The Role And Mechanism Of CircAMOTL1 In Epithelial-to-mesenchymal Transition Of Prostate Cancer

Prostate cancer(PCa)is one of the most common malignant tumors in the male genitourinary system.Poor prognosis and recurrence make it a serious threat to men's health.CircRNA is a novel class of non-coding RNA,which are widely expressed in eukaryocyte.They are usually found to be conserved,stable,and often exhibit cell type-specific or tissue-specific expression.Emerging evidences elicit that circRNAs are associated with several diseases,especially in the development of cancer.The function of circRNAs in prostate cancer hasn't been reported yet.EMT refers to the process by which cells transit from epithelial phenotype to mesenchymal phenotype.EMT is critical for tumor migration and invasion,however the underlying mechanism is little known so far.There is also no knowledge of circRNA in regulation of EMT.In this study,we aim to clarify the function of circAMOTL1 in prostate cancer and the mechanism in regulation of EMT,thus to lay an experimental foundation for further research.Through screening the tissues of PCa and BPH for circRNA,we found circAMOTL1 was downregulated in PCa tissues.Cells experiment showed that circAMOTL1 can modulate the expression of EMT-related proteins.In order to provide theoretical basis for applying circRNA into diagnosis and treatment of PCa,we will research circAMOTL1 expression in PCa and its molecular mechanism in EMT regulation in this study.Object: To clarify the expression of circAMOTL1 in PCa and the mechanism in regulation of EMT.Methods:1 We collected PCa tissue specimens and BPH tissue specimens and set the BPH specimens as control group.Then we performed RNA isolution and pathological morphology analysis separately.2 We used divergent primers and convergent primers for head-to-tail qPCR splicing assays after reverse transcription,thus to confirm the expression of those circRNA in PCa tissues and cells.Then we designed additional divergent primers to identify full-length circRNA.In parallel,we subjected clinical specimens to fluorescence in situ hybridization using a probe against circAMOTL1.3 We extracted the RNA of several non-cancerous and cancerous cell lines and performed q RT-PCR,thus to test the differential expression of circAMOTL1.We transfected GFP-circAMOTL1 or GFP-Ctl into cells and tested the expression levels of circAMOTL1.We also performed Transwell and wound healing assays after transfection.4 We transfected si-Ctl,si-circAMOTL1,and si-linear AMOTL1 into cells,then we detected the expression levels of circAMOTL1 and AMOTL1 mRNA.Transwell and wound healing assays were performed subsequently to test effects of.si-circAMOTL1 on PC3 cells migration and invasion.5 We transfected GFP-circAMOTL1 or GFP-Ctl or si-circAMOTL1 or si-Ctl into cells.We first examined the effects of circAMOTL1 on cell phenotype.Then we performed Western blotting to analyze the expression of EMT-related proteins.Results:1 To confirm the presence of circRNAs in PCa.Divergent primers amplify circRNAs in cDNA but not in genomic DNA(gDNA).Four kind circRNAs were expressed in PCa tissues,especially circAMOTL1.RT-PCR revealed that circAMOTL1 was accurately amplified and it has a length of 1072 nucleotides.2 Down-regulation of circAMOTL1 expression in PCa.The results of qRT-PCR analysis showed that circAMOTL1 expression was significantly downregulated in PCa,compared to BPH.Comparing with BPH,circAMOTL1 has a low expression in PCa in FISH.Taken together,our results demonstrated the downregulation of circAMOTL1 in PCa.3 Gain of circAMOTL1 inhibits PCa cell migration and invasion.We evaluated circAMOTL1 expression in several cancerous and non-cancerous cell lines and observed low levels of circAMOTL1 expression in three PCa cell lines,particularly PC3 cells,compared to normal RWPE-1 cells.The overexpression plasmids led to increased expression of circAMOTL1,compared with the control vector.The results of Transwell and wound healing assays show that circAMOTL1 overexpression significantly inhibited PC3 cells migration and invasion.4 Loss of circAMOTL1 promotes PCa cell migration and invasion.The qRT-PCR results show that si-circAMOTL1 reduced only the circAMOTL1 transcript,but did not affect linear AMOTL1 RNA.However,si-linear AMOTL1 effectively knocked down both transcripts.Results of Transwell and wound healing assays indicate that the downregulation of circAMOTL1 led to dramatically increased cell migration and invasion.5 CircAMOTL1 participates in modulating the expression of EMT-related proteins.The overexpression of circAMOTL1 induced an epithelial-like morphological feature,whereas silencing induced a mesenchymal morphological phenotype.An EMT-related protein analysis show that overexpression of circAMOTL1 increased expression of the epithelial markers E-cadherin and claudin1,but not ?-catenin,and reduced expression of the mesenchymal marker vimentin in PC3 cells.Conversely,downregulation of circAMOTL1 yielded the reverse effects(except ?-catenin).Conclusions:1 CircAMOTL1 expression was significantly downregulated in PCa.2 CircAMOTL1 can inhibit PCa cell migration and invasion.3 CircAMOTL1 reduces prostate cancer cell migration and invasion by modulating the expression of EMT-related proteins.