The Role And The Mechanism Research Of TLR3 On Pancreatic Cancer And Its Lymph Node Metastasis

BackgroundPancreatic cancer(PC)is one of the most common malignant tumors of the digestive system.The incidence of pancreatic cancer has been increasing year by year in both domestic and foreign countries.In the United States,PC ranks as the fourth leading cause of cancer related deaths,and the mortality of PC ranks as the sixth in malignant tumors.Because of the insidious initiation,the lack of specific clinical manifestations,high degree of malignancy,rapid progression,early to metastasis,and not easy to diagnose early,most patients are already in advanced stage.Curative pancreatic resection is the most effective way to treat pancreatic cancer currently,however,most patients have lost the chance of surgical resection at the time of diagnosis,leading to poor prognosis of pancreatic cancer.Therefore,exploring the molecular mechanism of PC development and progression,and then searching for new early diagnostic markers and potential therapeutic targets,have become the focus of pancreatic cancer research.At present,cancer is characterized by ten hallmarks,constituting sustaining proliferative signaling,evading growth suppressors,resisting cell death,enabling replicative immortality,inducing angiogenesis,activating invasion and metastasis,avoiding immune destruction,tumor-promoting inflammation,genomic instability and mutations,deregulating cellular energetics.Tumor-promoting inflammation is considered as the seventh feature of cancer.More and more researches have shown that inflammation is closely related to the occurrence and development of tumors.Inflammatory diseases can increase the risk of some tumors.Non-steroidal anti-inflammatory drugs can reduce the morbidity and mortality of some tumors.There are a large number of inflammatory cells,cytokines and chemokines in the micro-environment of distant organ metastases,indicating that innate immunity and adaptive immunity play an irreplaceable role in the development of cancer.Therefore,we hope to elucidate that how important molecules in innate immunity play a role in the development of cancer from the perspective of innate immunity.Pattern-recognition receptors(PRRs)are the main mediators of immune recognition and immune response to danger signals.Toll-like receptors(TLRs)are newly discovered in recent years as one of the pattern recognition receptors of immune response.Activation of TLRs can activate the downstream signaling pathway and regulate the transition from innate immune response to adaptive immune response.They play an important role in against the infection of foreign pathogenic microorganisms and are also receptors that have been studied thoroughly.As one of the important members of TLRs,the ligand of TLR3 is ds RNA virus and Poly I: C.In many immune cells(such as macrophages,dendritic cells)and some non-immune cells(such as endothelial cells,epithelial cells and tumor cells),TLR3 are expressed.The activation of TLR3 by ds RNA virus or exogenous Poly I: C can activate downstream signaling.Studies have shown that the downstream signaling pathways of TLR3 mainly include My D88-dependent pathway and My D88-independent pathway.My D88-dependent signaling pathway can induce the expression of some inflammatory cytokines,such as IL-1,IL-6,TNF-α and so on,participating in non-specific immune response.And My D88-independent signaling pathway can induce type I interferon and other cytokine expression inducing DC cell differentiation and maturation,which involved in antiviral response.More and more evidence suggests that there is an important link between TLR3 and tumors and TLR3 has a dual role in oncology.First of all,a large number of studies have shown that TLR3 can promote the apoptosis of tumor cells,such as breast cancer,prostate cancer,liver cancer,head and neck squamous cell carcinoma and other tumors.The activation of TLR3 can cause apoptosis of tumor cells.At the same time,the activation of TLR3 can promote the secretion of some inflammatory factors and interferons,and then enhance the anti-tumor effects of immune cells such as dendritic cells,natural killer cells and macrophages.Therefore,TLR3 agonists are used as the adjuvant immunotherapy drugs of certain types of tumors.In addition to anti-tumor effect,TLR3 has tumor-promoting effect.It has been reported that TLR3 can mediate tumor invasion and metastasis by increasing the metastatic potential of tumor cells.Such as the overexpression of TLR3 in oral squamous carcinoma is closely associated to peripheral nerve metastasis.Host lung epithelial cell TLR3 is critical for cancer lung metastasis via recruitment of neutrophils.We conducted transcriptome sequencing of primary PC and its lymph node metastases,and found that the expression of TLR3 in lymph node metastases increased compared to primary PC.At present,there are few studies on the occurrence,development and metastasis of pancreatic cancer,and the mechanism of TLR3 for PC is still unclear.Therefore,we hope to find a new marker to assist the diagnosis and predict the prognosis of PC by exploring the effect and mechanism of TLR3 in the progression of lymph node metastasis of pancreatic cancer.ObjectiveThe objective of this work is to investigate the clinical significance of TLR3 expression in PC,to further explore the molecular mechanism of TLR3 in promoting lymph node metastasis of PC,to find a new marker for the diagnosis and prognosis evaluation of PC,and to provide a potential target for treatment.Methods1.Transcriptome sequencing technology was used to detect genes’ expression in 3 cases of primary PC and their compared lymph node metastases.2.Immunohistochemistry was used to detect and compare the expression of TLR3 in primary PC and its lymph node metastases of 57 PC patients with lymph node metastases,and at the same time to detect the expression of TLR3 in 27 cases of primary PC without lymph node metastases.The correlation between the expression of TLR3 in primary PC and clinicopathological features was analyzed in all 84 PC patients.3.The expression of TLR3 in As PC-1,Bx PC-3,PANC-1 and MIA Pa Ca-2 was detected by real-time quantitative PCR(q RT-PCR)technology.4.PANC-1 cell line was selected as a tool of this research.Using Lipofectamine to transfect the plasmid(p UNO1-h TLR3-GFP)into PANC-1 cell to overexpress TLR3, and then the cells were screened with Blasticidin S to obtain a relatively stable cell line with TLR3 overexpression(PANC-1 TLR3).Fluorescence microscopy and qRT-PCR technology were used to verify the successful overexpression of TLR3 in PANC-1,and then conduct the follow-up experiments in vitro and in vivo.5.The use of PANC TLR3 in vitro cytology experiments was to observe the effect of TLR3 on the biological behavior of PC cells.The effect of TLR3 on the proliferation of PC cells was detected by CCK-8 assay.The effects of TLR3 on the migration and invasion of PC cells were detected by Transwell migration and invasion assay.6.PANC-1 cells were injected into the pancreas of nude mice to establish PC orthotopic implantation nude mice model in order to observe the effect of TLR3 on the proliferation of PC in vivo.PANC-1 cells were injected into the spleen of nude mice to construct the model of liver metastasis of PC in order to observe the effect of TLR3 on the metastasis ability of PC in vivo.7.In order to explore the molecular mechanism of TLR3 affect PC cell lymph node metastasis,we use q RT-PCR,Western blot and other molecular biology techniques to detect changes of key moleculars in some signal transduction pathways of pancreatic cancer cells after over-expressing TLR3.Results1.Results of Transcriptome sequencing showed that TLR3 expression in lymph node metastases of PC was higher than that of primary PC.2.According to results of immunohistochemistry,the expression of TLR3 in lymph node metastases was higher than that of the primary PC in 57 cases of pancreatic cancer with lymph node metastases(Wilcoxon signed-rank test,Z=-2.668,P=0.008).In all 84 patients with PC,the expression level of TLR3 in primary PC was correlated with tumor diameter and lymph node metastases(P=0.0383,P=0.0006,respectively).That was to say,compared with patients with low expression of TLR3,patients with high TLR3 expression had relatively larger tumors and were more likely to have lymph node metastases.3.The Kaplan-Meier survival analysis showed that the median survival time(12 months)of PC patients with high TLR3 expression level was significantly lower than that(20 months)of PC patients woth low TLR3 expression level(χ2= 7.1114,P=0.0077).Therefore,TLR3 can be used as an index to predict the prognosis of patients with PC.However,Cox’s proportional hazards regression model showed that TLR3 couldn’t be thought as an independent risk factor for the prognosis of PC.4.The results of cytology experiments in vitro showed that the proliferation ability of PANC-1 cells was enhanced after over-expressing TLR3.At the same time,the migration ability was significantly increased,but the invasive ability was not significantly affected.The results of PC orthotopic implantation nude mice model showed that the tumors in TLR3 overexpression group were significantly larger than those in control group.The model of hepatic metastasis in nude mice by injecting PC cells into spleen showed that compared with control group,the number of metastases to liver is significantly increased in TLR3 overexpression group.5.The results of molecular biology experiments showed that overexpression of TLR3 decreased the expression of E-cadherin and increased the expression of N-cadherin and the expression of β-catenin in PANC-1 cells,which were correlated with the progression of Epithelial-mesenchymal transition(EMT)and the expression of CD44 High,indicating TLR3 may be through the Wnt/β-catenin pathway regulation of pancreatic cancer cells EMT and upregulation of CD44 way to promote lymph node metastasis.Conclusion TLR3 expression in lymph node metastases of PC was higher than that of primary PC.Compared with patients with low expression level of TLR3 in primary PC,patients with high expression level of TLR3 in primary PC had a relatively larger tumor diameter,were more likely to metastasize to lymph nodes,and had a relatively poorer prognosis.Therefore,TLR3 was valuable in reflecting the patient’s condition and is also a prognosis index for PC patients.Over-expression of TLR3 could enhance the proliferation and the metastatic capacity of PC cells.Over-expression of TLR3 in PC cells could down-regulate the expression of E-cadherin and up-regulate the expression of N-cadherin,which induced PC cells occurred the process of epithelial-mesenchymal transition(EMT).This process may be regulated by the Wnt / β-catenin signaling pathway.TLR3 could also up-regulate the expression of CD44,which made it easier for pancreatic cancer cells to metastasize to the lymph nodes.