The Regulation Of ULK1 Expression In Prostate Cancer Tissues And Cells And The Effect Of MiR-372 In Prostate Cancer Cells

Prostate cancer is the second cause of tumor-related mortality of male and seriously threatens the health of prostate cancer patients.Nowadays,it attracts more and more concerns from the researchers worldwide.Conservatively existing in eukaryotic cells,autophagy process is precisely regulated by ATG genes and plays key role in intracellular degradation of proteins and organelles.The tumor cells are often confronted with nutrition stress for its rapid growth.Thus,autopahgy process is essential for the tumor genesis and proliferation.In mammals,serine/threonine kinase ULK1 is the homology gene of ATG1 in yeast.ULK1 is essential for initiation of autophagy process.microRNA(~22nt),a kind of noncoding RNA,inhibits gene expression through binding 3'UTR region of target gene.Through regulating large number of functional genes,microRNAs involve in diverse biological processes.Particularly,the abnormal expression of microRNA has been proved closely related with prostate cancer.Therefore,this research focused on the key ATG gene ULK1 in prostate cancer.Through analyzing prostate cancer cell and prostate cancer tissues,the research aimed to describe the characteristic of ULK1 expression in prostate cancer.Then,the research focused on miR-372/ULK1 regulation combining computational prediction with experimental verification.Furthermore,the research comprehensively analyzed the effects of miR-372 utilizing high-throughput mRNA microarray in prostate cancer cell.The research achieved results below.1.ULK1 expresses highly in prostate cancer cell and prostate cancer tissues.Quantitative PCR and Western blot shows that ULK1 expresses highly in prostate cancer cell lines LNCaP,DU-145 and PC-3,compared to prostate normal cell line RWPE-1.Especially,the expression of ULK1 is significant high in PC-3 cell.IHC staining of Prostate Cancer Tissues Microarray with ULK1 specific antibody also shows ULK1 highly expresses in prostate cancer tissues significantly.2.ULK1 affects prostate cancer cell proliferation,clone formation and migration.The over-expression of ULK1 enhances cell proliferation in PC-3 and RWPE-1 cells.Conversely,ULK1 knockdown stable cell line shows impairment of cell proliferation,clone formation and migration in PC-3 cell.ULK1 knockdown RWPE-1 stable cell line also shows that ULK1 is essential for RWPE-1 cell.The ULK1 inhibitors significantly repress proliferation and clonogenicity,affect cell cycle and induce apoptosis in PC-3 cell.3.miR-372 inhibits ULK1 expression and represses autophagy in PC-3 cell.Combined with bioinformatic prediction and testified with experiment,the research found that miR-372 could inhibit ULK1 expression by binding its 3'UTR region.miR-372 inhibits LC3-II conversion and autophagy in PC-3 cell by targeting ULK1.4.Transcription factor c-myc inhibits miR-372 expression.In PC-3,miR-372 can be regulated by c-myc.Through luciferase reporter assay and ChIP analysis,two c-myc binding sites were found in miR-372 promoter region,including BS1(-753)and BS2(-1369).c-myc inhibits miR-372 expression through binding these two sites in PC-3 cell.Furthermore,c-myc upregulates autophagy and highly expresses in PC-3 cell.Therefore,the research proposes the c-myc/miR-372/ULK1 regulation pathway.In PC-3 cell,highly expression of c-myc inhibits miR-372 level,and eventually results in ULK1 high expression.c-myc/miR-372/ULK1 regulation pathway may play crucial roles in PC-3 cell through contribution to autophagy process.5.Comprehensively analyze the effects of miR-372 utilizing high-throughput mRNA microarray in PC-3.Microarray results show that 71 significant downregualted genes(> 2.5 folds)and 65 significant upregualted genes(> 2.5 folds)in miR-372 treated PC-3 cell.Gene Ontology enrichment analysis of differential expressed genes shows that the molecular function of miR-372 is RNA binding,miR-372 affects cell metabolism through macormoecular complex disassembly and cellular comonent disassembly.The research comprehensively illustrates the effects of miR-372 in PC-3 cells.