| Object:NUPR1,a transcriptional regulation factor,is induced by stress and involved in cell proliferation and cell cycle process.Previous study indicates that NUPR1 is associated with regulation of tumor development.NUPR1 promotes ER stress of breast cancer cells,proliferation of glioma cells,malignant process of pancreatic cancer cells and so on.Moreover,NUPR1 is reported to regulate premature senescence and apoptosis of cancer cells,NUPR1 contributes to inhibition of cancer development.In cancer patients,the balance of autophagy process is purposed to promote tumor progress by regulating internal environment homeostasis.However,the mechanism remains unclear by which balance autophagy process involves in cancer.It is reported that NUPR1 is associated with regulation of autophagy.At present,it is rarely reported about autophagy of lung cancer by NUPR1.In this study,NUPR1 related survival rate is investigated in non-small cell lung cancer patient.And we also study the mechanism by which NUPR1 control autophagy process for further understanding lung cancer development.This may be helpful to target therapeutic strategy of lung cancer.Method: There are three sections in this work.In section one,the expression of NUPR1 is investigated in lung cancer patients.In section two,the phenotype is explored after changing NUPR1 expression.In section three,the transcriptional regulation mechanism by which NUPR1 regulates lung cancer is uncovered.Section one: The expression of NUPR1 is analyzed by IHC assay in lung cancer tissue chip.Statistical analysis indicates that NUPR1 associates with survival rate of lung cancer patients.The expression of NUPR1 is detected by RT-PCR and Western blot in NHBE and lung cancer cell lines.Section two: NUPR1 is down regulated by lentivirus mediated RNA interference system.Cell image of lung cancer cells is observed by inverted microscope.Live cell staining is analyzed using neutral red staining,AO staining and Lyso-tracker Red staining.Biomarkers are detected through Western blot.For exploring the function changes of lung cancer cells,we perform Brd U assay,cell cycle analysis,soft agar assay,cancer cells subcutaneous injection.Section three: Changes of transcriptome are analyzed between control group and NUPR1 knockdown group.Different expression genes are verified by RT-PCR.We confirm that NUPR1 directly regulates SNAP25 by Ch IP and luciferase assay.During autophagy process,SNAP25 works with other partners and SNAP25 partners are searched by IP/MS in this study.Results: Section one: Our data indicate that NUPR1 expresses higher in lung cancer cells compared to NHBE.Using IHC,we studied NUPR1 expression in 118 clinical lung cancer specimens and 22 adjacent tissues.Variable expressions of NUPR1 were found in lung tumor tissues,whereas cancer-adjacent lung tissues did not express significant levels of NUPR1.Subjects whose tumors had low NUPR1 expression had strikingly longer survival time than those whose tumors had high NUPR1 expression levels(P < 0.01).Section two: NUPR1-depleted cells exhibite a typical appearance of pronounced vacuolization of the cytoplasm,and cell death increases in NUPR1-depleted cells.Live cell staining data reveal that vacuoles are acid structures.Loss of NUPR1 impaires autolysosome efflux in lung cancer cells,and causes autolysosomal vacuolization.Deficiency of autophagy restrains vacuoles induced by NUPR1 knockdown.Down regulation of NUPR1 induces premature senescence of lung cancer cell,inhibits proliferation and tumorigenesis in vitro and in vivo.Section three: Knockdown of NUPR1 and subsequent RNA-seq analysis reveales that different expression genes involved in autophagy and lysosomal processes(ATG9B,LCN2,TPPP3,SNAP25,and SQSTM1).Knockdown of SNAP25 induces cytoplasmic vacuolization,cellular premature senescence and decreased proliferation similar to that observed with NUPR1 knockdown.Over expression of SNAP25 would partially rescue impaired function caused by NUPR1 knockdown.Finally,we reveal that SNAP25 is a target gene of NUPR1. |
