| BackgroundPancreatic cancer is a highly malignant tumor and its malignancy ranks among the top tumors.At present,surgery is still the main treatment for pancreatic cancer.However,because the majority of the tumors found in the advanced stage,the effect of surgical treatment is poor and the 5-year survival rate is less than 9%.Besides,other treatments such as radiotherapy and chemotherapy,as well as emerging treatment methods such as target drugs and cell therapy,are not suitable as the clinical intervention of pancreatic cancer.Therefore,the analysis of the molecular mechanism of pancreatic cancer and the identification of the new target of this tumor may provide a new way to improve the treatment of pancreatic cancer.CD8+ T cells play a critical role in anti-tumor immunity.However,tumors can escape immune attack through various mechanisms of immunosuppression,such as inhibiting the proliferation and activation of CD8+ T cells.Reactivating the anti-tumor responses of CD8+T cells via checkpoint blockade has recently been demonstrated to have notable therapeutic effects on cancer.However,not all cancers respond to checkpoint blockade.pancreatic cancer,one of the deadliest human malignancies,is characterized by an immunosuppressive tumor microenvironment that renders it largely refractory to immunotherapy.Although many pancreatic cancer patients exhibit CD8+ T cell tumor infiltration,immunotherapy has not led to clinical benefits;one major limiting factor is CD8+ T cell dysfunction or hypo-responsiveness.Recent clinical trials using a combination of immunoregulatory agents,such as angiogenesis inhibitors,have shown positive initial results.It is therefore of great clinical interest to develop other therapies to potentiate the anti-tumor activity of CD8+ T cells by modulating different pathways.However,the precise mechanism by which T cell dysfunction is established in tumors remains unclear.Up to now,there are various molecules that have been found to play important roles in pancreatic cancer.Among them,the gene transcription-related protein was reported to be highly correlated with the occurrence and the development of tumors.Several studies showed that the transcriptional cofactor 4(PC4)was elevated in many tumors,such as non-small cell lung cancer and breast cancer.Further,elevated PC4 expression was reported to be related to the malignancy of tumors,indicating that PC4 might be critical factor in the occurrence and the differentiation of many tumors.At 2007,Kiran found that PC4 protein interacted with p53,affecting the expression of the downstream genes p21.Such regulation promoted cell apoptosis and finally inhibited tumor growth Hence,PC4 is termed as a tumor suppressor factor.On the other hand,other reports revealed that knockdown of PC4 protein in non-small cell lung cancer inhibited the expression of XRCC4-like factor(XLF)at transcriptional level.XLF regulated the non-homologous end of joining(NHEJ)of cell DNA,thus elevating the radiotherapy sensitivity of tumors.In addition,the reduction of the protein in non-small cell lung cancer also suppressed tumor growth by the induction of cell cycle arrest and the increase of cell apoptosis.These data suggested that PC4 was also a promoting factor in tumors.PC4 in tumors demonstrated its characteristics of ’double-edged sword’ function and exhibited a strong ability to interfere the tumor growth.Therefore,PC4 may be a potential regulator for the growth of pancreatic cancer.In order to explore the expression and function of PC4 in pancreatic cancer,we identified that the expression of PC4 mRNA in pancreatic cancer via the bioinformatics analysis.Based on the above findings,we studied the role of PC4 and NPC1L1 in pancreatic cancer.Purpose1、To explore the function of PC4 in pancreatic cancer,as well as the relationship between this protein and the immune microenvironment of the tumor,we designed the following experiments,which might provide new ideas for the treatment of pancreatic cancer.2、To explore the function of PC4 regulatory protein NPC1L1 in pancreatic cancer and the relationship between NPC1L1 and the inhibitory immune microenvironment of pancreatic cancer.Method1.The public databases were used to explore the relationship between the expression level of PC4 mRNA and pancreatic cancer.The PC4 protein expression level was quantified by the analysis of immunohistochemical staining of pancreatic cancer samples collected by ourselves,and the relationship between its expression level and the prognosis of pancreatic cancer was analyzed.The PC4 knockdown cell line was established with the help of lentivirus infection,and the PC4 knockdown cell line was injected into nude mice to explore the effect of PC4 protein on the growth of pancreatic cancer.The potential relationship between PC4 protein and cells in the immune microenvironment of pancreatic cancer,and tumor stroma,as well as the relationship between the expression of PC4 and immune checkpoints in pancreatic cancer were analyzed by the bio-information analysis we also investigate the function of PC4 regulatory protein NPC1L1 in pancreatic cancer.Finally,the relationship between NPC1L1 and the immune microenvironment of pancreatic cancer was studied by bioassayResult1.The expression of PC4 was related to the prognosis of pancreatic cancerWe found that compared with corresponding adjacent tissues,the expression of PC4(gene name SUB1)mRNA was increased in the pancreatic cancer tissues via analysis of the GEPIA public database.This database also revealed that PC4 mRNA had no effect on the survival of pancreatic cancer patients.Then we detected the protein expression level of PC4 in our collected pancreatic cancer tissue samples and found that this protein was highly expressed in pancreatic cancer tumor tissues,little in normal tissue.Furthermore,PC4 was identified to be related to the differentiation of the pancreatic cancer,and the PC4 expression was abundant in tumors with low differentiation.In addition,we also found that the expression of PC4 protein was closely related to patient survival.2.The knockdown of PC4 significantly inhibited the growth of pancreatic cancer in vivoWe successfully constructed cell lines that stably knocks down PC4 with pancreatic cancer cell lines CFPAC-1 and ASPC-1.Next,the pancreatic cancer cell line was used to construct the tumor model.The results showed that the knockdown of PC4 significantly inhibited the growth of pancreatic cancer,while the tumor grew normally in the control group.The difference between the two groups was statistically significant.3.The expression of PC4 was related to the immunosuppressive microenvironment of pancreatic cancerWe found that the expression of PC4 in tumors was significantly related to the immune cells,interstitial components and immune checkpoints of the immune microenvironment of pancreatic cancer via the analysis of multiple databases and algorithms.The infiltration of regulatory T cell and M2 macrophage increased when the expression of PC4 expression increased.At the same time,PC4 expression was positively correlated with the immune score and interstitial score of pancreatic cancer,and there was statistical significance among multiple immune checkpoints.4.PC4 plays a regulatory role in NPC1L1 in pancreatic cancerThrough data base analysis,we found that in pancreatic cancer,the expression of PC4 was significantly correlated with the expression of NPC1L1,suggesting that PC4 may play a role on NPC1L1.5.NPC1L1 is highly expressed in pancreatic cancer and is significantly correlated with the prognosis of the pancreatic cancer.Through database analysis,we found that at the mRNA level,the expression of NPC1L1 in pancreatic cancer was higher than that in adjacent pancreatic cancer,which may be regulated by KRAS and demethyl enzyme TET-3,.At the protein level,we found that the expression of NPC1L1 in pancreatic cancer was higher than that in adjacent pancreatic cancer by tissue microarray staining,and it was significantly correlated with the patient’s prognosis.6.NPC1L1 plays an important role in regulating tumor immune response.Through the data base analysis,we found that pancreatic cancer patients with high or low expression of NPC1L1 had significant differences in tumor inflammatory response pathways.Furthermore,through multiple immunological databases,we found that NPC1L1 mainly affected CD8+ T cells in the tumor microenvironment.Conclusion1.We found that PC4 protein was highly expressed in pancreatic cancer and was closely related to the prognosis of patients via the analysis of bioinformatics and detection of the expression of the PC4 in pancreatic cancer tissues respectively.The knockdown of PC4 protein significantly inhibited tumor growth.In addition,the bioinformatics analysis showed that PC4 protein was closely related to the suppressive immune microenvironment.2.The PC4 regulatory protein NPC1L1 is highly ectopic expressed in pancreatic cancer,and the higher of expression,the worse the prognosis of patients.Further,we found that NPC1L1 is associated with the suppressive immune microenvironment of pancreatic cancer... |
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