Function Of BTN3A1 In Esophageal Squamous Cellcarcinoma And Its Mechanism Of Radiaresistance

BackgroundEsophageal cancer is one of the most common cancers and the sixth most common cause of cancer death in the world.Esophageal cancer can be categorized by 2 main histologic subtypes;adenocarcinoma(AC)and squamous cell carcinoma(SCC).The incidence of esophageal squamous cell carcinoma is the highest in Asia and Africa,accounting for 90%of the total incidence of esophageal carcinoma.ESCC is among the most aggressive forms of human squamous cell carcinoma and the 5-year survival remains poor at an estimated 15-20%.At present,radiotherapy and chemotherapy are the main treatment for patients with locally advanced or unresectable ESCC.Studies have shown that chemoradiotherapy has been shown to provide long-term survival in up to 27%of patients diagnosed with ESCC.Advances in updated techniques of delivering precision therapy have enhanced radiation target coverage and minimized exposure to non-target organs,improving toxicity profiles.However,radiotherapy resistance is a major cause of treatment failure in ESCC patients.Changes in the genome or epigenome of tumor cells can cause primary radiation resistance.Radiation therapy itself may also lead to secondary radiation resistance.Radiation can cause single or double strand DNA break of cancer cells.DNA damage can change genome without accurate repair,which may make some tumor cells resistant to radiation therapy.It is an important strategy to find effective radiosensitizing targets and design sensitizing drugs to deal with radiation resistance.Human butyrophilin subfamily 3 member A(BTN3A),also known as CD277,includes isoforms BTN3A1,BTN3A2,and BTN3A3 and is structurally associated with B7 costimulatory molecules.The extracellular regions of the three subtypes are structurally similar,sharing 95%homology.BTN3 A plays a role in T-cell activation and in the adaptive immune response and regulates the proliferation of activated T-cells.BTN3A regulates the release of cytokines by activated T-cells and mediates the response of T-cells toward infected and transformed cells that are characterized.In particular,the B30.2 intracellular domain plays an important role in this process.However,only the intracellular B30.2 domain of BTN3A1 directly binds phosphorylated antigens(pAgs)through a positively charged pocket to activate Vy9V82 T cells.In addition BTN3A1 is expressed in many tumors,such as ovarian cancer,bladder cancer,breast cancer,renal cell carcinoma and pancreatic ductal adenocarcinoma.However,its role in these tumors is unclear,and the prognostic role of BTN3A1 in different cancers varies substantially.Objectives1.To explore the BTN3A1 clinical significance and its functions in ESCC;2.To clarify the specific molecular mechanism of radiation up-regulation BTN3 A1 expression and explore the role and mechanism of BTN3A1 in ESCC radioresistance;3.To verify the regulatory effect of BTN3A1 on autophagy related gene ULK1 and its mechanism.Materials and methods1.Clinical specimen collection and analysis:Purchase tissue chips(Catalog ESC77,Superbiotek Pharmaceutical Technology Co.).BTN3A1 and LC3B immunohistochemistry(IHC)were assessed.To analyze the relationship between BTN3A1 expression and survival prognosis.2.Cell line construction:(1)Stable transfection:esophageal cancer cell lines were transfected with lentivirus vectors containing DNA encoding human BTN3A1 and shRNAs targeting BTN3A1 mRNA,shRNAs targeting HIF-1α mRNA and mRFP-GFP-LC3 lentivirus respectively,followed by purinamycin treatment.(2)Transient transfection:cell lines were transfected with BTN3A1 overexpressing plasmids,BTN3A1(shMT)overexpressing plasmids,BTN3A1 promoter mutant overexpressing plasmids and HIF-1α overexpressing plasmids,respectively.3.Cell irradiation treatment:When the cells were fused to 70%,the medium was changed and the cells were irradiated with different doses at a dose rate of 100 cGy/min using a highspeed linear accelerator,and a tissue compensation membrane of 1.5 mm was used.After irradiation,the cells were returned to the incubator for subsequent analysis and statistics according to the experimental requirements.4.Cell proliferation detection:(1)CCK-8 assay:Cells(3×103cells/well)were inoculated into 96-well plates.After 24 h incubation,10μl CCK-8 solution was added to each well for 2 h incubation.Measure the absorbance with a spectrophotometer.(2)Cloning formation assay:the cells were inoculated into the 6-well plate at a density of 1000 cells per well.After 10 days of incubation,cells were fixed with paraformaldehyde,stained with crystal violet and counted(clones of more than 50 cells were counted).(3)EdU incorporation assay:the cells were plated in 24-well plates at 1×105 cells/well density,incubated for 24h,stained with EdU kit and photographed with fluorescence microscope.5.Cell migration detection:(1)Cell scratch test:Draw straight lines on the cells gently with the standard 200μL pipette tip,and rinse off the detached cells with PBS.Then the cells were cultured for 24 hours and the scratches were photographed under a microscope.(2)Transwell assay:1×105 cells were added into 200 μL serum-free medium and inoculated into the upper chamber.500 μL medium containing 20%FBS was added in the lower chamber as the inducer.After 24 h,the cells were fixed with methanol,stained with 0.1%crystal violet and counted under a microscope.6.Chromatin immunoprecipitation:The ChIP kit produced by Epigentek was used for this part of the experiment.Anti-HIF-1α primary antibody,rabbit IgG was used as negative control,and anti-RNA polymerase Ⅱ(Epigentek)as positive control.The ChIP products were tested by RT-PCR and nucleic acid gel electrophoresis on 1.5%agarose gel.7.Double luciferase reporter gene assay:Construct wild-type or mutant BTN3Al promoter luciferase plasmid.Luciferase plasmids were co-transfected with HIF-1α plasmids using DNA transfection reagents.24 h after transfection,cell lysates were collected and luciferase activity was measured using Luc-PairTM Duo-Luciferase Assay Kit 2.0.8.BTN3A1 and ULK1 immunoprecipitation:Cells were lysed with lysis buffer containing protease inhibitors,and the extracted proteins were treated with corresponding antibodies:anti-ULK1 antibody,IgG antibody,anti-FLAG magnetic bead and protein A/G plus agarose bead.After the unbound protein was removed by washing with PBS,the remaining protein was suspended in 1×SDS sample buffer and boiled for 10 min.The product was analyzed by Western Blot.9.Subcutaneous tumor formation experiment:The 5-week-old BALB/c-nude male mice were randomly divided into groups.According to the experimental requirements,KYSE150 cells(1×107 cells)carrying corresponding vectors injected subcutaneously into the armpits of the mice.The tumor volume(V)was measured every 3 days by the following formula:V=(ab2)/2(a for long diameter,b for short diameter).When the tumor volume reached～200 mm3,radiation was given at a dose of 4 Gy on the 1st,3rd and 5th day.The tumor size was measured every 3 days for 3 weeks,and the nude mice were killed for follow-up experiments.10.Detection of protein molecule expression(immunofluorescence,immunohistochemistry):(1)Immunofluorescence:After fixing the cells with formaldehyde for 15 minutes,immerse them in 0.5%Triton X-100 osmotic solution for 20 minutes,then block and incubate the first antibody.Add the fluorescent secondary antibody of the corresponding species and incubate it at room temperature in dark for 1 h.After washing with PBS,repeat staining with DAPI for 15 minutes.Observe and take photos under a fluorescence microscope.(2)Immunohistochemistry:Tissue sections were baked at 65℃ for 2 h,dewaxed,and rehydrated.Block and incubate the first antibody.After removal,incubate the second antibody at room temperature for 60 minutes.Subsequently,DAB staining,hematoxylin counterstaining,and dehydration sealing were performed.11.Bioinformatics analysis:Transcriptome data of BTN3A1 were obtained from TCGA database and GEO database,and Autophagy related genes were downloaded from HADb Human Autophagy database.12.Statistical analysis:Statistical analysis was performed using SPSS 21.0 version and GraphPad Prism 8.0.All data were analyzed for normality and variance.Continuous variables were expressed as mean ± standard deviation,and data between the two groups were analyzed by T-test or Mann-Whitney U test.Kaplan-Meier method was used to evaluate OS and PFS,and compared with log-rank test.Immunohistochemical scores were analyzed by X-tile 3.6 software,and correlations were analyzed by Pearson test.P<0.05 was statistically analyzed.Results1.BTN3A1 upregulation is related to a poor prognosis:Postoperative pathological specimens of 118 patients with middle and advanced esophageal squamous cell carcinoma were collected.All patients received postoperative radiotherapy and chemotherapy.High BTN3 A1 expression was positively correlated with local recurrence rate and distant metastasis rate,while low BTN3A1 expression was correlated with longer OS and PFS.The relationship between BTN3A1 expression and prognosis in patients with early esophageal cancer was studied using ESCC tissue microarray.2.BTN3A1 promotes the growth and migration of ESCC cells:The proliferation of BTN3A1 overexpressed cells was accelerated and the migration ability was enhanced.After BTN3A1 knockdown,cell proliferation was prevented and cell movement was restricted.In vitro experiments further verified the carcinogenic effect of BTN3A1.3.HIF-la is the upstream target of radiation-induced BTN3A1 expression:Radiotherapy upregulates the expression of BTN3A1 in ESCC cells.Inhibition of HIF-1α can significantly inhibit the overexpression of BTN3A1 induced by ionizing radiation.HIF-la was increased under 8Gy irradiation,which acted as a transcription factor to bind to the BTN3A1 promoter and promote the transcription process of BTN3 A1.4.The expression of BTN3A1 affects the radiosensitivity of ESCC cells:BTN3A1 knockdown sensitizes ESCC cells to radiation.Cloning formation assay,cell proliferation curve and EdU cell proliferation assay showed that BTN3A1 knockdown induced radiosensitization of ESCC cells.Subcutaneous tumor formation in nude mice also verified that downregulation of BTN3A1 can sensitize radiotherapy.5.BTN3A1 promotes autophagy in ESCC cells:Cell death mode PCR array showed that the BTN3A1 had the most significant correlation with autophagy.BTN3A1 knockdown significantly decreased the levels of ULK1,LC3BII/LC3BI and increased the expression of P62.However,BTN3A1 did not affect the expression of APG7,ATG5,ATG16L1 and Beclinl.6.BTN3A1 knockdown decreases the level of autophagy in ESCC cells:The autophagy level of KYSE150 and ECA109 cells increased after 8 Gy irradiation.However,after BTN3A1 knockdown,the autophagy mediated by radiation was reversed.By transfecting KYSE150 cells with tandem mRFP-GFP-LC3 lentivirus vectors,Confocal microscopy showed that down-regulated BTN3A1 expression significantly reduced the formation of autophagosomes and autolysosomes,and inhibited the autophagy flux activated by radiotherapy.Transmission electron microscopy(TEM)was used to observe the ultrastructure of KYSE150 cells After irradiation with 8 Gy,the number of autophagy vacuoles in ESCC cells increased,and BTN3A1 knockdown significantly reduced the number of autophagy vacuoles in irradiated cells.7.BTN3A1 promotes cell radioresistance by activating autophagy:Inhibition of autophagy by 3-MA enhanced the radiation sensitivity of ESCC cells.KYSE150 cells treated with EBSS were less sensitive to radiation than control cells.When cells treated with EBSS were transfected lentivirus with a BTN3A1-specific shRNA,the sensitivity was increased to some extent and was not different from that of the control group.KYSE150 cells transfected with BTN3A1 plasmids were more resistant to IR than control cells,and the cells treated with 3MA,were resensitized to IR.IHC staining revealed that LC3B protein expression was positively correlated with BTN3A1 expression in tumor tissues.8.ULK1 is the downstream target of autophagy induced by BTN3A1:Combining TCGA database and GEO database analysis results,we speculated that BTN3A1 expression was related to ULK1.We performed coimmunoprecipitation(Co-IP)in KYSE150 cells,and the proteins pulled down by specific antibody were identified using mass spectrometry analysis.ULK1 was detected in only the BTN3A1 group but not in the vector group.BTN3A1mediated autophagy may be related to the ULK1 gene.BTN3A1 overexpression regulated the expression of ULK1 and promoted its phosphorylation at Ser555.Conclusion1.BTN3A1 upexpression was related to poor prognosis in patients with esophageal squamous cell carcinoma.BTN3A1 promotes the growth and migration of cancer cells.2.Radiation increases BTN3A1 expression through a process mediated by HIF-1α.BTN3A1 is associated with low radioreactivity.3.BTN3A1 promotes cell radioresistance by regulating autophagy;ULK1 is a downstream target of BTN3A1 induced autophagy.