| IntroductionPancreatic cancer is a highly invasive malignant tumor in the digestive system,with insidious onset,short course,rapid progress,high mortality and no typical early symptoms.Surgical resection is the only effective treat method for the pancreatic cancer.Most patients have been diagnosed at a late stage of the disease and lost the best treat time.Perineural invasion is a special transfer of pancreatic cancer,has a high incidence rate of 80%-100%,and has the poor prognosis and low survival rate in patients.Mechanism is still unclear but involves a variety of signal molecules and different signal pathways.At present,more and more studies have found that autophagy plays an important role in tumors,and closely related to the occurrence,development,differentiation and prognosis of pancreatic cancer.Microtubule-associated protein 1A/1B-light chain 3(LC3)is one of the common markers of autophagic corpuscles in mammalian cells.It participates in the formation of autophagy and is a key step of autophagy to form and insert into phagocytic membrane which directly related to poor prognosis in patients with pancreatic cancer.Studies showed that LC3 was highly expressed in the lymph nodes and the infiltrated nerve fibers in the patients with pancreatic cancer.Autophagy may play a role in the PNI and distant metastasis.Only those pancreatic cancer cells that remain viable in nerve tissue can eventually develop into a clinically visible pancreatic cancer PNI.One of the mechanisms that maintain the viability of cancer cells is probably autophagy.There is no study to confirm the association between autophagy related protein LC3 and PNI.Therefore,we collected clinical data firstly,and elucidated the relationship between autophagy protein LC3 and PNI in pancreatic cancer and their clinicopathological characteristics and survival prognosis by immunohistochemistry and survival analysis.Then the gene expression profiles of pancreatic cancer were analyzed based on LC3 guidance,to find the differential genes involved in autophagy of pancreatic cancer.The protein interaction network was constructed to cluster the differential genes.Cluster module is used to study the interaction between modules and find the key proteins(or genes)that interact with each other,and further explore the association between LC3 and PNI from the molecular level.It provided a theoretical basis for elucidating the pathophysiological mechanism of pancreatic cancer and provided the basis for the early diagnosis of pancreatic cancer and the search for therapeutic targets.Part I investigation of the relationship between autophagy and perineural invasion(PNI),clinical features and prognosis in patients with pancreatic cancerObjective:To investigate the relationship between autophagy and nerve invasion,clinical features and prognosis in patients with pancreatic cancer.Methods:Clinical and pathological data were retrospectively collected from 109 patients with pancreatic ductal adenocarcinoma who underwent resection at the First Affiliated Hospital of Zhengzhou University from January 2011 to August 2016.Expression levels of the autophagy-related protein microtubule-associated protein 1A/1B-light chain 3(LC3)and perineural invasion marker ubiquitin carboxy-terminal hydrolase(UCH)in pancreatic cancer tissues were detected by immuno-histochemistry.The correlations among LC3 expression,perineural invasion,and clinical pathological features in pancreatic cancer were analyzed.The patients were followed up for further survival analysis.Results:In 109 cases of pancreatic cancer,68.8%(75/109)had evidence of perineural invasion and 61.5%(67/109)had high LC3 expression.Perineural invasion was associated with lymph node metastasis,pancreatitis and CA19-9 levels(P<0.05).LC3 expression was related to lymph node metastasis(P<0.05)and was positively correlated with neural invasion(P<0.05,r=0.227).Multivariate logistic regression analysis indicated that LC3,lymph node metastasis,pancreatitis,and CA19-9 level were factors that influenced neural invasion,whereas only neural invasion itself was an independent factor of high LC3 expression.Univariate analysis showed that LC3 expression,neural invasion and CA19-9 level were related to the overall survival of pancreatic cancer patients(P<0.05).Multivariate COX regression analysis indicated that perineural invasion and LC3 expression were independent risk factors for poor prognosis in pancreatic cancer(P<0.05).Conclusion:Perineural invasion in patients with pancreatic cancer is positively related to autophagy.Neural invasion and LC3 expression are independent risk factors for pancreatic cancer with poor prognosis.Part II Analysis of autophagic gene expression profiles in pancreatic cancer based on LC3 guidanceObjective:To research the relationship between autophagy related protein LC3 and PNI in pancreatic cancer by the opinion of bioinformatics.Methods:From the GEO collection of two sets of pancreatic cancer/normal tissue gene expression data sets(GSE16515 and GSE15471),probe gene expression was calculated by Robust Multi-array Average(RMA)algorithm by Affymetrix expression spectrum of original data,annotate package of R language combined with chip annotate documentation to mark the corresponding gene in probe needle.Gene expression data were associated with clinical feature data(cancer samples and normal samples).Significance Analysis of Microarrays(SAM)algorithm was used to screen differentially expressed genes related to pancreatic cancer(|log2(fold change)|)>1,FDR(False Discovery Rate)<0.01.GO analysis and KEGG Pathway were used to analyze the functional enrichment of differential genes.The human protein interaction network data were downloaded from the STRING database,and the protein interaction data that only contain differentially expressed genes are screened.Then the interwork data of the differentially expressed genes were introduced into the Cytoscape,and excavated the module with the ClusterOne plug-in In the analysis,a module with a p value of less than 0.05 was selected as a significant result to annotate the differential genes.According to the information of function module and the reliable protein interaction data,the interaction relationship between modules was analyzed which is crosstalk and found pivot nodes between functional modules.Results:Based on the above two sets of pancreatic tissue total gene expression data set,6098 genes and 12928 differentially expressed genes were obtained,by Analysis of genes with higher phenotypic correlation.GO analysis of the differentially expressed genes showed that 14 significant functional items were closely related to autophagy in three aspects:molecular function,biological process and cell components.14 significant functional items were actin binding、protease binding、regulation of extrinsic apoptotic signaling pathway、extrinsic apoptotic signaling pathway in absence of ligand、extrinsic apoptotic signaling pathway、negative regulation of extrinsic apoptotic signaling pathway、negative regulation of protein ubiquitination、regulation of protein ubiquitination、response to metal ion、positive regulation of proteolysis、proteasome accessory complex、ion channel complex、proteasome complex and proteasome regulatory particle.A total of 986 differentially expressed genes were enriched in these functional items.After eliminating the autophagy related genes of human cancer cells defined by the gene functional annotation database,347 differentially expressed genes were obtained.In addition,the differentially expressed genes in KEGG were significantly analyzed.The pathway hsa04144 and Hsa04020 were related to the internal phagocytosis and calcium signaling pathway respectively,and both related to autophagy.Based on the protein interaction network database String,a protein interaction network was constructed,including 2256 differentially expressed genes and 12632 interacting pairs.Through module mining,65 cluster modules were screened,and LC3 gene was included in module 32.It was found that was interrelated with a number of genes associated with autophagy and could form functional modules with use of network tools to analyze the interaction of LC3 gene and its direct interaction.It also could connect these genes among the three autophagy related genes ATG5,ATG7,ATG4A.Found modules and pivot related autophagy,ubiquitin C(UBC)was used as a pivot node in functional modules to connect multiple modules related to cancer and autophagy.A new pathway,hsa04216(Ferroptosis),had significantly higher(p.adjust = 1.06E-07),was found by KEGG analysis.Conclusion:There are 347 genes that unclearly associated with autophagy in human cancer cells were concentrated.Several pathways related to autophagy have been found,which provide a new basis for future research.The ubiquitin C of the pivot gene was found by Crosstalk analysis based on autophagy and LC3.Combined with the key role of ubiquitin carboxyl terminal hydrolase in the development of PNI,it is considered that LC3 is highly likely to regulate the ubiquitin-protease system through ubiquitin C and then affect the PNI of pancreatic cancer.ConclusionThe clinical study found a significant positive correlation between the high expression of LC3 and the positive rate of PNI in the patients with pancreatic cancer.The level of PNI and LC3 is an independent risk factor for poor prognosis of pancreatic cancer.Gene expression profiles have been used to detect 347 genes that unclearly associated with the autophagy of human cancer cells.Enriched the key pathways involved in autophagy and provided new information for future research.It is also found that LC3 may affect pancreatic cancer PNI and poor prognosis through ubiquitin C. |
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