| Background and Study aims:Pancreatic cancer is one of the most malignant gastrointestinal tumors.Pancreatic cancer progresses rapidly and the treatment to it is limited.Worldwide,the incidence and mortality of pancreatic cancer rank 12 th and 7th among malignancies,respectively.The 5-year survival rate is less than 5%.The early diagnosis of pancreatic cancer is difficult and there is no specific early warning signs,and also there are a lack of screening processes for pancreatic cancer.So it is already in the advanced stage when the disease is diagnosed.Up to this day,radical surgery still offers the only hope of curing pancreatic cancer,but the prognosis remains disappointing.About 85% of surgically viable patients have a total survival time of less than 5 years due to th hidden metastasis preoperatively or recurrence postoperatively,and such metastasis is very rapid and unpredictable,often occurring and developing before screening,diagnosis and treatment.In addition,stromal cell and ECM interstitial pressure constrict blood vessels and reduce the flow of chemotherapeutic compounds.Therefore,the osmotic effect of chemotherapeutic drugs is limited.Furthermore,membranous adenocarcinoma cells are not sensitive to chemotherapeutic drugs,resulting in poor chemotherapeutic effect.Besides,the adenocarcinoma cells stemed from the membranous are not sensitive to chemotherapy drugs,resulting in the poor efficacy of chemotherapy.Although radiotherapy can alleviate the pain of patients to some extent,there is no definite evidence that it can extend the overall survival period.In summary,the characteristics of pancreatic cancer are that late detection,early metastasisr,easy recurrence and poor prognosis.The main factor that makes the treatment of pancreatic cancer difficult and poor prognosis is that metastasis has occurred in the early stage of the disease.Therefore,in view of the above behavioral characteristics of pancreatic cancer,it is of great significance and imperative to find key proteins and regulatory pathways to regulate its malignant biological behavior.To develop novel targeted drugs for this protein,or to block its related signaling pathways to regulate malignant biological behavior can provide new therapeutic methods for pancreatic cancer patients,which will improve the adverse prognosis of pancreatic cancer patients.In order to clarify the molecular mechanism involved in regulating invasion and metastasis of pancreatic cancer,we used BOP to induce Syrian hamsters in order to establish a non-dissociated pancreatic cancer cell line(PC-1)from the in situ focus of pancreatic cancer.PC-1 cells were implanted into another Syrian hamster of the same species by caudal vein injection,and liver metastases were taken to establish dissociated pancreatic cancer.Cell line(PC-1.0).These two pancreatic cancer cell lines are a pair of homologous cell lines with different invasion and metastasis potential.The distinction between them is that the invasive and metastatic ability of the dissociative pancreatic cancer cell line(PC-1.0)is relatively strong,while that of the non-dissociative pancreatic cancer cell line(PC-1)is relatively weak.This couple of homologous pancreatic cancer cell line mouldl is perfect to go deeply into the mechanism of invasion and metastasis.The differential proteins obtained by differential proteomics analysis of these two cells were highly correlated with the invasion and metastasis of pancreatic cancer.Further research of the most significantly differential expressed proteins can furnish a bertter targeted comprehension of the principles involved in invasion and metastasis of malignant pancreatic tumor.The biological characteristics of human pancreatic cancer cells AsPC-1 were similar to those of the dissociative pancreatic cancer cell line(PC-1.0),while the biological characteristics of human pancreatic cancer cells Capan-2 were similar to those of the non-dissociative pancreatic cancer cell line(PC-1).The screening of differential proteins in hamster pancreatic cancer cell lines and their roles in invasion and metastasis can be verified in these two human pancreatic cancer cell lines.Exosomes are 40-150 nm extracellular vesicles released by cells under normal and pathological conditions.It was first discovered in reticulocytes of sheep,which,in 1987,Johnstone named “exosome”.Exosome vesicles contain proteins,DNA,mRNA and noncoding RNA.These substances may mediate specific intercellular communications,activating signaling pathways in cells with which they can fuse or interact.Exosomes have been detected in tumor microenvironment,and many recent studies have confirmed that exosomes play an important role in promoting tumor angiogenesis,epithelialmesenchymal transformation and immunity,thus promoting the invasion and metastasis of cancer cells.Circulating blood exosomes can be used as non-invasive biomarkers to prompt early blood screening and postoperative recurrence warning for patients with related tumors.Tenascin-C(TNC),as a member of the extracellular matrix glycoprotein family,plays an important role in tissue damage repair,fibrosis,inflammatory reaction and partial tumor angiogenesis and metastasis.TNC is low or not expressed in normal human tissues,but it is highly expressed in many malignant tumors.Recently,the role of TNC as an exosomal protein in malignant tumors has attracted extensive attention.In particular,the role of exosome-derived TNC in the early diagnosis of pancreatic cancer,or whether it can indicate early metastasis and the effect indicating prognosis is unclear.This project intends to study the molecular mechanism of exosome-derived TNC in the occurrence and development of pancreatic cancer and the regulation of malignant biological behavior of pancreatic cancer? Methods:1.In this study,quantitative proteomics(iTRAQ)was used to analyze the dissimilarity proteins of the extracellular vesicles in the supernatant of homologous hamster pancreatic cancer cell lines(PC-1.0,PC-1).Then,Tenascin-C(TNC),which was markedly overexpressed in PC-1.0,was choosed for further study.Exosome proteins from the supernatant of PC-1.0 cells were extracted using Exoquick-TC kit,and morphological identification was performed using Transmission electron microscope(TEM).Western-blot was used to identify the specified protein markers of the extracted exosomes.Western blot was used to determine whether TNC was an exosome protein and the expression differences of TNC in PC-1.0 and PC-1,AsPC-1 and Capan-2 cell lines.2.In cell experiment,TNC-shRNA was transfected into pancreatic cancer cell lines PC-1.0 and AsPC-1 instantaneously,and the silencing efficiency was confirmed by Reverse Transcription-Polymerase Chain Reaction and Western-blot.After effective silencing of TNC gene,CCK-8 was used to detect the changes in the proliferation of PC-1.0 and AsPC-1 cells after the down-regulation of TNC.After staining with Annexin ?-FITC /PI apoptosis detection kit,flow cytometry was used to detect the early and late apoptosis of PC-1.0 and AsPC-1 after down-regulation of TNC.The changes of invasion ability in PC-1.0 and AsPC-1cell lines after the down-regulation of TNC were detected by Woundhealing.The alterations of migration and invasion capabilities of PC-1.0 and AsPC-1 cell lines after down-regulation of TNC were further tested by means of Transwell assay.3.Next,in order to further explore the molecular mechanism involved in the regulation of the above malignant biological behavior of pancreatic cancer by TNC,we employ Western-blot to explicit the effect of down-regulated TNC on NF-?B signalling pathway.At the same time,the effects of TNC on the epithelial interstitial transition(EMT)and the relativity between TNC and Wnt/?-catenin signalling pathway were clarified,so as to reveal the possible molecular mechanism of the biological function of TNC in pancreatic cancer.Results: 1.TEM results showed that most of the saucer-shaped vesicles with clear membrane structure were slightly less than 100 nm.It was suggested that the vesicle structure extracted from the PC-1.0 cell supernatant using Exoquick-TC kit are exosomes-like.Western-blot results showed that the protein markers of exosome,including CD81,CD63 and HSP70 were all positive expression.These results suggested that the substances extracted from the supernatant of PC-1.0 cells were exosomes.The expression level of exosomal TNC in PC-1.0 and PC-1,AsPC-1 and Capan-2 cell lines shows significant differences.The data were analyzed and studied.Exosomal TNC was significantly highly expressed in PC-1.0 and AsPC-1 cell lines which possess strong invasion ability,while it was low expressed in PC-1 and Canpan-2 cell lines with weak invasion ability.2.Exosomal protein TNC was expressed at relatively high levels in PC-1.0 and AsPC-1 cell lines.Transient transfection with shRNA distinctly down-regulated the expression of exosomal TNC in both cells.At the level of cell,CCK-8 assay was used to detect the effect of proliferation when exosomal TNC is down-regulated.Compared with the control groups-NC groups,the TNC knockdown groups of PC-1.0 and AsPC-1 show weaken ablity in proliferation.In PC-1.0 cells,compared with NC group,the proliferation of shRNA group at 72 hour were distinctly lower.(2.880±0.097 vs 3.513±0.230,P <0.001),compared with NC group,the proliferation of shRNA group at 96 hour were distinctly lower than NC group(3.148 ±0.06 3 vs 4.020 ±0.0.080,P <0.001).In AsPC-1 cells,compared with NC group,the proliferation of shRNA group at 72 hour were distinctly lower than NC group(2.861±0.052 vs 3.388±0.267,P <0.001),compared with NC group,the proliferation of shRNA group at 96 hour were distinctly lower than NC group(3.083 ±0.031 vs 4.318 ±0.162,P <0.001).After staining with Annexin ?-FITC /PI apoptosis detection kit,flow cytometry was used to detect the effect on apoptosis when down-regulated the expression of exosomal TNC,in PC-1.0 and AsPC-1 cells,the apoptosis rate of shRNA group is higher than that of the NC group,and the effect on early apoptosis was not obvious,but the effect on late apoptosis was statistically significant,with P = 0.0387,P = 0.0119,respectively.The Wound-healing assay was performed to detect the effect of cell invasion ability when down-regulated the expression level of exosomal TNC,in PC-1.0 cell,shRNA group showed lower invasion ability than NC group.In AsPC-1cell,shRNA group showed lower invasion ability than NC group.Transwell assay was performed to further detect the effect of cell invasion and migration ability when down-regulated the expression level of exosomal TNC,in PC-1.0 cell,invasive cells were less in the shRNA group than in the NC group(227.667 ± 13.283 vs 758.667 ± 40.761,P< 0.001).While in AsPC-1cell,invasive cells were less in the shRNA group than in the NC group(310.000 ± 17.436 vs 743.333 ± 76.790,P< 0.05).In PC-1.0 cell,migrational cells were less in the shRNA group than in the NC group(236.333 ± 10.414 vs 735.000 ± 23.643,P< 0.0001).While in AsPC-1cell,migrational cells were less in the shRNA group than in the NC group(289.667 ± 10.138 vs 723.000 ± 56.454,P< 0.05).In summary,the results showed that the down-regulation of exosomal TNC weakened the ability of pancreatic cancer cell migration and metastasis.3.In both PC-1.0 and AsPC-1 cells,transient transfection with shRNA was used to knock down the exosomal TNC in pancreatic cancer cells,and compared with the high expression of exosomal TNC in the untreated cells,the NF-?B signalling pathway was influenced.The expression level of pho-NF-?B-P65 was down-regulated while the level of intracellular NF-?B-P65 remains consistent.In both PC-1.0 and AsPC-1 cells,transient transfection with shRNA was used to knock down the exosomal TNC in pancreatic cancer cells,and compared with the high expression of exosomal TNC in the untreated cells,the Wnt/?-catenin signalling pathway was influenced.The intracellular expression level of pho-?-catenin was up-regulated,while the expression level of related target genes in the downstream of Wnt/?-catenin signalling pathway was down-regulated.In PC-1.0 and AsPC-1 cells,expression levels of EMT-related protein markers were changed.The level of E-cadherin was up-regulated,while the expression level of Ncadherin and Vimenin were down-regulated after transient transfection with shRNA to knock down exosomalTNC in pancreatic cancer cells,compared with the high expression level of exosomal TNC in the untreated cells.Conclusions: 1.In pancreatic cancer cell lines,TNC is an exosomal protein.As an exosomal protein,the expression level of TNC is much higher in hamster pancreatic cancer cell line PC-1.0 and human pancreatic cancer cell line AsPC-1 which are high invasive cells,while the expression level of TNC is much lower in hamster pancreatic cancer cell line PC-1 and human pancreatic cancer cell line Canpan-2 which are low invasive cells.These results suggest that exosomal TNC may play a role in promoting the invasion and metastasis ablilities of malignant tumors of the pancreas.2.Down-regulation of exosomal TNC can inhibit the proliferation of pancreatic cancer cells and increase the rate of late apoptosis,but it has little influence on the early apoptosis of the ductal adenocarcinoma cells.Down-regulation of exosome TNC can inhibit the invasion and migration of pancreatic cancer cells.Down-regulation of exosome TNC expression plays an important role in improving malignant phenotype of pancreatic cancer.3.The role of exosomal TNC in regulating the expression of NF-?B signalling pathway and Wnt/?-catenin signalling pathway is a possible molecular mechanism for its biological function in pancreatic cancer. |
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