Knockdown of LINC01087 inhibits gastric cancer malignant behavior by regulating the miR-135a-5p/CAAP1 axis.

Long noncoding RNAs play important roles in the occurrence and development of many malignant cancers. This study focuses on the effects of LINC01087 on gastric cancer and its underlying mechanism. In the present study, LINC01087 and CAAP1 were found to be upregulated, and miR-135a-5p was diminished in gastric cancer specimens and cells. Inhibition of LINC01087 resulted in cell proliferation inhibition and induced cell apoptosis through the intrinsic apoptosis signaling pathway, as evidenced by the activation of caspase-3 and caspase-9. An investigation of the signaling pathway revealed that the effects on proliferation and apoptosis following LINC01087 knockdown were mediated by suppression of CAAP1. Furthermore, application of a miR-135a-5p inhibitor or overexpression of CAAP1 could attenuate the apoptotic effect achieved by LINC01087 inhibition, confirming the involvement of miR-135a-5p/CAAP1 signaling in the occurrence of gastric cancer. In conclusion, the LINC01087/miR-135a-5p/CAAP1 axis modulates gastric cancer tumorigenesis and pathogenesis and presents new insight into gastric cancer targeted therapy.

A Panel of Three Biomarkers Identified by iTRAQ for the Early Diagnosis of Pancreatic Cancer.

PURPOSE: Due to a lack of early diagnostic markers, pancreatic cancer (PC) remains a lethal disease. Proteomic approaches are now being applied to identify novel PC biomarkers. EXPERIMENTAL DESIGN: In this study, iTRAQ and LC-MS/MS are used to perform comparative analyses of serum from PC patients and healthy controls (HC), to identify specific serum biomarkers for PC. Serum levels of candidate proteins are determined using ELISA. RESULTS: Among 869 proteins identified, 55 are potential biomarkers; Vitamin K-dependent protein Z (PROZ) and tumor necrosis factor receptor superfamily member 6b (TNFRSF6B) are selected for further analysis. Serum levels of PROZ and TNFRSF6B are significantly higher in PC patients than in HC or pancreatic benign controls (BC) (p < 0.01). The AUCs range from 0.816 to 0.971 for PROZ, TNFRSF6B, and carbohydrate antigen 19-9, either individually or in combination, in PC versus HC+BC, and from 0.711 to 0.932 in PC Stage I versus HC+BC. CONCLUSIONS AND CLINICAL RELEVANCE: It is demonstrated that PROZ and TNFRSF6B are novel serum biomarkers for detecting early stage PC, and for distinguishing PC from pancreatic benign tumor and healthy individuals. Additional large cohort studies are needed to develop PROZ and TNFRSF6B as clinical PC biomarkers.

Novel risk scoring system for prediction of pancreatic fistula after pancreaticoduodenectomy.

BACKGROUND: The available prediction models for clinically relevant postoperative pancreatic fistula (CR-POPF) do not incorporate both preoperative and intraoperative variables. AIM: To construct a new risk scoring system for CR-POPF that includes both preoperative and intraoperative factors. METHODS: This was a retrospective study of patients who underwent pancreaticoduodenectomy (PD) or pylorus-preserving PD (PPPD) between January 2011 and December 2016 at the First Affiliated Hospital of Soochow University. Patients were divided into a study (01/2011 to 12/2014) or validation (01/2015 to 12/2016) group according to the time of admission. POPF severity was classified into three grades: Biochemical leak (grade A) and CR-POPF (grades B and C). Logistic regression was used to create a predictive scoring system. RESULTS: Preoperative serum albumin ≥ 35 g/L [P = 0.032, odds ratio (OR) = 0.92, 95% confidence interval (CI): 0.85-0.99], hard pancreatic texture (P = 0.004, OR = 0.25, 95%CI: 0.10-0.64), pancreatic duct diameter ≥ 3 mm (P = 0.029, OR = 0.50, 95%CI: 0.27-0.93), and intraoperative blood loss ≥ 500 mL (P = 0.006, OR = 1.002, 95%CI: 1.001-1.003) were independently associated with CR-POPF. We established a 10-point risk scoring system to predict CR-POPF. The area under the curve was 0.821 (95%CI: 0.736-0.905) and the cut-off value was 3.5. Including drain amylase levels improved the predictive power of the model. CONCLUSION: This study established a 10-point scoring system to predict CR-POPF after PD/PPPD using preoperative and intraoperative parameters. Ultimately, this system could be used to distinguish between high- and low-risk populations in order to facilitate timely interventions after PD.

HIF-2α promotes the formation of vasculogenic mimicry in pancreatic cancer by regulating the binding of Twist1 to the VE-cadherin promoter.

Vasculogenic mimicry (VM) is a blood supply modality that occurs independently of endothelial cell angiogenesis. Hypoxia and the epithelial-mesenchymal transition (EMT) induce VM formation by remodeling the extracellular matrix. Our previous study demonstrated that hypoxia-inducible factor-2 alpha (HIF-2α) promotes the progress of EMT in pancreatic cancer; however, whether HIF-2α promotes VM formation in pancreatic cancer remains unknown. In this study, we investigated HIF-2α expression and VM by immunohistochemistry in 70 pancreatic cancer patients as well as the role of Twist1and Twist2 in HIF-2α-induced VM in vitro and in vivo. We found that the overexpression of HIF-2α and VM were correlated with poor tumor differentiation, late clinical stage and lymph node metastasis, and a poor prognosis in pancreatic cancer. Moreover, the upregulation of HIF-2α in SW1990 cells induced VM formation, whereas the opposite results were found after silencing HIF-2α in AsPC-1 cells. A mechanistic study indicated that HIF-2α might regulate the binding of twist1 to vascular endothelial cadherin (VE-cadherin) to promote VM formation in pancreatic cancer cells, and that the P1 (-421bp) and P4 (-2110bp) regions of the Twist1 binding sequences are positive regulatory elements for VE-cadherin. In addition, we confirmed that the overexpression of HIF-2α increased Twist1 expression and promoted tumor growth and VM formation in pancreatic cancer xenografts in nude mice. These findings indicated that HIF-2α might play a critical role in VM and that HIF-2α and the pathway of HIF-2α inducing VM formation are potential therapeutic targets for pancreatic cancer.

Expression and clinical significance of glucose transporter-1 in pancreatic cancer.

Increasing evidence has demonstrated that malignant cells exhibit increased glucose uptake, which facilitates survival and growth in a hypoxic environment. The glucose transporter-1 (GLUT-1) is overexpressed in a variety of malignant tumors. However, the association between GLUT-1 expression and clinicopathological factors, 18F-fluorodeoxyglucose uptake and tumor proliferation in pancreatic cancer has not been investigated to date. In the present study, the expression of GLUT-1 in 53 pancreatic cancer tissues was analyzed, which revealed that GLUT-1 was overexpressed in pancreatic tissue and correlated with poor prognosis and clinicopathological characteristics, including increased tumor size, clinical stage and lymph node metastasis, maximum standardized uptake value (SUVmax) and Ki-67 expression. The receiver operating characteristic curve analysis indicated that a cut-off SUVmax value of 4.830 was associated with optimal sensitivity (88%) and specificity (71.4%) for the detection of strong positive GLUT-1 expression. In addition, as the expression of GLUT-1 was found to correlate with Ki-67 expression, GLUT-1 may exhibit a significant effect on cell proliferation in pancreatic cancer. Overall, these findings indicate that GLUT-1 may represent a prognostic indicator, and a potential therapeutic target for pancreatic cancer.

Knockdown of angiopoietin-2 suppresses metastasis in human pancreatic carcinoma by reduced matrix metalloproteinase-2.

Angiopoietin-2 (Ang2) has been shown highly expressed in resected human pancreatic carcinoma samples, and has tightly combination with tumor angiogenesis, but the role in metastasis of it is less clear. We were, therefore, interested in exploring the effects of Ang2 silencing on the invasion and metastasis of pancreatic carcinoma. Lentivirus (LV)-mediated Ang2 small hairpin RNA (LV-RNAi) and mock lentivirus (LV-NC) were transfected into pancreatic carcinoma cell line MIA PaCa-2. Groups were designed in this study: the control group (MIA PaCa-2 cells), the LV-NC group (cells transfected with the LV-NC), the LV-RNAi-KD1 group (cells transfected with LV-RNAi of knock down sequence (1) and the LV-RNAi-KD2 group (cells transfected with LV-RNAi of knock down sequence (2). Boyden chamber transwell assay was used to detect the cell invasion change. The protein levels of Ang2, MMP-2, and MMP-9 gene and mRNA level of MMP-2, MMP-9 were detected by Western blot and real-time polymerase chain reaction, respectively. Orthotopic pancreatic carcinoma xenotransplantation model were successfully built with MIA PaCa-2 cells injection. After treatment with intraperitoneal injection of LV-RNAi-KD2 (LV-RNAi), mice growth, liver function test, tumor volume and peritoneal metastatic numbers were observed and counted. Moreover, expression of Ang2, MMP-2, MMP-9 were measured by immunohistochemistry. Ang2 expression were successfully knocked down in two LV-RNAi groups, especially in the LV-RNAi-KD2group. Compared with the control group and the LV-NC group, the mRNA and protein level of MMP-2 gene were downregulated significantly in LV-RNAi groups, also the invasion cell number decreased in boyden chamber transwell assay after LV-RNAi transfection. Meanwhile, no obvious MMP-9 gene expression changes were found among all the groups. LV-RNAi injection inhibited pancreatic carcinoma metastasis and growth in vivo by downregulating the expression of MMP-2 not MMP-9. Most importantly, LV-mediated gene therapy with Ang2 knockdown exhibited almost no toxicity in vivo. These findings demonstrate that Ang2 gene silencing exert an anti-metastasis effect in vitro and in vivo, and Ang2 targeted gene therapy has the potential to serve as a novel way for pancreatic carcinoma treatment.

Lentivirus-mediated shRNA interference targeting vascular endothelial growth factor inhibits angiogenesis and progression of human pancreatic carcinoma.

Angiogenesis is known to be essential to the survival, growth, invasion and metastasis of cancer cells. Vascular endothelial growth factor (VEGF) is an important factor regulating tumor angiogenesis. In the present study, we analyzed the effect of lentivirus-mediated shRNA interference targeting vascular endothelial growth factor (VEGF) on angiogenesis and progression in the pancreatic cancer cell line Patu8988 in vitro and in vivo. The study aimed to construct a recombinant lentivirus carrying targeted VEGF shRNA (LV-RNAi) to be used to transfect Patu8988 cells, and we investigated its anti-angiogenic and growth inhibitory effects on pancreatic cancer. VEGF expression was measured by RQ-PCR, western blotting and enzyme-linked immunosorbent assay (ELISA). In subcutaneous transplantation models, tumor volumes were determined, and the expression levels of VEGF and CD34 were assessed by immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was used to determine apoptosis. In the orthotopic transplantation models, tumor volume and liver metastasis were determined. We successfully constructed LV-RNAi and confirmed that it knocked down the VEGF gene at the mRNA and protein levels in Patu8988 cells. In the subcutaneous transplantation models, tumors with low levels of VEGF expression exhibited reduced pancreatic carcinoma angiogenesis and growth, and the apoptotic index was significantly higher. In the orthotopic transplantation models, tumors with low levels of VEGF expression exhibited significantly reduced pancreatic carcinoma growth, but no significant difference was observed between the three mouse groups, LV-RNAi, LV-NC and the control, in regards to liver metastasis. In summary, lentivirus-mediated RNAi silencing of VEGF inhibited tumor angiogenesis and growth, and increased apoptosis of the pancreatic cancer cell line Patu8988. VEGF targeted gene silencing approach has the potential to serve as a novel treatment for pancreatic cancer.

Silencing of B7-H3 increases gemcitabine sensitivity by promoting apoptosis in pancreatic carcinoma.

In numerous types of cancer, the expression of a novel member of the B7 ligand family, the B7-H3 immunoregulatory protein, has been correlated with a poor prognosis. In the present study, we investigated the role of B7-H3 in chemoresistance in pancreatic carcinoma. Silencing of B7-H3, through lentivirus-mediated delivery of stable short hairpin RNA, was observed to increase the sensitivity of the human pancreatic carcinoma cell line Patu8988 to gemcitabine as a result of enhanced drug-induced apoptosis. Overexpression of B7-H3 caused the cancer cells to be more resistant to the drug. Subsequently, we investigated the underlying mechanisms of B7-H3-mediated gemcitabine resistance, and found that the levels of survivin decreased in cells in which B7-H3 had been knocked down. In vivo animal experiments demonstrated that tumors in which B7-H3 had been knocked down displayed a slower growth rate compared with the control xenografts. Notably, gemcitabine treatment led to a strong antitumor activity in mice with tumors in which B7-H3 had been knocked down; however, this effect was only marginal in the control group. Furthermore, survivin expression was weak in gemcitabine-treated tumors in which B7-H3 had been knocked down and apoptosis was increased, as revealed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining. In summary, the present study demonstrated that B7-H3 induces gemcitabine resistance in pancreatic carcinoma cells, at least partially by downregulating survivin expression. These results provide novel insights into the function of B7-H3 and encourage the design and investigation of approaches targeting this protein in treating pancreatic carcinoma.

A study of the suppressive effect on human pancreatic adenocarcinoma cell proliferation and angiogenesis by stable plasmid-based siRNA silencing of c-Src gene expression.

The non-receptor protein tyrosine kinase c-Src regulates diverse biological processes by associating with multiple signaling and structural molecules. Overexpression of c-Src occurs in pancreatic cancer and can be predictive of poor prognosis. The aim of this study was to investigate the inhibitory effects of plasmid-based siRNAs targeting the human c-Src gene on proliferation and angiogenesis in the human pancreatic adenocarcinoma cell line Panc-1. Three siRNAs targeting the c-Src gene were transfected into the Panc-1 pancreatic adenocarcinoma cell line mediated by Lipofectamine. Transfection efficiency was assessed by flow cytometry. Real-time quantitative PCR (RQ-PCR) was employed to detect the expression of c-Src mRNA, and the most effective siRNA was chosen to be cloned into a plasmid. Two single-strand DNA templates were designed according to the most effective siRNA sequences. The short hairpin RNA (shRNA) plasmid targeting c-Src with pGPU6/green fluorescent protein (GFP)/Neo vector psiRNA-c-Src was constructed. Sequencing was performed to check whether the plasmid was constructed correctly. Panc-1 cells were transfected with psiRNA-c-Src and the negative control plasmid (psiRNA-N), respectively. Following selection with G418, the transfected monoclonal cells were chosen. GFP was evaluated by flow cytometry and fluorescence microscopy to estimate transfection efficiency. RQ-PCR and western blotting were used to detect c-Src silencing efficiency. To verify the effects of gemcitabine chemoresistance of c-Src expression, MTT assay was performed. ELISA was used to determine VEGF levels in culture supernatants. In a nude mouse model, tumor growth was studied, c-Src, VEGF expression and microvessel density in tumor tissue were measured by immunohistochemistry. The transfection efficiency of siRNA in the Panc-1 cell line was above 90%, the most effective siRNA could suppress expression of the c-Src gene with an inhibition efficiency of 86.1%. Sequencing confirmed that the c-Src siRNA plasmid was successfully constructed. MTT assay indicated that the effect of gemcitabine-induced cytotoxicity was markedly increased in the psiRNA-c-Src group (P<0.05). Meanwhile, the expression of VEGF in vitro was reduced significantly (P<0.05) in the psiRNA-c-Src group. In nude mice bearing tumors, c-Src, VEGF expression and MVD were decreased in tumors produced from psiRNA-c-Src transfected cells (P<0.05). In summary, the siRNA expression constructs targeting c-Src could specifically suppress c-Src expression, inhibit VEGF expression, inhibit cell proliferation and enhance gemcitabine chemosensitivity in vitro. C-Src gene silencingwas able to inhibit angiogenesis of tumors in vivo. These findings demonstrate that the c-Src targeting gene silencing approach has the potential to serve as a novel tool for pancreatic carcinoma treatment.

Clinical significance of expression and amplification of the DcR3 gene in pancreatic carcinomas.

This study aimed to investigate the clinical significance of expression and amplification of decoy receptor 3 (DcR3) in pancreatic carcinomas (PC). mRNA expression was detected by PQ-PCR, and amplification was determined. DcR3 protein expression was detected by immunohistochemistry and ELISA. Correlations between DcR3 expression and clinical pathological factors were analyzed. The relative amount of DcR3 in PC tissues and non-cancerous tissues showed a statistically significant difference, 21 cases displaying more than two fold DcR3 amplification, while no such amplification was found in normal pancreatic tissues. DcR3 positive cell staining was located in the cytoplasm. The positive rate of DcR3 in PC and non-cancerous tissues showed a significant difference. DcR3 mRNA expression was correlated with clinical staging, size of the tumor, lymph node metastasis and histological staging, while protein expression was correlated with clinical data like tumor size. DcR3 gene amplification only correlated with tumor size. The level of DcR3 in serum of the PC resectable group before operation was 72.2±10.2 pg/ml, showing a significant difference compared to gallbladder carcinoma group (GC) or pancreatic benign tumor (PBT) group (P <0.01). In conclusion, DcR3 amplification is correlated with DcR3 expression in PC tissues, especially those clinical pathological factors which reflect tumor progression. Assessment of DcR3 level in sera of PC patients may be helpful for the early diagnosis and prognostic judgement.

Clinicopathologic significance of slug expression in human intrahepatic cholangiocarcinoma.

AIM: To explore the expression and function of slug, a transcriptional repressor, in human intrahepatic cholangiocarcinoma (IHCC) and identify its role in IHCC progression. METHODS: Expression of slug was detected in 36 cases of IHCC and 12 cases of normal intrahepatic bile ducts and liver parenchyma by immunohistochemistry. The patients were divided into low slug expression group (< 20% of carcinoma cells stained) and high slug expression group (> or = 20% of carcinoma cells stained). Slug expression was correlated with clinicopathological parameters of IHCC patients. The patients were defined as short-term survivors if their survival time was < 12 mo and as long-term survivors if their survival time was > or = 12 mo. RESULTS: Slug was not expressed in normal liver epithelium samples, lowly expressed in 15 tissue samples (10 -, 5 +) and highly expressed in 21 tissue samples (16 ++; 5 +++) from IHCC patients. The survival rate of patients with a low slug expression was 33.3% (n = 5) and 66.7% (n = 10), respectively. The survival rate of patients with a high slug expression was 61.9% (n = 13) and 38.1% (n = 8), respectively (P = 0.02). Lymph node metastasis was found in 4 (26.7%) out of the 15 patients with a low slug expression and in 14 (66.7%) out of the 21 patients with a high slug expression, respectively. The incidence rate of lymph node metastasis increased with the increasing slug expression level (P = 0.003), and higher in patients with a high slug expression than in those with a low slug expression. Slug expression did not significantly correlate with the tumor size and stage or histologic grade, or with the gender and age of patients CONCLUSION: Slug expression is a novel prognostic marker for IHCC with lymph node metastasis.

Effect of endothelial PAS domain protein 1 and hypoxia inducible factor 1alpha on vascular endothelial growth factor expression in human pancreatic carcinoma.

BACKGROUND: Transcription factors hypoxia inducible factor 1alpha (HIF 1alpha) and endothelial PAS domain protein 1 (EPAS1) promote the transcription of vascular endothelial growth factor (VEGF). VEGF enhances angiogenesis and vascular permeability of tumours, which promotes tumour growth and facilitates entry of cancer cells into blood circulation and metastasizing. This study examined whether HIF 1alpha and EPAS1 stimulated angiogenesis through activation of VEGF in human pancreatic carcinoma. METHODS: Specimens from pancreatic carcinoma and healthy parts of same pancreas were taken from 60 patients. Real time quantitative reverse transcription polymerase chain reaction estimated expression of HIF 1alpha, EPAS1, and VEGF mRNAs. Western blotting and immunohistochemical, streptavidin peroxidase method assessed expression of HIF 1alpha, EPAS1, and VEGF proteins. Microvessel density (MVD) was assessed. RESULTS: Highly significant increases in expression of EPAS1, VEGF, and MVD were found in pancreatic carcinoma tissue but not in normal pancreatic tissue: VEGF at mRNA and protein levels (t = 17.32, P = 0.0001; t = 98.41, P = 0.0001); EPAS1 protein level (t = 22.51, P = 0.0001). Expression of HIF 1alpha was similar in pancreatic carcinoma and normal pancreatic tissues at both mRNA and protein levels. Significant correlations were observed between EPAS1 and VEGF (r = 0.736, P = 0.0041), between VEGF and MVD (r = 0.858, P = 0.0001), and between EPAS1 and MVD (r = 0.641, P = 0.0003). No significant correlations were observed between HIF 1alpha and VEGF, or between HIF 1alpha and MVD. MVD and expression of EPAS1 and VEGF were significantly related with TNM staging, so was EPASI and VEGF with size of tumour. CONCLUSIONS: EPAS1 and VEGF, but not HIF1alpha, are overexpressed in pancreatic carcinoma. The expression of EPAS1 is correlated with that of VEGF and MVD. EPAS1 may be involved in the angiogenesis of pancreatic carcinoma by upregulating the expression of VEGF. Targeting EPAS1 may be a new method of antiangiogenic tumour therapy for pancreatic carcinoma.