Effects of different preservation schemes on isolated rat artery.

Allogeneic blood vessels are regarded as one of the best natural substitutes for diseased blood vessels due to their good vascular compliance and histocompatibility. Since the supply and demand of allograft blood vessels do not always match in time and space, a good preservation scheme for isolated blood vessels is essential. The abdominal aortas of 110 male Sprague-Dawley (SD) rats were randomly divided into three groups, including cold storage group (4°C) (CSG), frozen storage group (FSG) and ambient storage group (25 ± 2°C) (ASG). Seven time points of preservation for 1, 3, 5, 7, 14, 30 and 90 days were set for detection. The changes in vascular physiological function were evaluated by MTT test and vasoconstriction ability detection, and the changes in vascular wall structure were evaluated by the tension tolerance test and pathological staining. The vascular function of CSG was better than FSG within first the 7 days, but the result was opposite since the 14th day. The vascular wall structure, collagen and elastic fibres of vessels, in CSG, showed oedema within 30 days, and continuous disintegration and rupture at 90 days. The vessel wall structure of FSG remained intact within 90 days. The tensile strength of the vessels in CSG was better than that in FSG within 5 days, and there was no statistical difference between the two groups between the 7th and 30th day, and then, the FSG was higher than CSG on the 90th day. Both cold storage and frozen storage could be applied as safe and effective preservation schemes for isolated rat artery within first 30 days. Cold storage is recommended when the storage time is <14 days, and then, frozen storage is better.

The coagulation-related genes for prognosis and tumor microenvironment in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a malignancy characterized by challenging early diagnosis and poor prognosis. It is believed that coagulation has an impact on the tumor microenvironment of PDAC. The aim of this study is to further distinguish coagulation-related genes and investigate immune infiltration in PDAC. METHODS: We gathered two subtypes of coagulation-related genes from the KEGG database, and acquired transcriptome sequencing data and clinical information on PDAC from The Cancer Genome Atlas (TCGA) database. Using an unsupervised clustering method, we categorized patients into distinct clusters. We investigated the mutation frequency to explore genomic features and performed enrichment analysis, utilizing Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) to explore pathways. CIBERSORT was used to analyze the relationship between tumor immune infiltration and the two clusters. A prognostic model was created for risk stratification, and a nomogram was established to assist in determining the risk score. The response to immunotherapy was assessed using the IMvigor210 cohort. Finally, PDAC patients were recruited, and experimental samples were collected to validate the infiltration of neutrophils using immunohistochemistry. In addition, and identify the ITGA2 expression and function were identified by analyzing single cell sequencing data. RESULTS: Two coagulation-related clusters were established based on the coagulation pathways present in PDAC patients. Functional enrichment analysis revealed different pathways in the two clusters. Approximately 49.4% of PDAC patients experienced DNA mutation in coagulation-related genes. Patients in the two clusters displayed significant differences in terms of immune cell infiltration, immune checkpoint, tumor microenvironment and TMB. We developed a 4-gene prognostic stratified model through LASSO analysis. Based on the risk score, the nomogram can accurately predict the prognosis in PDAC patients. We identified ITGA2 as a hub gene, which linked to poor overall survival (OS) and short disease-free survival (DFS). Single-cell sequencing analysis demonstrated that ITGA2 was expressed by ductal cells in PDAC. CONCLUSIONS: Our study demonstrated the correlation between coagulation-related genes and the tumor immune microenvironment. The stratified model can predict the prognosis and calculate the benefits of drug therapy, thus providing the recommendations for clinical personalized treatment.

Effects of different preservation methods of human iliac veins.

With the progress of vascular anastomosis technology, the radical resection surgery of cancer combining with vascular resection and reconstruction has been focused by surgeon. As a natural substitute material for blood vessel, vascular allografts have good vascular compliance and histocompatibility. Generally, the donated veins could not be used immediately, and need to be well preserved. So, it is greatly significant to do research in the preservation effects of different preservation methods on veins. In this study, the effects of different preservative methods of human iliac veins were compared and analyzed in terms of cell viability, vascular wall structure and tension resistance. The donated human iliac veins were randomly divided into three groups: Cold Storage Group (4 °C) (CSG), Frozen Storage Group (-186 °C) (FSG)and Fresh Control Group (FCG). Six detection time-points of preservation for 1, 3, 5, 7, 14, 28 days were set respectively. There are ten samples in each group and each time-point separately. Survival and apoptosis of vascular cell were evaluated by MTT assay and Tunel fluorescence staining. Tensile test was used to evaluate mechanical properties of vessels. The changes of vascular endothelial cells, smooth muscle cells, collagen fibers and elastic fibers were evaluated by HE staining, Masson staining and EVG staining. Furthermore, the changes of organelles were observed by transmission electron microscope. With the extension of preservation period, the vascular cell viability and tension resistance of two groups decreased, and the apoptotic cells increased gradually. The apoptosis index of CSG was higher than FSG at each time point (P < 0.05). In terms of cell viability, CSG was higher within 3 days (P < 0.05), both groups were same between 3 and 14 days, and then CSG lower than FSG after 14 days (P < 0.05). In terms of tension resistance, CSG was stronger than FSG (P < 0.05) in first 7 days, both groups were same in 2nd week, and then CSG was weaker in 4th week (P < 0.05). In terms of vascular wall structure, in CSG, vascular endothelial cells were damaged and shed, smooth muscle cells were edema after 14 days, but the cell membrane and intercellular connection were still intact. In 4th week, endothelial cells were completely damaged and shed, the boundary of smooth muscle cell membrane was unclear, intercellular connection was damaged. Moreover, organelles were destroyed and disappeared, perinuclear condensation of chromatin was observed, and some cells had incomplete nuclear membrane or nuclear fragmentation; However, there were no obvious changes in the FSG within 28 days. Finally, local exfoliation and destruction of endothelial cells and edema-like changes of organelles were observed; the collagen fibers and elastic fibers of blood vessels in the two groups had no obvious damage and change within 28 days. For excised human iliac vein, cold and frozen storage can effectively preserve the cell viability, wall structure and tension resistance of blood vessels. With the extension of preservation time, the related performance of vessels declined in varying degrees. Within first week, the effect of cold storage is better than frozen storage, but frozen storage is significantly better than cold storage after 2 weeks.

Long-term survival in patients with distal cholangiocarcinoma after pancreaticoduodenectomy combined with portal vein system resection and reconstruction.

PURPOSE: Pancreaticoduodenectomy combined with portal vein resection for distal cholangiocarcinoma is rarely reported because it is a rare disease. We developed a program to evaluate the vascular invasion type, operation procedure, and long-term survival of distal cholangiocarcinoma patients with portal vein invasion. METHODS: We retrospectively reviewed data for 123 distal cholangiocarcinoma patients after pancreaticoduodenectomy between January 2013 and December 2019. Portal vein system invasion was confirmed pathologically in 17 patients. RESULTS: Multivariable Cox regression identified tumor differentiation degree, portal vein system invasion, and lymph node metastasis as independent risk factors affecting long-term survival. The 1- and 2-year overall survival rates for patients without and with portal vein system invasion were 79.7% and 58.9%, and 48.6% and 10.8%, respectively. Median overall survival in patients without and with portal vein system invasion was 33 months and 12 months, respectively. CONCLUSION: Portal vein system invasion is an important independent risk factor affecting long-term survival in patients with distal cholangiocarcinoma. Pancreaticoduodenectomy combined with portal vein system resection and reconstruction did not increase the incidence of perioperative complications or mortality.

Prognostic impact of lymph node parameters in distal cholangiocarcinoma after pancreaticoduodenectomy.

BACKGROUND: Pancreaticoduodenectomy is the only definitively curative therapy for the long-term survival of distal cholangiocarcinoma patients. Lymph node metastasis is widely accepted as an important prognostic factor for distal cholangiocarcinoma. The latest American Joint Committee on Cancer (AJCC) TNM classification system for distal cholangiocarcinoma has divided the lymph node metastasis patients into N1 and N2 by lymph node metastasis number. However, some studies suggested that the lymph node metastasis ratio may be better than the lymph node metastasis number. Therefore, we develop a program to analyze the correlation between lymph node parameters (lymph node dissection number, lymph node metastasis number, and lymph node metastasis rate) and long-term prognosis. METHODS: We retrospectively reviewed 123 distal cholangiocarcinoma patients after pancreatoduodenectomy from January 2011 to December 2019. The patients were grouped according to lymph node metastases and tumor-free and overall survival rates which were investigated with the Kaplan-Meier analysis. The logistic regression models were used for multivariate analysis to determine the risk factors for lymph node metastases. And the X-tile program was used to calculate the cutoff values for the lymph node parameters that discriminated survival. RESULTS: The 1-year, 3-year, and 5-year overall survival rates of patients with distal cholangiocarcinoma after pancreatoduodenectomy were 75.2%, 37.1%, and 31.5%, respectively. And the 1-year, 3-year, and 5-year overall survival rates of patients without and with lymph node metastasis were 83.0%, 50.7%, and 42.5% and 63.5%, 19.0%, and 19.0% (p = 0.000), respectively. Logistic regression showed CA19-9 and portal vein system invasion as independent risk factors for lymph node metastases. The receiver operating characteristic curve showed the optimal cutoff value of CA19-9 to predict the lymph node metastases was 75.5 U/mL. Determined by the X-tile software, the optimal cutoff values of the lymph node dissection number were 24 (p = 0.021), the lymph node metastasis number were 1 and 7 (p = 0.504), and the lymph node metastasis rate were 0.13 (p = 0.002). CONCLUSION: Lymph node metastasis is an important factor affecting the long-term survival of distal cholangiocarcinoma patients.CA19-9 and portal vein system invasion are independent risk factors for lymph node metastasis. Besides, the lymph node dissection number and lymph node metastasis rate can predict the long-term survival better than lymph node metastasis number.

Immunoparesis recovery 1 year after ASCT is independently associated with favorable survival in patients with symptomatic multiple myeloma who undergo autologous stem cell transplantation.

Immunoparesis is defined as a reduction in the levels of one, two, or three uninvolved immunoglobulins. However, there are very limited data on the incidence and prognostic significance of immunoparesis recovery 1 year after autologous stem cell transplantation (ASCT) in MM. We reviewed medical records of de novo MM patients who received ASCT at Beijing Chao Yang hospital. One hundred eight MM patients were included in the study. Conventional chemotherapy was administered as induction regimen in 16 patients (14.8%), whereas novel agents were used in 92 patients (85.2%). Most patients had immunoparesis at diagnosis (89.1%) and at the moment of ASCT as well (75%). After a median follow-up of 49 months, in the group with immunoglobulin recovery 1 year after ASCT, there was a trend towards longer progression-free survival (PFS) than in the group with immunoparesis (P = 0.054). And overall survival (OS) was significantly longer in patients with immunoparesis recovery (P = 0.004). In multivariate analysis, immunoparesis recovery 1 year after ASCT was independently associated with improved OS (P = 0.016). In conclusion, lack of immunoparesis recovery 1 year after ASCT in MM patients is associated with significantly shorter OS and this group of patients needs new treatment strategy to improve the prognosis.

IL-10 dependent modulatory effect of regulatory B10 cells on local scar formation following Roux-en-Y choledochojejunostomy in a novel rat model.

Choledochojejunostomy has been common surgical treatment of biliary tract disease. Scar formation at anastomotic often results in postoperative complications associated with bleak post-operative recovery, in which local inflammation may be a potential target to modulate local scar formation. This study investigated the effect of regulatory B10 cells on local scar formation through interleukin-10 signal pathway following Roux-en-Y choledochojejunostomy (RCJS) in a novel rat model. Sprague-Dawley (SD) rats with RCJS were randomly divided into blank group, experimental group, IL-10 blocking group, control group, and received different interventions and duration. Injected through dorsal vein of penis, rats in different groups were treated respectively according to scheme. These interventions were performed during surgery, on 1st day, and 2nd day after surgery. Related indexes, including blood examination, specimen tissue of anastomotic detection, were recorded and compared in different interventional groups. Rats in experimental groups had more rapid recovery in liver function and inflammatory index, and higher in IL-10 level. Flow cytometry analysis showed that rats in experimental groups had highest content of B10 cells and lowest content of CD4+CD25- T cells in peripheral blood. Wider anastomotic by macroscopical observation, and slighter proliferation of collagen fiber and smooth muscle fiber, lower α-SMA and TGF-ß1 levels by pathological staining were detected in experimental groups. Higher expression of the IL-10 gene and lower expression of TGF-ß1 at anastomotic were detected in experimental groups. B10 cells may relieve local inflammation of anastomotic following RCJS in rats through IL-10-dependent modulatory effect, and improve local scar formation.

Impact of the diagnosis of metabolic dysfunction-associated fatty liver disease and non-alcoholic fatty liver disease in patients undergoing liver transplantation for hepatocellular carcinoma.

Purpose: Whether the diagnosis of non-alcoholic fatty liver disease or metabolic dysfunction-associated fatty disease has a different impact on liver transplant recipients with hepatocellular carcinoma is not yet clear. Methods: Data from a two-center retrospective cohort study were collected to compare and investigate the differences between non-alcoholic fatty liver disease and metabolic dysfunction-associated fatty liver disease in clinicopathologic parameters and prognosis among liver transplant recipients with hepatocellular carcinoma. Results: A total of 268 liver transplant recipients with hepatocellular carcinoma were included. The prevalence among pre- and post-transplant metabolic dysfunction-associated fatty liver disease was 10.82% and 30.22%, while for non-alcoholic fatty liver disease, it was 7.09% and 26.87%, respectively. The clinicopathological parameters were similar between the two pre-transplant groups. In contrast, the post-transplant group with metabolic dysfunction-associated fatty liver disease exhibited a higher prevalence of diabetes mellitus and a greater body mass index. However, the other parameters were similar between the two post-transplant groups (p > 0.05). Factors such as the largest tumor size > 4 cm, microvascular invasion, lack of tumor capsule, post-transplant metabolic dysfunction-associated fatty liver disease, and decreased post-transplant lymphocyte percentage were related to an increased risk of recurrence. Conclusion: In patients undergone liver transplantation for hepatocellular carcinoma, the diagnosis of metabolic dysfunction-associated fatty disease is more strongly associated with metabolic abnormalities than the diagnosis of non-alcoholic fatty liver disease and is an independent predictor of hepatocellular carcinoma recurrence.

Impact of metabolic dysfunction-associated fatty liver disease on liver transplant recipients with hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease was proposed by international consensus to redefine the metabolic abnormal condition. However, its impact on liver transplant recipients with hepatitis B virus-related hepatocellular carcinoma has not been explored. METHODS: A two-center retrospective cohort study on liver transplant recipients with hepatitis B virus-related hepatocellular carcinoma was performed to analyze the impact of metabolic dysfunction-associated fatty liver disease on the clinicopathologic parameters and prognosis. RESULTS: There were 201 liver transplant recipients enrolled from two hospitals in our study. The pre- and post-transplant prevalences of metabolic dysfunction-associated fatty liver disease were 9.95% and 28.86%, respectively. The clinicopathological parameters revealed a similarity between patients with and without pre-transplant metabolic dysfunction-associated fatty liver disease. In contrast, the group with post-transplant metabolic dysfunction-associated fatty liver disease was linked with older age, a higher hepatitis recurrence rate and incidence of cardiovascular disease, usage of calcineurin inhibitors, a greater body mass index and waist circumference, lower albumin and high-density lipoprotein cholesterol levels, and poorer tumor-free survival and overall survival. The multivariate analysis showed the largest tumor size >4 cm (95% confidence intervals: 0.06~0.63, p = 0.006), microvascular invasion (95% confidence intervals: 1.61~14.92, p = 0.005), post-transplant metabolic dysfunction-associated fatty liver disease (95% confidence intervals: 1.40~10.60, p = 0.009), and calcineurin inhibitors-based regimen (95% confidence intervals: 0.33~0.96, p = 0.036) were the independent risk factors for recurrent hepatocellular carcinoma. CONCLUSIONS: Our study suggests that post-transplant metabolic dysfunction-associated fatty liver disease is more closely to metabolic abnormalities and that it can help identify liver transplant recipients at high risk of recurrent hepatocellular carcinoma.

Protective Effects of Different Hypothermal Preservation Solutions on Structure and Function of Isolated Rat Arteries.

OBJECTIVE: With the increasing application of vascular reconstruction in surgical procedures, allogeneic vessels are becoming more popular in clinical practice due to their abundant sources, precise diameter matching, improved histocompatibility, and higher long-term patency rate. This study aimed to investigate the protective effect of various preservation solutions on the function and structure of the isolated rat abdominal aorta preserved under hypothermal conditions. METHODS: The study utilized a total of 150 Sprague-Dawley (SD) rats, with 144 rats allocated to the experimental groups and 6 rats allocated to the control groups. The abdominal aorta of the rats was chosen as the subject of our research. The aorta in the experimental groups were randomly assigned to 4 groups: University of Wisconsin (UW) solution group, histidine-tryptophan-ketoglutarate (HTK) solution group, normal saline (NS) group, and sodium lactate Ringer's solution (RS) group. Samples were subjected to examination after preservation periods of 1 day, 3 days, 5 days, 7 days, 14 days, 30 days, and 90 days. Evaluation of vascular physiological function involved detecting and assessing vasoconstriction ability and measuring cell viability through the MTT test. Evaluation of the vascular wall structure involved tension tolerance tests and pathological staining. RESULTS: The pathogen-positive rate in the HTK group and NS group at 1 month was 16.7%. Regarding the vascular skeleton structure, both the UW group and HTK group exhibited intact structures after 2 weeks of preservation, with slightly edematous collagen and elastic fibers, which was significantly better than that of the NS group and RS group. In terms of cell activity and contractile function, all preservation groups showed similar effects within 2 weeks. However, after 2 weeks, the UW group showed the most favorable preservation effect (P<0.05). In terms of vascular tension, different groups exhibited similar effects within 1 week. However, after 2 weeks, the UW group showed the best preservation effect (P<0.05). CONCLUSION: All 4 types of preservation solution had a preservation effect on the structure and function of isolated blood vessels during short-term hypothermal preservation. However, after 2-week preservation, the UW solution was found to be the most suitable solution for the preservation of blood vessels.

Spatial transcriptomics reveals the heterogeneity and FGG+CRP+ inflammatory cancer-associated fibroblasts replace islets in pancreatic ductal adenocarcinoma.

Background: Understanding the spatial heterogeneity of the tumor microenvironment (TME) in pancreatic cancer (PC) remains challenging. Methods: In this study, we performed spatial transcriptomics (ST) to investigate the gene expression features across one normal pancreatic tissue, PC tissue, adjacent tumor tissue, and tumor stroma. We divided 18,075 spatial spots into 22 clusters with t-distributed stochastic neighbor embedding based on gene expression profiles. The biological functions and signaling pathways involved in each cluster were analyzed with gene set enrichment analysis. Results: The results revealed that KRT13+FABP5+ malignant cell subpopulation had keratinization characteristics in the tumor tissue. Fibroblasts from adjacent tumor tissue exhibited a tumor-inhibiting role such as "B-cell activation" and "positive regulation of leukocyte activation." The FGG+CRP+ inflammatory cancer-associated fibroblasts replaced the islets in tumor stroma. During PC progression, the damage to pancreatic structure and function was heavier in the pancreatic exocrine (AMYA2+PRSS1+) than in the endocrine (INS+GCG+). Conclusion: Our results revealed the spatial heterogeneity of dynamic changes and highlighted the significance of impaired exocrine function in PC.

Prognosis of Liver Transplantation for Hepatocellular Carcinoma in Terms of Different Criteria: A Single Center Experience.

OBJECTIVE: Patients undergoing liver transplantation for hepatocellular carcinoma (HCC) within the Milan criteria have an excellent outcome. We developed a program to analyze and prove that the Milan criteria can be expanded safely and effectively. METHODS: We retrospectively reviewed 117 HCC patients treated with liver transplantation between January 2013 and December 2017. Patients were grouped according to the Milan criteria, the University of California, San Francisco (UCSF) criteria, Up-to-seven criteria and Hangzhou criteria. Tumor-free and overall survival rates were investigated with a Kaplan-Meier analysis. Multivariable regression Cox models produced survival estimates for the patients that exceeded the Milan criteria. RESULTS: The 1-year, 3-year and 5-year overall survival rates of patients fulfilling the Milan criteria (n=44) were 100%, 87.5% and 78.9%, respectively. Compared with the Milan criteria, the UCSF criteria (n=50), Up-to-seven criteria (n=51) and Hangzhou criteria (n=86) provided an expansion of 13.6%, 15.9% and 95.9%, respectively. The 1-year, 3-year and 5-year overall survival rates of patients fulfilling UCSF criteria, Up-to-seven criteria and Hangzhou criteria were 96.0%, 84.9%, 76.9%; 96.1%, 85.2%, 77.6% and 97.7%, 83.9%, 66.7%, respectively (P>0.05). Multifactor Cox regression showed that tumor diameter and microvascular invasion were independent risk factors for survival in patients that exceeded the Milan criteria. CONCLUSION: Compared with the Milan criteria, the Hangzhou criteria can safely expand the scope of liver transplantation for HCC to a certain extent. By contrast, the UCSF criteria and Up-to-seven criteria result in a limited number of patients which need further expansion. Tumor diameter and microvascular invasion were the independent risk factors for survival in patients that exceeded the Milan criteria.

Establishment of rat allogenic vein replacement model and pathological characteristics of the replaced vessels.

Background: With the advancement of vascular anastomosis techniques in recent years, radical surgery for tumors combined with venous vascular resection and reconstruction has been widely used. This study intends to establish two different rat vein replacement models, and further analyze the pathological changes of blood vessels after replacement. Methods: Brown-Norway (BN) rats were selected as donors and recipients, randomly divided into control group, cuff group (1-week group, 2-week group, and 4-week group), and suture group (1-week group, 2-week group, and 4-week group), with 6 rats in each group. The perioperative conditions, inner diameter, flow velocity and histopathological changes of the replaced vessels at different time points were analyzed. Results: Both cuff group and suture group can safely establish the rat vein replacement model. From the surgical operation, the operation time and venous cross-clamp time in the cuff group were shorter than those in the suture group (P < 0.05). At 2 and 4 weeks after operation, the diameter of suture group was wider than that of cuff group, and the flow rate was faster (P < 0.05). With prolonged postoperative survival, the wall of the replaced vessels underwent infiltration of CD4+ and CD8+ lymphocytes and high TGF-ß1 gene expression. This leads to the proliferation of blood vessels and intimal layer. The results of vascular pathological staining showed that the infiltration degree of CD4+ lymphocytes at 2 weeks after operation and CD8+ lymphocytes at 4 weeks after operation in the suture group was lighter than that in the cuff group (P < 0.05). Meanwhile, TGF-ß1 gene content at 4 weeks after operation in suture group was significantly lower than that in cuff group (P < 0.05). Conclusion: Compared with cuff method, suture method is more suitable for the study of long-term pathological changes after vein replacement in rats. The main pathological changes in the long term after venous replacement in syngeneic background may be vascular fibrosis caused by inflammatory cell infiltration.

MiR-1301-3p Inhibits Epithelial-Mesenchymal Transition via Targeting RhoA in Pancreatic Cancer.

Micro(mi)RNAs play an essential role in the epithelial-mesenchymal transition (EMT) process in human cancers. This study aimed to uncover the regulatory mechanism of miR-1301-3p on EMT in pancreatic cancer (PC). The miRNA profilings from Gene Expression Omnibus data sets (GSE31568, GSE41372, and GSE32688) demonstrated the downregulation of miR-1301-3p in PC tissues, which was validated with 72 paired PC tissue samples through qRT-PCR detection. The low level of miR-1301-3p was associated with a poor prognosis for PC patients from the PC cohort of The Cancer Genome Atlas and the validation cohort. Gene Ontology analyses indicated that the target genes of miR-1301-3p were involved in cell cycle and adherent junction regulation. In vitro assays revealed that miR-1301-3p suppressed the proliferation and migration abilities of PC cells. Western blotting and luciferase reporter assays suggested that miR-1301-3p inhibited RhoA expression by targeting its 3'-untranslated region; RhoA upregulated N-cadherin and vimentin levels; however, it downregulated the E-cadherin level. In conclusion, our study showed that miR-1301-3p could serve as a prognostic biomarker for PC and suppress PC cell malignancy by targeting the RhoA-induced EMT process.

Dipeptidyl peptidase like 6 promoter methylation is a potential prognostic biomarker for pancreatic ductal adenocarcinoma.

BACKGROUND: Hypermethylation of gene promoters plays an important role in tumorigenesis. The present study aimed to identify and validate promoter methylation-driven genes (PMDGs) for pancreatic ductal adenocarcinoma (PDAC). METHODS: Based on GSE49149 and the PDAC cohort of The Cancer Genome Atlas (TCGA), differential analyses of promoter methylation, correlation analysis, and Cox regression analysis were performed to identify PMDGs. The promoter methylation level was assessed by bisulfite sequencing polymerase chain reaction (BSP) in paired tumor and normal tissues of 72 PDAC patients. Kaplan-Meier survival analyses were performed to evaluate the clinical value of PMDGs. RESULTS: In GSE49149, the ß-value of the dipeptidyl peptidase like 6 (DPP6) promoter was significantly higher in tumor compared with normal samples (0.50 vs. 0.24, P<0.001). In the PDAC cohort of TCGA, the methylation level of the DPP6 promoter was negatively correlated with mRNA expression (r = -0.54, P<0.001). In a multivariate Cox regression analysis, hypermethylation of the DPP6 promoter was an independent risk factor for PDAC (hazard ratio (HR) = 543.91, P=0.002). The results of BSP revealed that the number of methylated CG sites in the DPP6 promoter was greater in tumor samples than in normal samples (7.43 vs. 2.78, P<0.001). The methylation level of the DPP6 promoter was moderately effective at distinguishing tumor from normal samples (area under ROC curve (AUC) = 0.74, P<0.001). Hypermethylation of the DPP6 promoter was associated with poor overall (HR = 3.61, P<0.001) and disease-free (HR = 2.01, P=0.016) survivals for PDAC patients. CONCLUSION: These results indicate that DPP6 promoter methylation is a potential prognostic biomarker for PDAC.

Triptolide inhibits pancreatic cancer cell proliferation and migration via down-regulating PLAU based on network pharmacology of Tripterygium wilfordii Hook F.

Tripterygium wilfordii Hook F (TwHF) exhibits anti-tumor efficacy in pancreatic ductal adenocarcinoma (PDAC), however the pharmacological mechanisms are unclear due to complicated formulae and target genes. Using Traditional Chinese Medicine Systems Pharmacology and GeneCards databases, we performed a network pharmacology (NP) of TwHF and screened out 22 ingredients and 25 target genes associated with PDAC. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the 25 target genes were performed. Using STRING database, protein-protein interaction network of the 25 target genes was constructed, and indicated that triptolide (TL)-plasminogen activator urokinase (PLAU) as a potential target for PDAC treatment. Hence, in vitro experiments were performed and validated that TL inhibited PDAC cell proliferation and migration by suppressing PLAU expression. The results of Western blot suggested that PLAU activated endothelial-mesenchymal transition (EMT) progression. In two Gene Expression Omnibus datasets (GSE16515 and GSE28735), PLAU was up-regulated in tumor tissues, and PLAU overexpression was associated with poor overall survival of PDAC cohort of The Cancer Genome Atlas (P < 0.01). Immunohistochemistry illustrated that overexpression of PLAU protein was related to lymph node metastasis in 20 PDAC patients (P < 0.01). Based on NP of TwHF, we identified and validated that TL-PLAU could serve as a potential target for PDAC treatment.