and vasculogenic mimicry formation of gastric cancer cells; opposing results were achieved in CEACAM6-silenced groups. Moreover, we found that mosaic vessels formed by HUVEC cells and gastric cancer cells were observed in vitro by 3D-culture assay. Overexpressed CEACAM6 in gastric cancer cells promoted tumor growth, VEGF expression and vasculogenic mimicry structures formation in vivo. In accordance with these observations, we found that phosphorylation of FAK and phosphorylation of paxillin were up-regulated in CEACAM6-overexpressing gastric cancer cells, and FAK inhibitor Y15 could reduce tubule and vasculogenic mimicry formation. These findings suggest that CEACAM6 promotes tumor angiogenesis and vasculogenic mimicry formation via FAK signaling in gastric cancer and CEACAM6 may be a new target for cancer anti-vascular treatment.

Stat6 cooperates with Sp1 in controlling breast cancer cell proliferation by modulating the expression of p21Cip1/WAF1 and p27Kip1

BackgroundThe signal transducer and activator of transcription 6 (Stat6), a member of the family of DNA-binding proteins, has been identified as a critical cell differentiation modulator in breast cancer cells. As of yet, the mechanisms underlying this function have remained largely unknown. To further elucidate the role of Stat6 in breast cancer development, we investigated the consequences of exogenous Stat6 expression.MethodsProliferation assays and flow cytometry assays were conducted to evaluate the putative role of Stat6 on cell proliferation. To this end, we produced synchronized cells after a double thymidine block, as confirmed by FACS analysis. mRNA levels of Stat6 were measured by RNase protection analysis. To confirm the interaction among proteins, we employed GST pull-down assays and immunoprecipitation assays. Luciferase assays and ChIP assays were used to assess the transcriptional activity.ResultsCompared to control breast cancer cells, we found that exogenous Stat6 expression plays a critical role in controlling cell proliferation. Also in different breast tumor cell lines, endogenous Stat6 expression was found to be positively related to a lower proliferation rate. Interestingly, in human breast cancer cells Stat6 functions in G1/S cell cycle progression, and the growth-inhibitory effect of Stat6 was shown to be mediated by induction of the G1 cyclin-dependent kinase inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Simultaneously, G1-related cyclin/cyclin-dependent kinase activities and pRB phosphorylation were markedly reduced, and cell cycle progression was blocked in the G1 phase. Stat6 knockdown resulted in enhanced cell proliferation and a decrease in p21 and p27 mRNA levels in the steroid-responsive and non-responsive T-47D and MDA-MB-231 cell lines, respectively. In addition, the stimulatory effect of Stat6 on p21 and p27 gene transcription was found to be associated with interaction of Stat6 with the transcription factor Sp1 at the proximal Sp1-binding sites in their respective promoters.ConclusionsTogether, these results identify Stat6 as an important cell differentiation regulatory protein functioning, at least in part, by interacting with Sp1 to activate the p21 and p27 gene promoters in breast cancer cells.

A Novel Plant Homeodomain Finger 10-Mediated Antiapoptotic Mechanism Involving Repression of Caspase-3 in Gastric Cancer Cells

The mechanisms governing tumorigenesis of gastric cancer have been an area of intense investigation. Currently, plant homeodomain (PHD) finger (PHF) proteins have been implicated in both tumor suppression and progression. However, the function of PHF10 has not been well characterized. Here, we show that various levels of PHF10 protein were observed in gastric cancer cell lines. Alteration of PHF10 expression, which is associated with tumor cell growth, may result in apoptosis in gastric cancer cells both in vitro and in vivo. Knockdown of PHF10 expression in gastric cancer cells led to significant induction of caspase-3 expression at both the RNA and protein levels and thus induced alteration of caspase-3 substrates in a time-dependent manner. Moreover, results from luciferase assays indicated that PHF10 acted as a transcriptional repressor when the two PHD domains contained in PHF10 were intact. Combined with previous findings, our data suggest that PHF10 transcriptionally regulates the expression of caspase-3. Finally, by using systematic reporter deletion and chromatin immunoprecipitation assays, we localized a region between nucleotides −270 and −170 in the caspase-3 promoter that was required for the efficient inhibition of caspase-3 promoter activity by PHF10. Collectively, our findings show that PHF10 repressed caspase-3 expression and impaired the programmed cell death pathway in human gastric cancer at the transcriptional level. Mol Cancer Ther; 9(6); 1764–74. ©2010 AACR.