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PDF file - 11375K, A. Sorting of lentivirally transduced cell cultures to highest purity based on GFP expression. B. Western blot analysis of Tiam1 protein expression. C. Real-time RT-PCR analysis of Tiam1 mRNA expression. D. Rac1 activity determined by G-LISA assays. E. Western blotting for β-catenin in the cytoplasm and nucleus of panc-1 cells after transfection with si-rac1 or si-β-catenin or stimulation with NSC23766. F. CCK-8 analysis of cell proliferation after transfection with si-rac1 or si-β-catenin or treatment with NSC23766. G. EDU analysis of cell proliferation after transfection with si-rac1 or si-β-catenin or treatment with NSC23766.

Tiam1 is a rac1-specific guanine nucleotide exchange factor, and Tiam1-rac1 is involved in a number of cellular processes. Rac1 and RhoA act as molecular switches that cycle between GTP- and GDP-bound states to balance the activities of rac1 and RhoA. The downregulation of rac1 activity leads to upregulation of RhoA activity, which promotes invasion and migration of pancreatic cancers cells. At present, however, the role of Tiam1-rac1 and RhoA in pancreatic cancers is not fully understood. We found that Tiam1 was upregulated in pancreatic cancers and was significantly expressed in tumors without lymph node involvement or distant metastasis compared with cancers where there was involvement. Although Tiam1-rac1 signaling promoted pancreatic cancer cell proliferation and tumor growth via the Wnt signaling pathway in vitro and in vivo, inhibiting Tiam1-rac1 signaling did not prolong the overall survival time in vivo. This provided evidence that there was a balance between rac1 and RhoA activities in pancreatic cancers. Furthermore, only the combined inhibition of Tiam1-rac1 and RhoA had a beneficial effect on the growth of pancreatic cancers in vivo. Taken together, these results suggest that the progression of pancreatic tumors is partially controlled by the balance between Tiam1-rac1 and RhoA. Mol Cancer Res; 11(3); 230–9. ©2012 AACR.

PDF file - 9152K, A. Wound healing assay for BxPC-3 cells after Tiam1 knock-down. B. Determination of BxPC-3 cell migration and invasiveness after Tiam1 knock-down using transwell assays. C. Determination of Panc-1 cell migration and invasiveness after Tiam1 knock-down using transwell assay. D. Transwell assays of invasiveness after treatment with NSC23766 and/or Y27632.

Figure S6: IL-18 promotes proliferation and invasion of PC cells via the NF-κB pathway.

Purpose: We sought to find new immune-based treatments for pancreatic cancer.Experimental Design: We detected IL18 expression in plasma and specimens from patients with pancreatic cancer. We then investigated whether IL18 had a therapeutic effect for pancreatic cancer in vitro and in vivo and any underlying mechanisms.Results: Higher plasma IL18 was associated with longer overall survival (OS), but higher IL18 in pancreatic cancer tissues was associated with shorter OS and increased invasion and metastasis. Recombinant IL18 alone had no antitumor effect in the syngeneic mice with orthotopically transplanted tumors and promoted tumors in immunocompromised mice; it also facilitated immune responses in vitro and in vivo by augmenting the activity of cytotoxic T cells and NK cells in peripheral blood and lymph nodes. However, IL18 promoted the proliferation and invasion of pancreatic cancer cells, in vitro and in vivo, through the NF-κB pathway. Nevertheless, by coadministrating IL18 with BAY11-7082, an NF-κB inhibitor, we were able to prevent the procancerous effects of IL18 and prolong the survival time of the mice.Conclusions: IL18 has both cancer-promoting and cancer-suppressing functions. Although its single-agent treatment has no therapeutic effect on pancreatic cancer, when combined with the NF-κB pathway inhibitor, IL18 improved survival in a murine pancreatic cancer model. Our study implies the possibility of a combinational immunotherapy that uses IL18 and targets NF-κB pathway. Clin Cancer Res; 22(23); 5939–50. ©2016 AACR.

Supplemental Material and Methods

Background This study aimed to identify new protein markers that can evaluate the metastatic ability of Pancreatic neuroendocrine tumors (PanNETs) and predict prognosis together with Ki-67. Methods Multicentric series analysis was performed to evaluate the effect of metastasis on overall survival (N = 1,109). Single-cell RNA sequencing, proteomics (32 PanNETs and 10 normal pancreas tissues), and immunohistochemical staining (152 PanNETs) were conducted to screen proteins associated with PanNET metastatic capability. Associations between expression of screened proteins and survival were examined using Cox regression. In vitro experiments were performed to verify the function of candidate protein. A survival signature was constructed using recursive partitioning and survival forest analyses. Results The combination of G grade and metastasis predicted long-term survival more accurately though retrospective analysis. Single-cell RNA-seq highlighted the PanNET characteristics of metastasis that do not correspond with G grade and identified high metastatic capability subgroups in PanNET. Five proteins (PIN1, POSTN, SEMA4F, ASPN, and KCDT12), which may be related to the metastatic capability of PanNETs, were identified by proteomics. Random survival forest analysis showed that PIN1 could be a marker of PanNET metastatic ability. PIN1 overexpression promoted metastasis through LAMIN in PanNET cells. Finally, a survival signature with a consistency index of 0.921 and strong calibration was established, with good ability to predict patient survival. Conclusion In summary, we found that PIN1 can precisely evaluate the metastatic potential of PanNETs and, together with the proliferation marker Ki-67, can accurately predict the survival of PanNET patients.

AJCC TNM stage and WHO grade (G) are two widely used staging systems to guide clinical management for pancreatic neuroendocrine neoplasms (panNENs), based on clinical staging and pathological grading information, respectively. We proposed to integrate TNM stage and G grade into one staging system (TNMG) and to evaluate its clinical application as a prognostic indicator for panNENs. Accordingly, 5254 patients diagnosed with panNENs were used to evaluate and to validate the applicability of TNMG to panNENs. The predictive accuracy of TNMG system was compared with that of each separate staging/grading system. We found that TNM stage and G grade were independent risk factors for survival in both the Surveillance, Epidemiology, and End Result (SEER) and multicenter series. The interaction effect between TNM stage and G grade was significant. Twelve subgroups combining the TNM stage and G grade were proposed in the TNMG stage, which were classified into five stages TNMG. According to the TNMG staging classification in the SEER series, the estimated median survival for stages I, II, III, IV, and V were 203, 174, 112, 61, and 8 months, respectively. The predictive accuracy of TNMG stage was higher than that of TNM stage and G grade used independently. The TNMG stage classification was more accurate in predicting panNEN patient's prognosis than either the TNM stage or G grade.

In this work, the extended Debye model or Fourier transform is adopted to convert the polarization and depolarization current test results from the time domain into the frequency domain at low frequencies, thereby shortening the test time but producing large errors when the oil-paper insulation exhibits nonlinear characteristics. The voltage-current response characteristics of samples with three different moisture contents are tested to study the influence of moisture on the nonlinear characteristics. Hammerstein-Wiener (H-W) equivalent mathematical model is proposed for time-frequency domain conversion, and the results from this model and two formal methods are compared. The findings show that the errors of the two conventional methods increase with nonlinearity. The H-W model accurately depicts the nonlinear dielectric response of oil-paper insulation and thus can provide high-precision time-frequency domain conversion data.

Background: Pancreatic cancer was prone to metastasis, which leaded to the poor survival and low quality of life. Here, we found that FAM83H-AS1 was highly correlated with metastasis of pancreatic cancer and further explored the functions and mechanisms involving in the malignant progression of pancreatic cancer. Method: We profiled and analyzed the aberrant expression of lncRNA (long non-coding RNA) in TCGA database and screened FAM83H-AS1 most effective in promoting the migration of pancreatic cancer cells. Firstly, the expression level of FAM83H-AS1 in serum, tissues and cells were detected by RT-qPCR, and the distribution of it in pancreatic cancer cells was tested by FISH and nuclear/cytosol fractionation RT-qPCR. Next, a series of functional assays were conducted to elucidate the relationship between FAM83H-AS1 and pancreatic cancer in vivo and vitro. The regulation relationship between FAM83H-AS1 and FAM83H was verified by protein and RNA degradation assays respectively. A series of Co-immunoprecipitation assays was utilized to explore the potential mechanism of FAM83H and β-catenin. Rescue assays were used to illustrate the regulation axis of FAM83H-AS1/FAM83H/ Wnt/β-catenin in pancreatic cancer.Results: FAM83H-AS1 was highly expressed in cancer tissues and serum of PDAC patients, and was correlated with poorer survival. FAM83H-AS1, as an oncogene, promoted the proliferation, invasion and metastasis of PDAC cells by functional assays and bioinformatics analysis. FAM83H, the homologous gene of FAM83H-AS1, also manifested the same biological behaviour as FAM83H-AS1 in PDAC. Also, FAM83H-AS1 protected FAM83H mRNA from degradation and FAM83H directly bound β-catenin. Mechanically, FAM83H-AS1 facilitated the expression of FAM83H by stabilizing its mRNA and promoted FAM83H to decrease the ubiquitylation of β-catenin, which activated the effect genes of Wnt/β-catenin signalling.Conclusions: This study revealed the function of FAM83H-AS1 involving in the progression of PDAC, and might provide a new prognostic indicator and therapy target for PDAC.

Purpose. This study was aimed at investigating the roles of plasma miR-181b, miR-196a, and miR-210 in the diagnosis of pancreatic cancer (PC). Methods. Plasma samples were isolated from 40 patients with PC and 40 healthy individuals, respectively. The expression of miR-181b, miR-196a, and miR-210 was detected by qRT-PCR. The level of carbohydrate antigen 199 (CA199) was measured by an electrochemiluminescence (ECL) assay. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of miR-181b, miR-196a, miR-210, CA199, and their combinations in PC. Results. The expression of plasma miR-181b, miR-196a, and miR-210 was significantly upregulated in PC patients. The plasma level of CA199 was also significantly increased in PC patients. The expression of miR-181b, miR-196a, and miR-210 was closely associated with lymph node metastasis, clinical stage, and vascular invasion but not correlated with age, gender, and tumor size. miR-181b, miR-196a, and miR-210 have lower AUC than CA199 in the diagnosis of PC. miR-181b+miR-210 and miR-196a+miR-210 also have lower AUC than CA199. It is worth noting that miR-181b+miR-196a+miR-210 has a higher AUC than CA199 in the diagnosis of PC. Conclusion. The combination of plasma miR-181b, miR-196a, and miR-210 had a good diagnostic value for PC.

Cancer stem cells are the main reason for drug resistance and tumor relapse, and screening the targets for cancer stem cells is essential for tumor therapy. Here, we studied the role and regulatory mechanism of a G protein-coupled receptor named as G protein-coupled receptor 87 (GPR87) in the expansion of pancreatic ductal adenocarcinoma (PDA) stem cells. We found that GPR87 was an independent prognostic factor for PDA patients: patients with high GPR87 had a poor outcome. GPR87 significantly promoted the sphere formation ability, increased side population (SP) cell number, increased the expression of PDA stem cell markers, and increased the tumor initiation ability, suggesting that GPR87 promotes the expansion of PDA stem cells. Mechanism analysis suggested that signal transducer and activator of transcription 3 (STAT3) directly bound to the promoter of GPR87 to increase GPR87 expression; inversely, GPR87 also activated STAT3. Further analysis suggested that GPR87 activated Janus kinase 2 (JAK2), which can activate STAT3, inhibiting JAK2 activation in GPR87-overexpressing PDA cells, which significantly inhibited the expansion of PDA stem cells; these findings suggested that GPR87, JAK2, and STAT3 formed a positive feedback loop increasing PDA stem cell population. In PDA specimens, GPR87 expression is positively correlated with the phosphorylation level of STAT3 and JAK2, confirming GPR87 promoted PDA stem cell expansion through activating JAK2/STAT3. In summary, we found that GPR87, together with JAK2 and STAT3, formed a positive feedback loop to promote the expansion of PDA stem cells., Graphical Abstract

Purpose This study aimed to explore the regulatory effect of long noncoding RNA (lncRNA) ribonuclease mitochondrial RNA processing gene (RMRP) on hepatocellular carcinoma (HCC). Methods The expression of RMRP in HCC tissues and cell lines was assessed by qRT-PCR. Kaplan-Meier method was utilized to analyze the correlation between RMRP expression and the survival of HCC patients. MHCC97H and HuH7 cells were transfected with pcDNA3.1-RMRP or pcDNA3.1, respectively. MTT and flow cytometry assays were conducted to examine the proliferation and apoptosis of HCC cells, respectively. The migration and invasion of HCC cells were assessed using wound healing and transwell assays , respectively. StarBase3.0 and dual-luciferase reporter gene assay were used to identify the target relationship between miR-766 and RMRP. A xenografted tumor model was established in rats to evaluate the effect of RMRP in vivo. Results RMRP was down-regulated in HCC tissues and cells. Low expression of RMRP was correlated with poor survival of HCC patients. The A495 value and colony number were significantly decreased in pcDNA3.1-RMRP-transfected MHCC97H and HuH7 cells. The apoptosis rate was significantly increased in pcDNA3.1-RMRP-transfected MHCC97H and HuH7 cells. The migration rate and the number of invasive cells were significantly decreased in pcDNA3.1-RMRP-transfected MHCC97H and HuH7 cells. MiR-766 was a target of RMRP and eliminated the anti-tumor effect of RMRP on MHCC97H cells. The up-regulation of RMRP suppressed the growth of xenograft tumors in rats. Conclusion Overexpression of RMRP suppressed the tumorigenesis of HCC by targeting miR-766.

Laparoscopic duodenum-preserving total pancreatic head resection (LDPPHRt) is used for treating benign or low-grade malignant tumors of the pancreatic head. However, preservation of the duodenum and biliary tract integrity remains challenging. We present a new approach for LDPPHRt and evaluate its feasibility and safety.From April 2020 to December 2020, 30 patients successfully underwent LDPPHRt using the intracapsular approach in our center. Their medical records were reviewed for relevant clinical characteristics, pathologic findings, postoperative complications, and survival.The median diameter of the lesions was 3.6 cm (range, 2.0-5.5 cm). The median operative time was 234.7 min (range, 195-310 min). The median blood loss was 66.7 ml (range, 20-250 ml). The morbidity rate was 26.7%, including POPF, hemorrhage, lymphatic leakage, wound infection, pulmonary infection, and delayed gastric emptying. Five patients developed pancreatic fistula type A, and two patients had type B, classified according to the International Study Group on Pancreatic Fistula. No biliary tract injury or duodenal leakage was observed. The median postoperative hospital stay was 11.5 days (range, 6-25), and the operative mortality rate was 0%.The intracapsular approach is a feasible and safe surgical procedure in LDPPHRt for patients with benign or low-grade malignant tumors, especially those without severe pancreatic head fibrosis or peripancreatic adhesions.

Gemcitabine is the first-line treatment for patients with pancreatic cancer (PC), yet most patients develop resistance to gemcitabine. Recent studies showed that circular RNAs (circRNAs) have important regulatory roles in PC progression and chemoresistance. In this study, the ability of circRNA circ_0092367 to enhance gemcitabine efficacy was tested and the underlying molecular mechanism of circ_0092367 was investigated. The expression levels of circ_0092367, miR-1206, and ESRP1 were measured using qRT-PCR experiments. The effects of circ_0092367, miR-1206, and ESRP1 on PC cell lines exposed to gemcitabine were examined by CCK-8 assays. We performed luciferase assays to determine the relationship between circ_0092367 and miR-1206 and between miR-1206 and ESRP1. We demonstrated that circ_0092367 was significantly downregulated in PC tissues and cell lines, and a high expression of circ_0092367 was associated with improved survival in patients with PC. Gain- and loss-of-function assays revealed that circ_0092367 inhibited epithelial–mesenchymal transition (EMT) phenotypes and sensitized PC cells to gemcitabine treatment in vitro and in vivo. Cytoplasmic circ_0092367 could directly repress the levels of miR-1206 and thus upregulate the expression of ESRP1, thereby inhibiting EMT and enhancing the sensitivity of PC cells to gemcitabine treatment. Our findings show that circ_0092367 plays a crucial role in sensitizing PC cells to gemcitabine by modulating the miR-1206/ESRP1 axis, highlighting its potential as a valuable therapeutic target in PC patients.

Validity of the laparoscopic approach in pancreatic head lesion remains debatable. This study aims to compare the safety and effectiveness of laparoscopic pancreatoduodenectomy (LPD) and open pancreatoduodenectomy (OPD) and investigate the source of heterogeneity from surgeons' and patients' perspectives.We searched PubMed, Cochrane, Embase, and Web of Science for studies published before February 1, 2021. Of 6578 articles, 81 were full-text reviewed. The primary outcome was mortality. Three independent reviewers screened and extracted the data and resolved disagreements by consensus. Studies were evaluated for quality using ROB2.0 and ROBINS-I. According to different study designs, sensitivity and meta-regression analyses were conducted to explore the heterogeneity source. This meta-analyses was also conducted to explore the learning curve's heterogeneity. This study was registered with PROSPERO, CRD42021234579.We analyzed 34 studies involving 46,729 patients (4705 LPD and 42,024 OPD). LPD was associated with lower (P = 0.025) in unmatched studies (P = 0.017). No differences in mortality existed in randomized controlled trials (P = 0.854) and matched studies (P = 0.726). Sensitivity analysis found no significant difference in mortality in elderly patients, patients with pancreatic cancer, and in high- and low-volume hospitals (all P 0.05). In studies at the early period of LPD (40 cases), higher mortality (P 0.001) was found (all P 0.05).LPD showed non-inferiority in length of stay, complications, and survival outcomes in all analyses.In high-volume centers with adequate surgical experience, LPD in selected patients appears to be a valid alternative to LPD with comparable mortality, LOS, complications, and survival outcomes.

In contrast to the steady increase in survival observed for most cancer types, advances have been slow for pancreatic cancers. Current chemotherapy has limited benefits for patients with pancreatic cancer. Therefore, there is an urgent need for effective pancreatic cancer treatment strategies. At present, targeting the autophagic pathway is regarded as a promising new strategy for cancer treatment. Danthron (1,8-dihydroxyanthrquinone), a component from Rheum palmatum L. (polygonaceae), has several biological activities. However, the inhibition of autophagy by danthron has never been recognized, previously.Here we find that danthron may prevent autophagy, inhibit proliferation and induce apoptosis in pancreatic cancer cells in vitro. Autophagy induced by doxorubicin plays a protective role in pancreatic cancer cells and inhibition of autophagy by chloroquine or silencing autophagy protein 5 (Atg5) may chemosensitize pancreatic cancer cell lines to doxorubicin. Similarly, inhibition of autophagy by danthron also enhances toxicity of doxorubicin to pancreatic cancer cells. These results indicate that danthron has an anticancer effect and can sensitize the chemotherapeutic effect of doxorubicin on pancreatic cancer cells. These findings also suggest that inhibition of autophagy may be an effective way to promote the chemotherapy of pancreatic cancer.

BACKGROUND: Clinically relevant postoperative pancreatic fistula (CR-POPF) continues to be a major contributor to morbidity after pancreaticoduodenectomy (PD), but it remains unclear what risk factors can precisely predict the development of CR-POPF after laparoscopic pancreatoduodenectomy (LPD). We thus aimed to identify the risk factors for predicting CR-POPF after LPD. METHODS: A total of 388 consecutive patients who underwent LPD at our institution between July 2014 and December 2018 were identified. All data, including pre-, intra-, and postoperative risk factors associated with CR-POPF defined by the International Study Group of Pancreatic Fistula, were collected retrospectively. To evaluate the predictive performance of the risk factor models, areas under the receiver operating characteristic curve (ROC) were determined. RESULTS: CR-POPF was observed in 31 patients (8.0%) with significant association observed with body mass index (BMI), visceral fat area (VFA), subcutaneous fat area (SFA), total fat area (TFA), intra-abdominal fat thickness, main pancreatic duct width, soft pancreatic texture, operative time, underlying pathology, and albumin (Alb) on postoperative days (POD) 1­–3. Multivariate analyses revealed that VFA >82 cm(2) [odds ratio (OR) =11.088; P=0.029], main pancreatic duct width 320 min (OR =6.061; P221 cm(2) (OR =8.637; P=0.001) and VFA >82 cm(2) (OR =7.009; P82 cm(2), main pancreatic duct width 320 min were independent predictive risk factors of CR-POPF for LPD.

Background: Due to the significant heterogeneity in previous studies, concerns persist regarding the safety and effectiveness of laparoscopic pancreatoduodenectomy (LPD). Therefore, with the publication of new researches, a comprehensive systematic review is needed to evaluate the potential benefits of laparoscopic pancreatoduodenectomy. We aimed to compare safety and effectiveness of laparoscopic and open pancreatoduodenectomy (OPD) from the published studies, and investigate the source of heterogeneity from the surgeons’ and patients’ perspectives. Methods: We searched PubMed, Cochrane, Embase, and Web of Science databases for relevant studies published before February 1, 2021 comparing laparoscopic and open pancreatoduodenectomy. The database search yielded a total of 6,578 articles, and 81 articles were full-text reviewed. Studies of laparoscopic-assisted surgery were not included. The primary outcome was mortality. Three independent reviewers screened and extracted the data, and resolved disagreements by consensus. Studies were evaluated for quality using ROB2·0 and ROBINS-I. Meta-analyses were conducted in random-effect and fixed-effect models. Different meta-analyses were conducted according to the different designs of the studies. The sensitivity analyses and meta-regression were conducted to explore the source of heterogeneity. This study was registered with PROSPERO, ID CRD42021234579. Findings: We analyzed 34 studies involving 46,729 patients (4,705 LPD and 42,024 OPD). LPD was associated with lower mortality in overall studies (odds ratio [OR] 0·800, 95% confidence interval [CI] 0·659–0·973, P=0·025, I2=0·0%) and unmatched studies (OR 0·774, 95%CI 0·628–0·954, P=0·017, I2=0·0%). However, no differences in mortality were seen in RCTs (randomized controlled trial) (risk ratio 1·080, 95%CI 0·473–2·466, P=0·854, I2=13·4%) and matched studies (OR 0·904, 95%CI 0·515–1·587, P=0·726, I2=0·0%). Sensitivity analysis found that mortality was also similar between the two groups in elderly patients, patients with pancreatic ductal adenocarcinoma (PDAC), high-volume hospitals, and low-volume hospitals (all P >0·05). However, LPD showed non-inferiority in length of stay (LOS), severe complications, clinical relevant-postoperative pancreatic fistula, clinical relevant-biliary leak, intra-abdominal infection, readmission, overall survival, and 2-year relapse. Elderly patients benefited marginally from LPD, while patients with PDAC and patients in high-volume hospitals benefit significantly. Meta-regression confirmed that baseline differences including the proportion of males, age, proportion of biliary drainage, diagnosis of PDAC, and lesion size between LPD and OPD groups had a significant impact on LOS, severe complications, ICU stay, estimated intraoperative blood loss, and lymph node harvest (all P

Background:There is no widely-accepted staging system for pancreatic neuroendocrine tumors (pNETs). The aim of this study was to validate the American Joint Committee on Cancer (AJCC) 8th edition staging system for well-differentiated (G1/G2) pNETs.Methods:A multicenter dataset (n=1086) was used to evaluate the application of the AJCC 7th and 8th, the European Neuroendocrine Tumor Society (ENETS), and the modified ENETS (mENETS) staging systems for well-differentiated pNETs.Results:The proportion of patients with stage III tumors was extremely low (1.1%) according to the AJCC 7th staging system. For the ENETS staging system, patients with stage IIIA disease had worse estimated mean survival than patients with stage IIIB disease (78.9 vs. 107.3 months). When comparing with patients in stage I, the AJCC 7th, ENETS, and mENETS staging systems showed good performance in discriminating between stages; however, there was no significant difference in some stages when the reference was defined as the earlier stage. When the reference was defined as stage I or the earlier stage, there was a significant inter-stage difference in the AJCC 8th staging system.Conclusions:The AJCC 8th staging system is more suitable for pNETs than other TNM staging systems and may be adopted in clinical practice.

BackgroundPancreatic cancer is one of the most lethal human cancers.N6-methyladenosine (m6A), a common eukaryotic mRNA modification, plays critical roles in both physiological and pathological processes. However, its role in pancreatic cancer remains elusive.MethodsLC/MS was used to profile m6A levels in pancreatic cancer and normal tissues. Bioinformatics analysis, real-time PCR, immunohistochemistry, and western blotting were used to identify the role of m6A regulators in pancreatic cancer. The biological effects of methyltransferase-like 14 (METTL14), an mRNA methylase, were investigated using in vitro and in vivo models. MeRIP-Seq and RNA-Seq were used to assess the downstream targets of METTL14.ResultsWe found that the m6A levels were elevated in approximately 70% of the pancreatic cancer samples. Furthermore, we demonstrated that METTL14 is the major enzyme that modulates m6A methylation (frequency and site of methylation). METTL14 overexpression markedly promoted pancreatic cancer cell proliferation and migration both in vitro and in vivo,via direct targeting of the downstreamPERPmRNA (p53 effector related to PMP-22) in an m6A-dependent manner. Methylation of the target adenosine lead to increasedPERPmRNA turnover, thus decreasing PERP (mRNA and protein) levels in pancreatic cancer cells.ConclusionsOur data suggest that the upregulation of METTL14 leads to the decrease of PERP levels via m6A modification, promoting the growth and metastasis of pancreatic cancer; therefore METTL14 is a potential therapeutic target for its treatment.

For decades researches of genomic transcription of all kinds of species have demonstrated that the important role of Long non-coding RNAs (LncRNAs) in whole process of life entity has been more and more attached. Owing to constant developing of advanced technology, especially the emerge of next generation sequencing, researchers could explore further in the depth and breadth of LncRNAs. Given that the unique RNA loci location with its corresponding sense gene, antisense long noncoding RNAs (AS-lncRNAs), which are one of the main categories of LncRNAs classification, would have existed an identified close connection between them in a natural physiological state. This review characterizes the patterns of regulation between AS-lncRNAs and corresponding sense genes during the process of cancer progression in human, with emphases on the regular modulation ways of the potential molecular mechanism of AS-lncRNAs and the summary of underlying treatment targets in human cancers.

Purpose: Cancer stem cells (CSCs) initiate and maintain tumorigenesis due to their unique pluripotency. However, pancreatic stem cell gene signatures are not completely revealed yet. Here, we isolated pancreatic cancer stem cells (P-CSCs) and exploited their distinct genome-wide mRNA and miRNA expression profiles using microarrays. Methods: CD24+ CD44+ ESA+ cells were isolated from two pancreatic xenograft cells by the flow cytometry and identified the stem cell-like properties by the tumor formation, self-renew and chemoresistance. Microarrays and qRT-PCR were used to exploit their distinct Genome-wide mRNA and miRNA expression profiles. The function and candidate target genes of key microRNA were detected after Ectopic restoration in the pancreatic cancer cell lines MIA Paca-2 (CSChigh) and BxPC-3 (CSClow). Results: In this study, we isolated P-CSCs from two xenografts cells. Genome-wide profiling experiments showed 479 genes and 15 microRNAs specifically expressed in the P-CSCs, including genes involved in TGF-β and p53 signaling pathways and particularly miR-146b-3p as the most significantly downregulated miRNA. We confirmed miR-146b-3p as a downregulated signature in pancreatic cancer tissues and cell line MIA Paca-2 (CSChigh) cells. Ectopic restoration of miR-146b-3p expression with pre-miR reduced cell proliferation, induced apoptosis, increased G1 phase and reduced S phase in cell cycle in MIA Paca-2 (CSChigh), but not in BxPC-3 (CSClow). Re-expression of miR-146b-3p with lentivirus significantly inhibited tumorigenicity in vivo in MIA Paca-2, but slightly in BxPC-3. Furthermore, we demonstrated that miR-146b-3p directly targeted MAP3K10 and might activate Hedgehog pathway as well through DYRK2 and GLI2. Conclusions: These results suggest that P-CSCs have distinct gene expression profiles. MiR-146b-3p inhibits proliferation and induced apoptosis in P-CSCs high cells lines by targeting MAP3K10. Targeting P-CSCs specific genes may provide novel strategies for therapeutic purposes.

Additional file 6: Table S1. Association between clinicopathological features and m6A mRNA levels.

The lysosome is emerging as a central regulator of the autophagic process, which plays a critical role in tumor growth and chemoresistance. Alantolactone, which is a natural compound produced by Inula helenium, has been shown to induce apoptosis in numerous cancer types. However, the mechanism by which alantolactone regulates apoptosis is still poorly understood. In this work, we observed that alantolactone caused the accumulation of autophagosomes due to impaired autophagic degradation and substantially inhibited the activity and expression of CTSB/CTSD proteins that when depleted caused lysosomal dysfunction. Furthermore, we found that alantolactone inhibited the proliferation of pancreatic cancer cells in vitro and in vivo and enhanced the chemosensitivity of pancreatic cancer cells to oxaliplatin. In addition, a reduction in TFEB levels was a critical event in the apoptosis and cell death caused by alantolactone. Our data demonstrated that alantolactone, which impaired autophagic degradation, was a pharmacological inhibitor of autophagy in pancreatic cancer cells and markedly enhanced the chemosensitivity of pancreatic cancer cells to oxaliplatin.

Purpose This study aimed to explore the regulatory effect of long noncoding RNA (lncRNA) ribonuclease mitochondrial RNA processing gene (RMRP) on hepatocellular carcinoma (HCC). Methods The expression of RMRP in HCC tissues and cell lines was assessed by qRT-PCR. Kaplan–Meier method was utilized to analyze the correlation between RMRP expression and the survival of HCC patients. MHCC97H and HuH7 cells were transfected with pcDNA3.1-RMRP or pcDNA3.1, respectively. MTT and flow cytometry assays were conducted to examine the proliferation and apoptosis of HCC cells, respectively. The migration and invasion of HCC cells were assessed using wound healing and transwell assays , respectively. StarBase3.0 and dual-luciferase reporter gene assay were used to identify the target relationship between miR-766 and RMRP. A xenografted tumor model was established in rats to evaluate the effect of RMRP in vivo. Results RMRP was down-regulated in HCC tissues and cells. Low expression of RMRP was correlated with poor survival of HCC patients. The A495 value and colony number were significantly decreased in pcDNA3.1-RMRP-transfected MHCC97H and HuH7 cells. The apoptosis rate was significantly increased in pcDNA3.1-RMRP-transfected MHCC97H and HuH7 cells. The migration rate and the number of invasive cells were significantly decreased in pcDNA3.1-RMRP-transfected MHCC97H and HuH7 cells. MiR-766 was a target of RMRP and eliminated the anti-tumor effect of RMRP on MHCC97H cells. The up-regulation of RMRP suppressed the growth of xenograft tumors in rats. Conclusion Overexpression of RMRP suppressed the tumorigenesis of HCC by targeting miR-766.

BACKGROUND Inflammation plays an important role in initiation and development of severe acute pancreatitis (SAP). Curcumin exerts potent anti-inflammatory effects in many diseases, including acute pancreatitis. However, the specific molecular mechanisms are not clear. MATERIAL AND METHODS Intra-biliopancreatic duct injection of taurocholate was used to establish an animal model of SAP. Curcumin was administrated to animals as pre-treatments. Concentrations of cytokines in serum and ascites were measured by enzyme-linked immunosorbent assay (ELISA). A colorimetric method was used to determine the amylase activity. Western blotting was used to examine the expression levels and phosphorylation levels of proteins. Immunoprecipitation was used to assess the molecular association between apoptosis signal- regulating kinase 1 (ASK1) and thioredoxin (Trx). RESULTS Pre-treatment with curcumin reduced the concentrations of interleukin (IL6) and tumor necrosis factor (TNFα) in serum and ascites, as well as the ascites volume and amylase activity in SAP rats. Pre-treatment with curcumin reduced the expression level of TNF receptor-associated factor 1 (TRAF1), IL6, and TNFa in pancreas in SAP rats. Moreover, the phosphorylation levels of mitogen-activated protein kinase (MAPK) kinase 4 (MKK4), MKK7, and c-Jun NH(2)-terminal protein kinase (JNK) were reduced by curcumin pre-treatment. The molecular association between ASK1 and Trx was recovered by curcumin pre-treatment. As a result, the nuclear translocation of nuclear factor kappa B (NF-κB) was suppressed in pancreases from SAP rats. CONCLUSIONS Activation of the TRAF1/ASK1/JNK/NF-κB signaling pathway is involved in the inflammation of SAP. Curcumin exerts anti-inflammatory effects by suppressing this proinflammatory pathway.

Pancreatic cancer (PC) is highly resistant to current therapies; thus, there is an urgent need to develop new treatment strategies. The proteasome is crucially important for proteostasis, which is involved in cell proliferation and survival, making it an attractive therapeutic target in cancer. However, recent studies have indicated that bortezomib, a highly selective proteasome inhibitor, has limited effects in solid tumors including PC. Thus, more mechanistic insights into chemo-sensitization strategies for bortezomib are urgently needed. Herein, we demonstrate that bortezomib induced apoptosis and autophagy via a mechanism involving endoplasmic reticulum (ER) stress in PC cells. Additionally, bortezomib treatment led to increased levels of intracellular reactive oxygen species (ROS), which play critical roles in bortezomib-induced ER stress and apoptosis. Moreover, autophagy functions as a compensatory mechanism to eliminate bortezomib-induced ROS and resists ER stress-mediated apoptosis. Additionally, the Nrf2-mediated antioxidative response, which works against with bortezomib-induced autophagy, also protected cells against bortezomib-induced ROS production. Finally, the dual inhibition of autophagy and Nrf2 signaling cooperatively enhanced bortezomib-induced apoptosis by elevating ROS levels and ER stress. Together, these data demonstrate that activation of autophagy and the Nrf2 antioxidant system, which lowers intracellular ROS, are mechanistically how PC cells overcome bortezomib treatment. In summary, combining proteasome inhibitors with drugs targeting autophagy and Nrf2 signaling could be a promising therapeutic approach for PC treatment.

Background: Pancreatic cancer is highly malignant and characterised by rapid and uncontrolled growth. While some of the important regulatory networks involved in pancreatic cancer have been determined, the cancer relevant genes have not been fully identified. Methods: We screened genes that may control proliferation in pancreatic cancer in seven pairs of matched pancreatic cancer and normal pancreatic tissue samples. We examined KIF15 expression in pancreatic cancer tissues and the effect of KIF15 on cell proliferation in vitro and in vivo. The mechanisms underlying KIF15 promotion of cell proliferation were investigated. Results: mRNA microarray and functional analysis identified 22 genes that potentially play an important role in the proliferation of pancreatic cancer. High-content siRNA screening evaluated whether silencing these 22 genes affected proliferation of pancreatic cancer. Notably, silencing KIF15 exhibited the most potent inhibition of proliferation compared with the rest of the 22 genes. KIF15 was upregulated in human pancreatic cancer tissues, and higher KIF15 expression levels correlated with shorter patient survival times. Upregulation KIF15 promoted pancreatic cancer growth. KIF15 upregulated cyclin D1, CDK2, and phospho-RB and also promoted G1/S transition in pancreatic cancer cells. KIF15 upregulation activated MEK–ERK signalling by increasing p-MEK and p-ERK levels. MEK–ERK inhibitors successfully inhibited cell cycle progression, and PD98059 blocked KIF15-mediated pancreatic cancer proliferation in vivo and in vitro. Conclusions: This study identified KIF15 as a critical regulator that promotes pancreatic cancer proliferation, broadening our understanding of KIF15 function in tumorigenesis.

// Xiuhui Shi 1, * , Hang Zhang 1, * , Min Wang 1, * , Xiaodong Xu 1 , Yan Zhao 1 , Ruizhi He 1 , Min Zhang 1 , Min Zhou 1 , Xu Li 1 , Feng Peng 1 , Chengjian Shi 1 , Ming Shen 1 , Xin Wang 1 , Xingjun Guo 1 and Renyi Qin 1 1 Department of Biliary-Pancreatic Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China * These authors have contributed equally to this study Correspondence to: Renyi Qin, email: ryqin@tjh.tjmu.edu.cn Xingjun Guo, email: xjguo@tjh.tjmu.edu.cn Keywords: long noncoding RNA, AFAP1-AS1, cholangiocarcinoma, proliferation, metastasis Received: February 07, 2017 Accepted: March 15, 2017 Published: April 06, 2017 ABSTRACT We investigated the role of actin filament associated protein 1 antisense RNA1 (AFAP1-AS1) lncRNA in promoting cholangiocarcinoma (CCA). qRT-PCR analysis of patient samples showed that AFAP1-AS1 expression was higher in CCA tumors than matched adjacent non-tumor tissue. AFAP1-AS1 levels were also higher in CCA cell lines (HuCCT1 and TFK-1) than a normal biliary epithelium cell line (HIBEpic). AFAP1-AS1 knockdown in CCA cell lines using shAFAP1-AS1 reduced cell proliferation and colony formation in CCK-8 and colony formation assays, respectively. Cell cycle analysis demonstrated that AFAP1-AS1 knockdown resulted in G0/G1 cell cycle arrest and inhibition of S-G2/M transition compared to the controls. CCA cells transfected with shAFAP1-AS1 also exhibited reduced metastasis and invasiveness in Transwell and wound healing assays. This was further confirmed in xenograft experiments with nude mice using CCA cells transfected with shAFAP1-AS1 or control shRNA. AFAP1-AS1 knockdown cells produced smaller tumors, demonstrating that AFAP1-AS1 promotes tumor growth in vivo . AFAP1-AS1 knockdown also increased expression of actin filament associated protein 1 (AFAP1) and reduced cell stress filament integrity, as determined from western blot and immunofluorescence assays, respectively. These findings indicate that AFAP1-AS1 exerts oncogenic effects in CCA. We postulate that AFAP1-AS1 is a potentially useful diagnostic and prognostic biomarker and therapeutic target for CCA.

Background This study aimed to investigate the effect of combination of plasma miR-181b, miR-196a and miR-210 on early diagnosis of pancreatic cancer (PC).Methods In our study, the plasma was isolated from patients with PC and healthy individuals, respectively. The expressions of miR-181b, miR-196a and miR-210 were detected by qRT-PCR. Moreover, the level of carbohydrate antigen 199 (CA199) was measured by electrochemiluminescence (ECL) assay. Furthermore, the area under the receiver operating characteristic (ROC) curve (AUC) was used to analyze the diagnostic efficacy of miR-181b, miR-196a, miR-210 and CA199, as well as the combination of thses miRNAs in early PC patients and healthy individuals.Results The expressions of miR-181b, miR-196a and miR-210 were significantly upregulated in PC patients. In addition, the level of CA199 was also significantly upregulated in the plasma of PC patients. The expressions of miR-181b, miR-196a and miR-210 were closely associated with lymph nodes metastasis, clinical stage and vascular invasion, but had no correlation with the patient's age, gender and tumor size. Moreover, miR-181b, miR-196a and miR-210 have lower AUC than CA199 in PC patients. The combinations miR-181b + miR-196a, miR-181b + miR-210, miR-196a + miR-210 also have lower AUC than CA199 in PC patients. It is worth noting that the combinations miR-181b + miR-196a + miR-210 have higher AUC than CA199 in PC.Conclusions Our study demonstrated that the combination of plasma miR-181b, miR-196a and miR-210 had good value for PC early diagnosis.

Background Accumulation evidence indicates the vital role of long non-coding RNAs (lncRNAs) in tumorigenesis and the progression of malignant tumors, including pancreatic cancer (PC). However, the role and the molecular mechanism of long non-coding RNA 00976 is unclear in pancreatic cancer. Methods In situ hybridization (ISH) and qRT-PCR was performed to investigate the association between linc00976 expression and the clinicopathological characteristics and prognosis of patients with PC. Subsequently, linc00976 over-expression vector and shRNAs were transfected into PC cells to up-regulate or down-regulate linc00976 expression. Loss- and gain-of function assays were performed to investigate the role of linc00976 in proliferation and metastasis in vitro and vivo. ITRAQ, bioinformatic analysis and rescue assay were used to illustrate the ceRNA mechanism network of linc00976/miR-137/OTUD7B and its downstream EGFR/MAPK signaling pathway. Results linc00976 expression was overexpressed in PC tissues and cell lines and was positively associated with poorer survival in patients with PC. Function studies revealed that linc00976 knockdown significantly suppressed cell proliferation, migration and invasion in vivo and in vitro, whereas its overexpression reversed these effects. Based on Itraq results and online database prediction, Ovarian tumor proteases OTUD7B was found as a downstream gene of linc00976, which deubiquitinated EGFR mediates MAPK signaling activation. Furthermore, Bioinformatics analysis and luciferase assays and rescue experiments revealed that linc00976/miR137/OTUD7B established the ceRNA network modulating PC cell proliferation and tumor growth. Conclusion The present study demonstrates that linc00976 enhances the proliferation and invasion ability of PC cells by upregulating OTUD7B expression, which was a target of miR-137. Ultimately, OTUD7B mediates EGFR and MAPK signaling pathway, suggesting that linc00976/miR-137/OTUD7B/EGFR axis may act as a potential biomarker and therapeutic target for PC.