Combined ultrasound endoscopy-guided fine-needle aspiration with DNA methylation of SHOX2 and RASSF1A genes to enhance the auxiliary diagnostic precision of pancreatic cancer.

The purpose of this study was to assess the influence and the clinical effectiveness of the short stature homeobox 2 (SHOX2) and ras association domain family 1A (RASSF1A) genes by tissue sampling through ultrasound endoscopy-guided fine-needle aspiration (EUS-FNA) as auxiliary diagnostic tools for pancreatic cancer (PC). Methylation markers were detected in 96 patients using real-time fluorescence quantitative PCR (qPCR), and the performance of this diagnostic assay was compared with CA19-9, CEA, and puncture fluid-based exfoliative cytology using receiver operating characteristic curve (ROC) analysis. The PC group exhibited higher methylation rates for SHOX2, RASSF1A, and the combined assay of both genes compared to the control group (95.7 % vs. 54.0 %, 78.3 % vs. 36.0 %, and 73.9 % vs. 16.0 %, P < 0.05). The areas under the ROC curve (AUC) for CA19-9, CEA, liquid-based exfoliative cytology, SHOX2, RASSF1A, the combination of SHOX2 and RASSF1A, the combination assay with CEA, CA19-9, and liquid-based exfoliative cytology were 0.827, 0.692, 0.767, 0.770, 0.732, 0.870, 0.870, 0.933, and 0.900, respectively. Therefore, the methylation assay based on the combined SHOX2 and RASSF1A genes in EUS-FNA puncture fluid is more effective than using a single gene, liquid-based exfoliative cytology, or intravenous tumor markers for diagnosing PC. Combining the conventional marker CA19-9 enhances the diagnostic value, making it a promising approach to complement histology and cytology.

Macroscopic on­site quality evaluation of biopsy specimens to improve the diagnostic accuracy of endoscopic ultrasound­guided fine needle aspiration using a 22­gauge needle for solid lesions: A single­center retrospective study.

The present study aimed to evaluate the clinical value of macroscopic on-site evaluation (MOSE) of solid masses by endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) using a standard 22-gauge needle and to explore the cut-off length of macroscopic visible core (MVC) required to obtain an accurate histopathological diagnosis. In total, 119 patients who satisfied the inclusion and exclusion criteria and underwent EUS-FNA were divided into conventional FNA and FNA combined with MOSE groups. In the MOSE group, the presence of MVC was examined and its total length measured, after which the pathological results of FNA were compared with the final diagnosis. The diagnostic sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of FNA in the two groups were calculated and the effect of MOSE on the FNA result was analyzed. The MOSE group had a higher diagnostic sensitivity (75.0% vs. 89.8%; P=0.038) and accuracy (74.5% vs. 90.6%; P=0.026). MVC was observed in 98.4% (63/64) of patients in the MOSE group. The median length of MVC was 15 mm. The optimal cut-off length of MVC for obtaining an accurate histological diagnosis was 13 mm, with a sensitivity of 90.2%. No statistically significant significance was observed in the specificity, PPV and NPV between the groups. Thus, MOSE helps to improve the diagnostic ability of FNA for solid masses and may be a useful alternative to assess the adequacy of puncture specimens in units where rapid on-site evaluation cannot be performed.

Long noncoding RNA STCAT16 suppresses cell growth and its expression predicts prognosis in patients with gastric cancer.

Gastric cancer (GC) is a leading cause of cancer­associated mortality worldwide. Previous studies demonstrated that long noncoding RNAs (lncRNAs) may be dysregulated in GC and may serve important roles in cancer progression. The present study aimed to investigate the role of the novel lncRNA stomach cancer­associated transcript 16 (STCAT16; Assembly Gene ID G038291) in the development and progression of GC. The present data suggested that the expression level of STCAT16 was decreased in GC tissues. The expression level of STCAT16 was identified to be associated with lymph node and tumour node metastasis stages. Furthermore, the expression level of STCAT16 was identified to be significantly associated with poor survival and prognosis. Knockdown of STCAT16 promoted proliferation, colony formation, migration and invasion of BGC­823 cells. In contrast, these features were suppressed in AGS cells following overexpression of STCAT16. In vivo, tumour growth was significantly decreased following STCAT16 overexpression. Collectively, the present data suggested that the lncRNA STCAT16 may act as a tumour suppressor and may inhibit GC tumour cell growth and migration. Additionally, the decreased expression level of STCAT16 was identified to be associated with poor prognosis in patients with GC.

Dermcidin as a novel binding protein of lncRNA STCAT3 and its effect on prognosis in gastric cancer.

Long non­coding RNAs (lncRNAs) perform distinct biological functions by regulating gene expression through various molecular mechanisms under normal physiological and pathological conditions. However, the function of the stomach cancer­associated transcript­3 (STCAT3) lncRNA, including its prognostic significance and role as a binding protein in gastric cancer (GC), remain unclear. In the present study, 56 potential binding proteins of STCAT3 were screened using through mass spectrometry and bioinformatics analysis. Among these, dermcidin, GAPDH, annexin, calmodulin­like protein, cathepsin­D and suprabasin were demonstrated to be candidate binding proteins using a literature search. RNA­protein interaction prediction was used to confirm these six proteins. Finally, dermcidin was identified as the binding protein of STCAT3 by comparing the mRNA and protein levels of the candidate genes and their correlations with STCAT3 in plasmid­transfected BGC­823 GC cell lines, as well as by validating the interplay between dermcidin and STCAT3 in other GC cell lines. Immunohistochemical analysis of tissues from 98 patients with GC further confirmed the interaction between dermcidin and STCAT3. The results of the present study also revealed that STCAT3 and dermcidin and independent predictors of overall survival in patients with GC. Furthermore STCAT3 and dermcidin are positively correlated with lymph node metastasis and tumor/node/metastasis score. In summary, the present study suggests that dermcidin is a novel binding protein of lncRNA STCAT3, which serves an important role in the progress and clinical outcome of GC.

Nuclear transcription factor CDX2 inhibits gastric cancer­cell growth and reverses epithelial­to­mesenchymal transition in vitro and in vivo.

The epithelial­to­mesenchymal transition (EMT) has been noted as a critical event in the early step of cancer metastasis. Recent studies showed that nuclear transcription factor caudal type homeobox transcription factor 2 (CDX2) is a prognostic factor, which acts as a marker of good outcome in gastric cancer (GC) patients. However, the association between CDX2 expression and EMT has remained to be fully elucidated. The present study reported that forced overexpression of CDX2 in MKN45/CDX2 cells inhibited GC­cell growth and proliferation, and attenuated migration and invasion in vitro. Furthermore, MKN45/CDX2 cells exhibited a significant upregulation of E­cadherin protein and a significant downregulation of vimentin protein expression. These results were further supported by in vivo tumorigenicity assays, which showed that CDX2 suppressed gastric tumor xenograft growth and inhibited EMT in nude mice. These results indicated that CDX2 is capable of inhibiting GC­cell growth and invasion. CDX2 may participate in the process of EMT of GC cells by regulating the expression of the epithelial and mesenchymal proteins E­cadherin and vimentin.

microRNA-32 inhibits the proliferation and invasion of the SGC-7901 gastric cancer cell line in vitro.

microRNAs (miRNAs) are a class of endogenously expressed, small non-coding RNAs, which suppress their target mRNAs at the post-transcriptional level. miRNAs play key roles in tumor metastasis. The aim of the present study was to investigate the expression of miRNA-32 (miR-32) on the biological behavior of the human gastric cancer cell line, SGC-7901. SGC-7901 cells were transfected with miR-32-mimic, miR-32-inhibitor and empty plasmid vectors using Lipofectamine™ 2000. The expression of GFP was observed by fluorescent microscopy and miR-32 gene expression was detected by quantitative polymerase chain reaction. The cell counting kit-8 assay was performed to evaluate the effect of miR-32 expression on cell proliferation in vitro. Alterations in the migration and metastatic potential of SGC-7901 cells, prior to and following miR-32 gene transfection, were assayed by cell chemotactic migration and invasion tests. The results of the current study showed that the proliferation rate of the transfected SGC-7901 cells overexpressing miR-32 is reduced and cell chemotactic migration and invasion potentials is markedly reduced following miR-32-mimic transfection (P<0.05). In addition, the results demonstrated that overexpression of miR-32 greatly inhibits the proliferation and decreases the migration and invasion capabilities of SGC-7901 cells in vitro.

Reactivation of the homeotic tumor suppressor gene CDX2 by 5-aza-2'-deoxycytidine-induced demethylation inhibits cell proliferation and induces caspase-independent apoptosis in gastric cancer cells.

The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) is widely used as an anticancer drug for the treatment of leukemia and solid tumors. Gastric cancer (GC) patients who were positive for caudal type homeobox transcription factor 2 (CDX2) expression showed a higher survival rate compared with those who were CDX2 negative, which suggests that CDX2 performs a tumor suppressor role. However, the molecular mechanisms leading to the inactivation of CDX2 remain unclear. In the present study we demonstrated that the expression levels of CDX2 and DNA methyltransferase enzyme 1 (DNMT1) mRNA were significantly higher in GC compared with distal non-cancerous tissue. The expression of CDX2 mRNA was significantly correlated with Lauren classification, TNM stage and lymph node metastasis. DNMT1 mRNA expression was significantly correlated with TNM stage, pathological differentiation and lymph node metastasis. The expression of CDX2 mRNA was inversely correlated with that of DNMT1 mRNA in GC. Hypermethylation of the CDX2 gene promoter region, extremely low expression levels of CDX2 mRNA and no expression of CDX2 protein were the characteristics observed in MKN-45 and SGC-7901 GC cell lines. Following the treatment of MKN-45 cells with 5-aza-CdR, the hypermethylated CDX2 gene promoter region was demethylated and expression of CDX2 was upregulated, while DNMT1 expression was downregulated. Furthermore, a concentration- and time-dependent growth inhibition as well as increased apoptosis were observed. Caspase-3, -8 and -9 activities increased in a concentration-dependent manner following exposure to different concentrations of 5-aza-CdR. Therefore, our data show that the overexpression of DNMT1 and methylation of the CDX2 gene promoter region is likely to be responsible for CDX2 silencing in GC. 5-Aza-CdR may effectively induce re-expression of the CDX2 gene, inhibit cell proliferation and enhance the caspase-independent apoptosis of MKN-45 cells in vitro.

[Expression of GC-C mRNA in peripheral blood mononuclear cells and its clinical roles in determining state of gastric cancer].

OBJECTIVE: To detect the expression of GC-C mRNA in peripheral blood of gastric carcinoma patients and determine its potential candidacy as a molecular biological marker for predicting the micrometastasis and determining the status of gastric cancer. METHODS: GC-C mRNA from peripheral blood samples of gastric carcinoma (n = 60), dysplastic (n = 21), intestinal metaplasia (n = 15) and healthy cases (n = 20) from November 2009 to August 2010 at Affiliated Hospital of Nantong University were assessed by real-time fluorescent quantitative PCR (RFQ-PCR). RESULTS: The expressions of GC-C mRNA in peripheral blood from patients with dysplasia, intestinal metaplasia and healthy controls were absent or very low. And a high level of GC-C mRNA was detected in the patients with gastric carcinoma than those with dysplastic and intestinal metaplasia, and the positive rate were 48.3% (29/60), 9.5% (2/21), 20.0% (3/15), respectively (all P < 0.05). The levels of GC-C mRNA were significantly correlated with Lauren typing, clinical stage, tumor differentiation degree, depth of invasion and lymph node metastasis (all P < 0.05). The GC-C mRNA expressions were positively correlated in peripheral blood and gastric carcinomas tissues (r = 0.4009, P = 0.0015). CONCLUSIONS: The over-expression of GC-C mRNA is found in peripheral blood from gastric carcinoma patients. Due to its close correlation with clinical stage and lymph node metastasis, it may become a potential prognostic marker of gastric carcinoma.

Ectopic expression of guanylyl cyclase C and endogenous ligand guanylin correlates significantly with Helicobacter pylori infection in gastric carcinogenesis.

The molecular mechanisms leading to gastric carcinogenesis still remain unclear. Recently, several studies demonstrated that over-expression of guanylyl cyclase C (GCC) has been detected in intestinal-type gastric cancer (GC) and precursor lesions. Our objective was to explore the expression levels of GCC and endogenous ligands guanylin (GN) and uroguanylin (UGN) and the correlation between Helicobacter pylori (H. pylori) and GCC, GN, and UGN expressions in patients at different stages from normal mucosa to superficial gastritis, atrophic gastritis, intestinal metaplasia (IM), dysplasia, and finally adenocarcinoma. The expression of GCC and GN was absent in the distal normal gastric tissues and superficial gastritis in all cases, whereas they were measured in IM, dysplasia, and GC. The expression of GCC and GN was closely related to intestinal-type GC. From superficial gastritis to gastric carcinomas, the H. pylori positive rate was 19.7, 33.3, 69.6, 80.0, and 82.1%, respectively. The positive correlation was found between GCC and GN in IM, dysplasia, and GC. Also, the positive correlation was found between GCC, GN, and H. pylori infection in them. These results demonstrate that the detection of GCC and GN will be beneficial to diagnosis human gastric carcinoma and precancerous lesions. Ectopic expression of GCC and GN in human gastric mucosa and H. pylori infection may play an important role in the carcinogenesis of the intestinal-type GC.

Lentivirus-mediated short hairpin RNA targeting the APRIL gene suppresses the growth of pancreatic cancer cells in vitro and in vivo.

RNA interference (RNAi) is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Herein, we constructed a short hairpin RNA (shRNA) lentiviral expression vector targeting a proliferation-inducing ligand (APRIL) gene in the CFPAC-1 cell (a type of cell strain of human pancreatic cancer) in order to observe the inhibitory effect of APRIL gene's shRNA on the growth of the CFPAC-1 cell in vitro and in vivo. The results showed that lentivirus-mediated RNAi effectively inhibited the expression of APRIL mRNA and protein in CFPAC-1 cells. Moreover, it can inhibit the growth of pancreatic cancer cells in vitro and in vivo. Our study indicates that lentivirus-mediated gene therapy is an attractive strategy in the treatment of pancreatic cancer and justifies the use of lentivirus in cancer gene therapy studies.

[Expression of guanylyl cyclase-c and caudal type homeobox transcription factor 2 in human gastric cancer and precursor lesions].

OBJECTIVE: To investigate the expression of guanylyl cyclase-C (GC-C) and caudal type homeobox transcription factor 2 (CDX2) in human gastric mucosa at different stages and the significance thereof. METHODS: An Immunofluorescence method was used to detect the expression of GC-C and CDX-2 in 23 specimens of gastric carcinoma and matching noncancerous tissues. Western blotting was used to detect the protein expression of GC-C and CDX2 in the gastric carcinoma tissues and matching noncancerous tissues too. RESULTS: The GC-C and CDX2 expression rates were 39.1% and 39.1% respectively in the intestinal metaplasia specimens, 55.6% and 55.6% respectively in the dysplasia specimens, and 56.7 % and 60.0% in the gastric carcinoma specimens, all significantly higher than those in the normal mucosa specimens (all P = 0.000) without significant differences in the expression of GC-C and CDX-2 among the 3 pathological groups. The GC-C and CDX-2 expression was positively correlated with Lauren classification, The expression levels of GC-C and CDX-2 were significantly higher in the intestinal-type than in the diffuse-type gastric carcinoma (P < 0.05). The GC-C expression was positively correlated with the expression of CDX-2 in intestinal metaplasia and gastric carcinoma. CONCLUSION: Ectopic expression of GC-C and CDX2 in human gastric mucosa may play an important role in the carcinogenesis of intestinal-type gastric carcinoma. Detection of GC-C and CDX2 helps diagnose gastric carcinoma and precursor lesions.