Research On Blocking CyclinB1 Regulates Autophagy Of Nasopharyngeal Carcinoma Cells And Underlying Mechanism

Background: Nasopharyngeal carcinoma(NPC)is a highly malignant carcinoma in head and neck tumor and prones to metastasis.The incidence and mortality of NPC in China account for 38.29% and 40.14% of overworld,respectively.Although early stage of NPC is sensitive to chemoradiotherapy,70%-80% of patients are at advanced stage when diagnosised,and the effect of traditional chemoradiotherapy is dissatisfied.Therefore,it is urgent to find new therapeutic targets to improve the efficacy of NPC.Cyclin B1 is a crucial protein involved in mitosis.Overexpression of cyclin B1 frequently occurs in various neoplasms including NPC.Furthermore,overexpression of cyclin B1 indicates a poor prognosis.These indicate that cyclin B1 may be an oncogene and targeting cyclin B1 can be an attractive strategy for NPC therapy.Autophagy is a process of intracellular self-digestion,and its influence on tumor is two edges.On the one hand,autophagy can inhibit the tumor cells in the early stage.On the other hand,in the late stage of tumor,autophagy can provide energy for tumor cells by degrading misfolded proteins or damaged organelles,thus maintaining the homeostasis.This project aims to explore the influence of cyclin B1 downregulation on cell autophagy,to elucidate the underlying molecular mechanism,and to determine cyclin B1 as a potential therapeutic target.Objective: To elaborate the influence of cyclin B1 silencing on cell autophagy,to unveil the underlying molecular mechanism,and to determine cyclin B1 as a potential therapeutic target.Methods: NPC cells CNE-1 and CNE-2 were synchronized to the S phase by the dual blocking of thymidine nucleoside(Td R);The si RNA transfection efficiency of CNE-1 and CNE-2 cells was determined by fluorescence microscope and flow cytometry,and the down-regulating efficiency was assessed by western blot;The autophagosomes were observed by RFP-GFP-LC3 dual fluorescence labeling,LC3 immunofluorescence,dansylcadaverine(MDC)staining and transmission electron microscopy;The expressions of autophagy related proteins LC3-I,LC3-II and p62 were detected by western blot;Transcriptome sequencing was performed to evaluate the expression of different genes after silencing cyclin B1;Bioinformatics analysis of the enrichment of differentially gene-related signal pathways and their biological functions;RT-PCR and western blot were applied to further verify the transcriptome sequencing results;The intracellular ROS and mitochondrial m ROS levels were measured by flow cytometry;ATP level was determined by chemiluminescence;ROS inhibitors suppressed intracellular ROS level;Si RNA down-regulated the expression of AMPK;The Cancer Genome Atlas(TCGA)database was used to investigate the expression of cyclin B1 in different tumors and its relationship with prognosis;CCK-8 and crystal violet assay were applied to detect NPC cell proliferation;Annexin V-FITC/PI and Tunel assay were performed to detect apoptosis;Cell senescence was determined by ?-galactosidase staining.Results:1.Double Thymidine(Td R)2.5mmol/L blocking could efficiently synchronize the cells to S phase.Then,the cells viability was desirable and harvested for transfection experiments.2.Three si RNAs(si CB1,si CB2,si CB3)are designed against the open reading frame of cyclin B1 m RNA.Western blot showed the protein level of cyclin B1 is apparently declined after transfected with each of the cyclin B1 si RNAs and si CB2 effect is best(P<0.05).3.The si RNA2 against cyclin B1 of CNE-1 and CNE-2 cells markedly elevated the amount of LC3B-II and MDC,but attenuated p62(P<0.05).Then,TEM,RFP-GFP-LC3 B dual-reporter assay and LC3 B immunofluorescence assays,which represented endogenous autophagosomes,displayed the noticeable accumulation of double-membrane electron-dense autophagosomes and LC3 puncta,respectively(P<0.05),in nasopharyngeal carcinoma cells treated with cyclin B1 si RNA2.4.Transcriptome sequencing determined a total of 2721 differentially expressed genes after silence of cyclin B1,and up-regulated genes were enrichment in lysosome and autophagy signal pathway.Its biological function mainly involved in ubiquitination,protein binding,membrane assembly.5.Downregulation of cyclin B1 could activate AMPK.The activated AMPK phosphorylated both ULK1 at Ser555 site and beclin1 at Ser93 site,ultimately potentiated activity of the class III phosphatidylinositol(Ptd Ins)3-kinase complex.Besides,an elevated ATG4 a level was observed in our research,which cleaved the C-terminal part of MAP1LC3 allowing the release of form I(LC3-I).Then,a subpopulation of form I was subsequently converted to a form II.Eventually,these kinases and ATG proteins were able to orchestrate multiple steps of autophagy progression and rationalize the complexities of autophagic program.6.ATP levels were almost constant between si CB1 and control group,supporting that ATP was not involved in autophagy induced by silencing of cyclin B1(P>0.05).Instead,cellular and mitochondrial ROS levels was evidently elevated.These led to an activation of AMPK,which permitted persistent firing of the downstream signal transduction(P<0.05).7.ROS is vital to autophagy induced by cyclin B1 knockdown.NAC remarkablely attenuated AMPK phosphorylation and LC3-II level in cyclin B1 knockdown cells,indicating that ROS was responsible for cyclinB1 knockdown-induced autophagy8.AMPK is imperative for autophagy in response to cyclin B1 knockdown-induced autophagy.Double knockdown of AMPK and cyclin B1 evidently abrogated cyclin B1 silencing-induced autophagy,implying that AMPK was also involved in autophagy mediated by cyclin B1 downregulation.9.Cyclin B1 is overexpressed in various carcinomas including NPC.We performed a comprehensive analysis on 33 types of tumors from TCGA.Of which,24 kinds of tumors overexpressed cyclin B1 including Adrenocortical carcinoma,Bladder Urothelial Carcinoma,Breast invasive carcinoma,Cervical squamous cell carcinoma and endocervical adenocarcinoma,Colon adenocarcinom,Lymphoid Neoplasm Diffuse Large B-cell Lymphoma,Esophageal carcinoma,Glioblastoma multiforme,Head and Neck squamous cell carcinoma,Liver hepatocellular carcinoma,Lung adenocarcinoma,,Lung squamous cell carcinoma,Ovarian serous cystadenocarcinoma,Pancreatic adenocarcinoma,Rectum adenocarcinoma,Skin Cutaneous Melanoma,Stomach adenocarcinoma,Testicular Germ Cell Tumors,Uterine Corpus Endometrial Carcinoma,Uterine Carcinosarcom.10.Overexpression of cyclin B1 indicates a poor prognosis including Adrenocortical carcinoma,Kidney renal clear cell carcinoma,Kidney renal papillary cell carcinoma,Liver hepatocellular carcinoma,Brain lower grade glioma,Lung adenocarcinoma,Mesothelioma,Pancreatic adenocarcinoma,Skin Cutaneous Melanoma.11.Downregulation of cyclin B1 inhibits proliferation.Cell viability and proliferation were assessed by:(i)The CCK-8 assay,(ii)crystal violet staining assay.The results implied that cell viability was repressed after 48 h and 72 h si RNA intervention(P<0.05).12.Downregulation of cyclin B1 induces apoptosis and senescence.Cell Cell apotosis was detected by both Annexin V-FITC/propidium iodide(PI)and Colorimetric TUNEL Apoptosis Assay.The results demonstrated downregulation of cyclin B1 could induce apotosis(P<0.05),and ?-Galactosidase staining showed that cyclin B1 depletion triggered senescence(P<0.05).13.Inhibition of autophagy preeminently facilitated the antiproliferation triggered by cyclin B1 knockdown.Conclusions:1.Specific downregulation of cyclinB1 induces autophagy in CNE-1 and CNE-2 cells.2.Cyclin B1 downregulation mediate autophagy via AMPK-ULK-1-dependent pathway.3.Downregulation of cyclin B1 constrains the growth of NPC cells4.CyclinB1 downregulation in concert with autophagy inhibition induces antiproliferation in CNE-1 and CNE-2,which open a novel avenue of anti-tumor proliferation.