MiR-143 Targeting TAK1 Attenuates Pancreatic Ductal Adenocarcinoma Progression via MAPK and NF-κB Pathway In Vitro.

BACKGROUND: Transforming growth factor (TGF)-ß-activated kinase 1 (TAK1) is one of the major regulators of inflammation-induced cancer cell growth and progression. MiR-143 dysregulation is a common event in a variety of human diseases including pancreatic ductal adenocarcinoma (PDA). AIMS: To identify the interaction between TAK1 and miR-143 in PDA. METHODS: Data mining of TAK1 expression in PDA patient gene profiling was conducted. QRT-PCR and western blot were performed to detect the expression of TAK1 in PDA tissues and cell lines. Ectopic miR-143 and TAK1 were introduced to PDA cells. Cell growth, apoptosis and migration were examined. Xenograft models were used to examine the function of TAK1 in vivo. Western blot and luciferase assay were carried out to investigate the direct target of miR-143. RESULTS: PDA patient gene profiling data (GSE15471 and GSE16515) showed that TAK1 mRNA was aberrantly up-regulated in PDA tissues. TAK1 protein levels were overexpressed in PDA tissues and cell lines. Overexpression of TAK1 was strongly associated with positive lymph node metastasis. Inhibition of TAK1 suppressed cell growth, migration, and induced cell apoptosis in vitro and in vivo. Further studies demonstrated that TAK1 was a direct target gene of miR-143. MiR-143 also inhibited PDA cells proliferation and migration, induced apoptosis and G1/S arrest. Moreover, TAK1 depletion inactivated MAPK and NF-κB pathway, mimicking the function of miR-143. CONCLUSIONS: The study highlights that miR-143 acts as a tumor suppressor in PDA through directly targeting TAK1, and their functional regulation may provide potential therapeutic strategies in clinics.

Silencing of GATA6 suppresses SW1990 pancreatic cancer cell growth in vitro and up-regulates reactive oxygen species.

BACKGROUND/AIMS: Pancreatic cancer has the worst prognosis of any gastrointestinal cancer with a mortality rate approaching its incidence. Previous studies have indicated that GATA6 plays a key role in organ development and function, and that abnormal expression of GATA6 may induce tumorigenesis. Meanwhile, it has been reported that generation of reactive oxygen species contributes to carcinogenesis. In this study, we set out to study the role of GATA6 expression on proliferation and apoptosis of pancreatic cancer cells and the role of reactive oxygen species. METHODS: Four target miRNA sequences against GATA6 mRNA were synthesized and used to transfect SW1990 cells. Then, GATA6 expression in SW1990 cells was examined by western blot and quantative real-time polymerase chain reaction. Cell proliferation was examined by WST-8 and colony formation assay. Cell cycle progression and apoptosis were measured by flow cytometry. We also measured the generation of reactive oxygen species by immunofluorescence and flow cytometry. RESULTS: RNA interference against GATA6 successfully inhibited mRNA and protein expression of GATA6 in the SW1990 pancreatic cancer cell line. Silencing of GATA6 by RNA interference inhibited cell proliferation and increased apoptosis of SW1990, and enhanced the expression of reactive oxygen species. CONCLUSIONS: These results suggest that the RNA interference approach against GATA6 may be an effective therapeutic approach for treatment of pancreatic cancer.

Characterization of a cancer stem cell-like side population derived from human pancreatic adenocarcinoma cells.

AIMS AND BACKGROUND: To identify and partially characterize the side population cells derived from three human pancreatic adenocarcinoma cell lines. METHODS: Side population cells were sorted from the human pancreatic adenocarcinoma cell lines SW1990, Capan-2, and BxPC-3 using flow cytometry and then analyzed for cell proliferation, clone formation, differentiation, chemoresistance, invasive potential, and tumorigenicity in a mouse model. RESULTS: Human pancreatic carcinoma cell lines SW1990, Capan-2, and BxPC-3 contain 2.7% +/- 0.35%, 3.6% +/- 1.2%, and 2.8% +/- 0.8% side population cells, respectively. We further investigated cancer stem cell characteristics with the moderately differentiated human pancreatic adenocarcinoma cell line SW1990. Flow cytometry analysis revealed that side population cells could differentiate into side population and non-side population cells and could exhibit differentiation potential. Using a clone formation assay, side population cells were shown to have a higher proliferation than non-side population cells. Compared to non-side population cells, side population cells were also more resistant to gemcitabine, a commonly used anti-cancer agent against pancreatic carcinoma, and were more invasive. Importantly, the CD133 level in side population cells was significantly higher than that in non-side population cells. The enhanced tumorigenecity was further confirmed in a male diabetic/severe combined immunodeficiency mouse model. As few as 3 x 10(3) side population cells were sufficient to induce tumor formation in the mouse model, compared to 10(7) non-side population or unsorted cells. CONCLUSIONS: Side population cells isolated from human pancreatic adenocarcinoma cell lines harbor cancer stem cell-like properties that may be related to the invasive potential and therapeutic-resistance of pancreatic adenocarcinoma.

[Experimental study of MAT1 gene silencing mediated by siRNA in pancreatic cancer].

OBJECTIVE: To investigate the inhibitory effect of gene silencing mediated by MAT1-siRNA constructed in vitro transcription for pancreatic cancer in vivo and in vitro. METHODS: 21-nt double strand siRNA targeting MAT1 gene was constructed and labeled with Cy3 fluorescent labeling reagent. Human pancreatic cancer cells of the line BxPC3 were cultured and divided into 4 groups: MAT1-siRNA transfected group, negative siRNA control group, lipid control group, and blank control group. The rate of cell duplication was determined by counting the cells for three consecutive days. Cell cycle profiles were determined by flow cytometry. Semi-quantitative analysis of the level of MAT1-mRNA expression was performed using the RT-PCR technique. The level of MAT1 protein expression was analyzed by Western-blotting. 18 nude mice were injected subcutaneously with BxPC3 cells to establish mouse tumor models, and then divided randomly into 3 equal groups: MAT1-siRNA group undergoing injection of MAT1-siRNA directly into the tumors 2 times a week for 4 weeks, blank control group, and negative MAT1-siRNA group. 4 weeks later the mice were killed to observe the weight and size of tumor and to calculate the tumor inhibition rate. RESULTS: Two of the 4 designed MAT1-siRNAs significantly suppressed the growth of the BxPC3 cells. 72 h after transfection the cell duplication was inhibited by 34.9% in the MAT1-siRNA transfection group. The cell cycle profile showed 83.9% of the MAT1-siRNA transfected cells were in the G0/G1 phase, a rate significantly higher than that in the blank control group (59.86%, P < 0.01). 48 h later the content of MAT1-mRNA of the MAT1-siRNA transfected cells was significantly reduced by 80.12%, and 72 h after the transfection the content of MAT1 protein was reduced by 50.12%, a rate significantly higher than those of the 2 control groups (both P < 0.01). The weight and volume of the transplant tumors in the MAT1-siRNA injected nude mice were significantly reduced compare with the negative siRNA injected control nude mice and blank control nude mice (both P < 0.05). The inhibition rate was 42.53%. CONCLUSION: MAT1 gene silencing mediated by siRNA significantly suppresses the growth of pancreatic cancer cells in vitro, and significantly achieves an anti-tumor effect on the subcutaneously transplanted pancreatic tumor in vivo.

The novel long intergenic noncoding RNA UCC promotes colorectal cancer progression by sponging miR-143.

The human genome contains thousands of long intergenic noncoding RNAs (lincRNAs). However, the functional roles of these transcripts and the mechanisms responsible for their deregulation in colorectal cancer (CRC) remain elusive. A novel lincRNA termed upregulated in CRC (UCC) was found to be highly expressed in human CRC tissues and cell lines. UCC levels correlated with lymph node metastasis, Dukes' stage, and patient outcomes. In SW480 and SW620 cells, knockdown of UCC inhibited proliferation, invasion, and cell cycle progression and induced apoptosis in vitro. Xenograft tumors grown from UCC-silenced SW620 cells had smaller mean volumes and formed more slowly than xenograft tumors grown from control cells. Inversely, overexpression of UCC in HCT116 promoted cell growth and invasion in vitro. Bioinformatics analysis, dual-luciferase reporter assays, and RNA immunoprecipitation assays showed that miR-143 can interact with UCC, and we found that UCC expression inversely correlates with miR-143 expression in CRC specimens. Moreover, mechanistic investigations showed that UCC may act as an endogenous sponge by competing for miR-143, thereby regulating the targets of this miRNA. Our results suggest that UCC and miR-143 may be promising molecular targets for CRC therapy.

[A meta-analysis of clinical efficiency of two methodologies of cholangiopancreatography].

OBJECTIVE: To compare the efficiency of endoscopic retrograde cholangiopancreatography (ERCP) and magnetic resonance cholangiopancreatography (MRCP) in patients with suspected biliary tract or pancreatic diseases. METHODS: Find those prospective comparison trials about the efficiency of ERCP and MRCP in patients with suspected biliary tract or pancreatic diseases from many kinds of database, such as MEDLINE, EMBASE and so on. According to inclusion criteria, two operant choose suitable papers for this study. Collect corresponding original data and make a meta-analysis to compare the sensitivity and specificity of ERCP and MRCP in choledocholithiasis, strictures and malignant tumor. RESULTS: Finally we get 6 articles from 302 ones. To diagnose choledocholithiasis, strictures and malignant tumor, the difference of sensitive between ERCP's and MRCP's is not significant. When it comes to the specificity of ERCP and MRCP in those diseases, ERCP is better than MRCP only in strictures, OR is 6.17 (95% CI 1.35-20.24), P = 0.02. However, we find ERCP is better than MRCP not only in total sensitivity but specificity of biliary tract or pancreatic diseases, OR is 1.72 (95% CI 1.04-2.85) and 4.05 (95% CI 1.32-12.42) respectively, P = 0.04, 0.01. CONCLUSIONS: ERCP is better than MRCP, to biliary tract or pancreatic diseases, in not only sensitivity but specificity. Doctors should think much about patients' situation, tolerance and cost-effectiveness, when they make a decision which examination should patients take.

Noncoding RNAs and pancreatic cancer.

Noncoding RNAs (ncRNAs) represent a class of RNA molecules that typically do not code for proteins. Emerging data suggest that ncRNAs play an important role in several physiological and pathological conditions such as cancer. The best-characterized ncRNAs are the microRNAs (miRNAs), which are short, approximately 22-nucleotide sequences of RNA of approximately 22-nucleotide in length that regulate gene expression at the posttranscriptional level, through transcript degradation or translational repression. MiRNAs can function as master gene regulators, impacting a variety of cellular pathways important to normal cellular functions as well as cancer development and progression. In addition to miRNAs, long ncRNAs, which are transcripts longer than 200 nucleotides, have recently emerged as novel drivers of tumorigenesis. However, the molecular mechanisms of their regulation and function, and the significance of other ncRNAs such as piwi-interacting RNAs in pancreas carcinogenesis are largely unknown. This review summarizes the growing body of evidence supporting the vital roles of ncRNAs in pancreatic cancer, focusing on their dysregulation through both genetic and epigenetic mechanisms, and highlighting the promise of ncRNAs in diagnostic and therapeutic applications of pancreatic cancer.

[The effect of recombinant adenovirus encoding antisense MAT1 on cell cycle of human pancreatic cancer cells BxPC-3].

OBJECTIVE: To investigate the regulatory effect of the human MAT1 gene on the cell cycle G(1)/S transition of human pancreatic cancer BxPC-3 cells. METHODS: To construct the replication deficient recombinant adenovirus of antisense MAT1 gene using homologous recombination by AdEasy system. Cell growth assay was carried out by counting alive cells after trypan blue exclusion. The protein expressions of MAT1, cyclin D1, cyclin E, and Rb were detected by western blotting. The cell cycle status was determined by flow cytometry. RESULTS: The recombinant adenovirus encoding antisense MAT1 fragment Ad-MAT1AS was obtained with the titer of expression was 5 x 10(10) pfu/ml. The expression of MAT1 of BxPC-3 was significantly reduced after Ad-MAT1AS infection. In this case BxPC-3 cell cycle was arrested in G(1) phase. The estimated proportion of G(0)/G(1) phase cells in the control for blank and vector cultures ranged from 40% to 44%. In contrast, 79% of the Ad-MAT1AS cells were in G(0)/G(1) phase. Cyclin E and pRb gene expression changes were observed in the infected cells. CONCLUSION: The results suggest that MAT1 gene may play an important role in the regulation of cell cycle G(1)-->S transition of BxPC-3 cells.

Clinical analysis of primary small intestinal disease: A report of 309 cases.

AIM: To evaluate the major clinical symptom, etiology, and diagnostic method in patients with primary small intestinal disease in order to improve the diagnosis. METHODS: A total of 309 cases with primary small intestinal disease were reviewed, and the major clinical symptoms, etiology, and diagnostic methods were analyzed. RESULTS: The major clinical symptoms included abdominal pain (71%), abdominal mass (14%), vomiting (10%), melaena (10%), and fever (9%). The most common disease were malignant tumor (40%). diverticulum (32%) and benign tumor (10%). Duodenal disease was involved in 36% of the patients with primary small intestinal diseases. The diagnostic rate for primary small intestinal diseases by double-contrast enteroclysis was 85.6%. CONCLUSION: Abdominal pain is the most common clinical symptom in patients with primary small intestinal disease. Malignant tumors are the most common diseases. Duodenum was the most common part involved in small intestine. Double-contrast enteroclysis was still the simplest and the most available examination method in diagnosis of primary small intestinal disease. However, more practical diagnostic method should be explored to improve the diagnostic accuracy.

Inhibition of hedgehog signaling depresses self-renewal of pancreatic cancer stem cells and reverses chemoresistance.

Pancreatic cancer stem cells play a crucial role in tumorigenesis and chemoresistance. The Hedgehog signaling pathway is a key regulator in pancreatic tumorigenesis and drug resistance. To identify pancreatic cancer stem cells, tumorspheres derived from the PANC-1 pancreatic cancer cell line were cultured under a floating-culture system. PANC-1 tumorspheres possessed properties of self-renewal, differentiation, higher tumorigenesis and chemoresistance. It was observed that Hedgehog pathway is active in PANC-1 tumorspheres as shown by expression of hedgehog components Smo, Gil 1 and Gli 2, detected by quantitative RT-PCR and western blotting. After cyclopamine-mediated blockade of hedgehog, a decrease in proliferation of PANC-1 tumorspheres and G0/G1 transition were observed, as well as a decreased expression of Bmi-1 in PANC-1 tumorspheres. Cyclopamine reversed chemoresistance to gemcitabine, resulting in decreased expression of ABCG2 in PANC-1 tumorspheres. Taken together, our data indicate that PANC-1 tumorspheres have 'stemness' potential, and hedgehog signaling pathway plays an important role in the regulation of self-renewal and reversal of chemoresistance in cancer stem cells in pancreatic adenocarcinoma.

Thymidylate synthase genetic polymorphisms and cancer risk: a meta-analysis of 37 case-control studies.

BACKGROUND: Several studies have evaluated the association between polymorphisms of thymidylate synthase (TS) and cancer risk in diverse populations but with conflicting results. By pooling the relatively small samples in each study, it is possible to evaluate the association using a meta-analysis. METHODS: A comprehensive search was conducted to identify all case-control studies on TS on a 28-bp tandem repeats in 5'untranslated region (5'UTR) and a 6-bp insertion (ins) and deletion (del) mutation in 3'UTR of the gene and cancer risk. Meta-analysis was conducted using a fixed and random effect model. RESULTS: Our meta-analysis on a total of 13 307 cancer cases and 18 226 control subjects from 37 published case-control studies showed no significant association between the risk of cancer and the 5'UTR 28-bp tandem repeats polymorphism (3R/3R vs. 2R/2R: OR = 1.06, 95%CI, 0.93 - 1.20) or the 3'UTR 6-bp ins/del polymorphism (del6/del6 vs. ins6/ins6: OR = 0.93, 95%CI, 0.81 - 1.08) with significant between-study heterogeneity. In the cancer type- and ethnic subgroup-stratification analyses, we did not find any association between TS polymorphisms and cancer risk either. CONCLUSION: TS 5'UTR 28-bp tandem repeats and 3'UTR 6-bp ins/del polymorphisms may not be associated with cancer risk.

[Effect of NK4 gene transfection on tumor growth of human pancreatic cancer xenograft in nude mice].

BACKGROUND & OBJECTIVE: NK4 is not only an antagonist of hepatocyte growth factor but also an angiogenesis inhibitor. Studies have confirmed that NK4 can inhibit tumor growth and metastasis, but its effect on pancreatic cancer remains unknown. This study was designed to observe the effect of NK4 gene on human pancreatic cancer in nude mice and the possible mechanisms. METHODS: The nude mouse model of pancreatic cancer was established with human pancreatic cancer cell line SW1990. The eukaryotic expression vector of NK4 gene was constructed and transfected into the tumors. The mice weight, tumor size and volume were measured before and after transfection. The apoptotic cells, microvessel density (MVD), and the expression of proliferating cell nuclear antigen (PCNA) in the tumors were observed using immunohistochemistry and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. RESULTS: Four weeks after NK4 gene transfection, the tumor volume and weight was significantly smaller in NK4-transfected group than in PBS control group and empty vector group [(1.39+/-0.33) cm(3) vs. (2.06+/-0.55) cm(3) and (1.90+/-0.36) cm(3), P<0.01; (1.30+/-0.81) g vs. (3.45+/-1.88) g and (3.14+/-1.51) g, P<0.01]; the inhibition rate was 62.29%. The tumor cell apoptotic index was significantly higher in NK4-transfected group than in the rest 2 groups (9.34+/-0.91 vs. 4.13+/-0.79 and 3.94+/-1.03, P<0.001); the MVD was significantly lower in NK4-transfected group than in the rest 2 groups (12.24+/-4.63 vs. 20.13+/-7.00 and 19.70+/-6.15, P<0.05); the expression of PCNA in NK4-transfected group was not different from those of the rest 2 groups (53.88+/-4.30 vs. 56.24+/-4.03 and 54.33+/-5.41,P>0.05). CONCLUSION: NK4 gene transfection may inhibit the growth of human pancreatic cancer in mouse model through suppressing angiogenesis and enhancing the apoptosis of pancreatic cancer cells.