| BackgroundPancreatic ductal adenocarcinoma(PDAC)is a very high degree of malignancy of digestive tract tumor,has the characteristics of difficult to diagnose early cases,lack of effective treatment and high mortality,which is worthy of the name of "king of cancer".Autophagy is a kind of eukaryotic programmed cell death mode in parallel with apoptosis.In the special tumor microenvironment of hypoxic and poor blood supply of pancreatic cancer,the autophagy level of pancreatic cancer cells is significantly increased,and may affect the occurrence and development of pancreatic cancer and the response to drug treatment through some unknown mechanisms.Our team has found that EHF(ETS homologous factor/epithelium-specific ETS factor family member 3,ESE3)plays a role of tumor suppressor transcription factor in pancreatic cancer,which can negatively regulate the EMT(epithelial mesenchymal transition)process of pancreatic cancer,suggesting that EHF may also have a negative relationship with the stemness characteristics of pancreatic cancer cells.In the pre experiment,RNA sequencing showed that there was a positive correlation between the expression of EHF and P62(also known as sqstm1).The expression of P62 was closely related to the level of autophagy.Autophagy was also considered to affect the stemness of tumor cells in the existing research.But whether EHF can affect the stemness expression of pancreatic cancer cells,and whether autophagy is involved in this process,and what role it plays,have not been studied.Therefore,we designed this experiment to explore whether autophagy and EHF can affect the level of stemness expression of pancreatic cancer cells,and whether there is a relationship between them in this process,and to clarify the mechanism of action.On this basis,we further sought a new way to promote the treatment of pancreatic cancer by influencing the role of autophagy and EHF.Method1.Six human pancreatic cancer cell lines were cultured in vitro,and their protein and RNA were collected.Two kinds of cells with relatively low and high EHF expression were selected by Western blot and RT-qPCR experiments,and then the virus solution with over and down expression of EHF was synthesized.The corresponding stable over and down expression of EHF expression was performed on the previously selected cells.Subsequently,Western blot and RT-qPCR were used to verify the stable cell line.After the validation,flow cytometry was used to detect the percentage of positive cells of ESA,CD24 and CD44 in pancreatic cancer cells,and the influence of EHF expression level on the stemness of pancreatic cancer cells was analyzed by suspension cell spheroidization and soft agar cloning.2.The RNA of EHF overexpression cell lines and control cell lines were extracted and sent to the company of biological sequencing for RNA sequencing experiment.The relationship between the expression of EHF and P62 at mRNA level was analyzed.The wax blocks of tumor tissue from 96 patients who underwent radical operation of pancreatic cancer in Tianjin Medical University Cancer Institute&Hospital were collected.Immunohistochemistry staining experiment was used to quantify the staining in histochemistry section.The expression of EHF and P62 in tumor tissue was analyzed by chi square test,and the corresponding clinicopathological and prognostic information of patients was collected.Kaplan Meier survival curve was used to analysis the relationship between the expression level of EHF and P62 and the relapse free and the overall survival.Meanwhile,the distribution of EHF and P62 in cells was observed.Eight pairs of matched tissues were randomly selected from the tumor tissue bank of pancreatic cancer,and the protein and RNA were extracted respectively.The relationship between EHF and P62 was studied at the tissue level by Western blot and RT-qPCR.The protein and RNA of EHF overexpression and down expression cell lines and corresponding control cell lines were extracted,and the relationship between EHF and P62 was also studied at the cell level by Western blot and RT-qPCR.Finally,we used chip experiment and double luciferase reporter gene experiment to explore whether EHF as a transcription factor affects P62 expression through transcriptional regulation.3.Western blot and RT-qPCR were used to study the relationship between EHF and autophagy of pancreatic cancer cells.4.The stable cell lines with over and down expression of P62 were constructed,on the basis of which,the stable cell lines with over and down expression of EHF and P62,together with the stable cell lines with over and down expression of EHF and the control cell lines which had been successfully constructed before,were extracted from the protein and RNA of these cell lines by western Block and RT-qPCR experiments were used to verify the effect of stable line construction at the cell level and to further explore the upstream and downstream relationship between EHF and P62.On this basis,flow cytometry,suspension cell spheroidization and soft agar cloning were used to analyze the effect of P62 on the stemnes of pancreatic cancer cells and its possible feedback effect on EHF.5.Immunofluorescence staining experiments were carried out on pancreatic cancer tissue sections and pancreatic cancer cells in vitro slides,respectively.By staining EHF and P62 at the same time,it was observed whether there was co localization relationship between them at the tissue level and the cell level in space.The interaction between EHF and P62 was studied by co-IP experiment.Whether P62 can affect the distribution of EHF in the cells was analyzed by the nucleoplasm protein separation experiment.Whether P62 can affect the transcription activity of EHF was explored by double luciferase reporter gene experiment.6.GST pull-down experiment was used to investigate whether there was direct interaction between EHF and P62 in vitro.NCBI database was used to search the base sequences of the main domains of EHF and P62,and foreign character particles with HA or flag tags were constructed for these domains.The plasmids containing each domain and tag protein were transferred into tool cell 293T by transient transfection.Using co-IP experiment and Western blot experiment to analyze which domain is responsible for the interaction between EHF and P62.7.The autophagy of pancreatic cancer cells was induced by rapamycin,an inhibitor of mTOR,and the autophagy was detected by Western blot,GFP-RFP-LC3 double labeled fluorescent autophagy flow monitoring and flow cytometry.Western blot and nucleoplasm protein separation experiments were used to analyze the effect of autophagy on the total amount of EHF and P62 and their distribution in cells.The effects of autophagy on the stemness of pancreatic cancer cells were analyzed by flow cytometry,cell suspension and soft agar cloning.Whether autophagy can affect the transcription activity of EHF was explored by double luciferase reporter gene experiment.8.In the case of autophagy induced by rapamycin,P62 overexpression plasmid was transiently transfected to form a recovery model.In parallel,the expression of P62 and EHF were effectively reduced by small interfering RNA,respectively,to form a blocking model.Western blot and RT-qPCR experiments were used to verify whether the model was constructed successfully.Western blot and GFP-RFP-LC3 double labeled fluorescent autophagy flow were used Monitoring experiment and flow cytometry to detect autophagy induction.Flow cytometry,suspension cell spheroidization,soft agar cloning and double luciferase reporter gene assay were used to analyze whether autophagy really affects the stemness expression of pancreatic cancer cells through P62-EHF pathway.9.KEGG Pathway analysis and GSEA gene enrichment analysis of bioinformatics were used to find the signal pathway of EHF regulating the sternness of pancreatic cancer cells.Western blot and RT-qPCR were used to verify the upstream and downstream of the selected pathway.After the successful construction of the blocking model,flow cytometry,suspension cell spheroidization and soft agar cloning were used to analyze whether EHF really affects the stemness expression level of pancreatic cancer cells through the selected signal pathway.10.The stable cell line and normal control cell line of pancreatic cancer cell line Pan02 overexpressed EHF were constructed by using EHF overexpression virus liquid.C57BL/6 mice were treated by subcutaneous tumorigenesis in groin area.Then normal saline and ULK-101 were given to treat the tumor respectively.The therapeutic effect of autophagy inhibitor on pancreatic cancer and whether EHF can be a sieve were observed and analyzed biomarkers were selected for patients with better response to autophagy inhibitors.On this basis,the subcutaneous tumorigenesis experiment of NGS mice were carried out with PDX cell line.Then,the normal saline,gemcitabine,the autophagy inhibitor ULK-101 and the combination of gemcitabine and ULK-101 were given respectively.The effect of the combination of autophagy inhibitor and common chemotherapy drugs in the treatment of pancreatic cancer was observed and analyzed.The results1.Through the detection of EHF expression in six human pancreatic cancer cell lines,PANC-1 and MIAPaCa2 cells with relatively low EHF expression were selected to construct stable cell lines of EHF overexpression,and BXPC-3 and SW1990 cells with relatively high EHF expression were selected to construct stable cell lines of EHF down expression.By flow cytometry,suspension cell spheroidization and soft agar colony formation,we can find that EHF has the ability of negatively regulating the cell stemness of pancreatic cancer.2.Through the analysis of RNA sequencing results,it was found that there was a positive correlation between the expression of EHF and P62 at mRNA level.Through the analysis of immunohistochemical staining results,it was found that EHF and P62 were distributed in the cell nucleus and plasma,and both had the trend of increasing and decreasing staining intensity,through the statistical analysis of staining score,the positive correlation between the two is also statistically significant,and both play a protective role in the survival of patients.The protein and RNA levels of pancreatic cancer tissue in vivo and pancreatic cancer cells in vitro suggest that there is a positive correlation between EHF and P62.Through database analysis,it is found that there is a potential binding area of EHF in P62 promoter region.Chromatin immunoprecipitation experiment shows that EHF indeed binds to P62 promoter region.Double luciferase reporter gene experiment shows that the spatial binding of EHF and P62 promoter region will ultimately enhance the transcription activity of P62.3.EHF did not affect the autophagy level of pancreatic cancer cells.4.EHF as a transcription factor does affect the expression of P62,but P62 as a downstream gene does not affect the expression of EHF.P62 also has the ability to negatively regulate the cell stem characteristics of pancreatic cancer,but its effect is not as strong as that of EHF.Considering that P62 may negatively regulate the cell stem characteristics of pancreatic cancer by assisting the role of EHF.5.By immunofluorescence analysis of the sections of pancreatic cancer tissue and the slides of pancreatic cancer cells in vitro,it was found that EHF and P62 were indeed distributed in the cell nucleus and cytoplasm,and their expression levels were positively correlated,and there was co localization of the two proteins in space.The protein interaction between EHF and P62 in pancreatic cancer cells was further confirmed by co-IP experiments.It was found that P62 could affect the distribution of EHF in cells without affecting the total amount of EHF protein.6.The GST pull-down experiment confirmed that there was a direct protein interaction between EHF and P62.Through the construction of plasmids with special labeled proteins that constitute the main domains of EHF and P62,and the co IP experiment after transfection of 293T tool cells,it was found that EHF binds to the ZZ domain of P62 through the PNT domain.7.Western blot,GFP-RFP-LC3 double labeled fluorescence autophagy flow monitoring and flow cytometry confirmed that rapamycin,an inhibitor of mTOR,can effectively improve the autophagy level of pancreatic cancer cells.However,the increase of autophagy level will not affect the total amount of EHF,but will reduce the total amount of P62 and then affect the distribution of EHF in cells,and will reduce the transcriptional regulation ability of EHF,and finally positively regulate the sternness of pancreatic cancer cells.8.The recovery and blocking experiments showed that autophagy affected the dryness of pancreatic cancer cells through P62-EHF pathway.9.Through bioinformatics analysis and Western blot experiments,EHF can indeed negatively regulate the activity of Wnt/β-catenin signaling pathway.Blocking experiments show that EHF can effectively regulate the dry characteristics of pancreatic cancer cells through Wnt/β-catenin signaling pathway.10.It is found that autophagy inhibitor can effectively control the development of pancreatic cancer cells in vivo through the treatment experiment of C57BL/6 mice after tumor implantation.This control effect is more obvious in tumor tissues with high expression of EHF,and the combination of autophagy inhibitor and traditional pancreatic cancer chemotherapy drug gemcitabine shows the effect of increasing the control of pancreatic cancer.Conclusions1.EHF is a transcription factor with antitumor effect in pancreatic cancer,which can protect the survival of patients and negatively regulate the sternness of pancreatic cancer cells.2.EHF can regulate the expression of P62 by forward transcription,and P62 will affect the distribution of EHF in the nucleus and the transcription activity of EHF through protein interaction,and finally form a positive and negative feedback loop to jointly affect the stemness of pancreatic cancer cells.3.Autophagy can reduce the expression of P62 and the distribution of EHF in the cell nucleus.At last,autophagy can enhance the dryness of pancreatic cancer cells by weakening the transcription activity of EHF and activating the downstream Wnt/β-catenin signaling pathway.4.Autophagy inhibitors can effectively control the development of pancreatic cancer cells.The combination of autophagy inhibitors and gemcitabine is more effective.The response of pancreatic cancer cells with over expression of EHF to this treatment is more obvious. |
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