| Background:Colon cancer(CC)is a common type of malignant tumor worldwide,ranking fifth in cancer mortality of China.Though recent researches have linked gene mutation,diet,microorganisms and their metabolites to CC,the detailed pathogenesis of CC is not yet clear.The most common sign and symptom of early CC is actually nothing at all.However,advanced-stage CC has a poor prognosis.Consequently,there is an urgent need to develop a stable biomarker for diagnosis of CC.Circular RNAs(circRNAs)belong to a new type of nc RNAs with a covalently closed circular configuration,exhibiting tissue specificity and biological stability,and taking part in physiological or pathological regulation.Currently,with the development of RNA sequencing and circRNAs microarray technologies,circRNAs have been shown to function as an oncogene or tumor suppressor gene by acting as miRNA sponges,regulators of gene transcription,protein-binding scaffolds,as well as protein sponges,recruiters and translators in the field of CC research.Although lots of CC-specific circRNAs have been discovered,the network of molecular interactions is intricate,more circRNAs and related molecular mechanism remain to be elucidated.The present study have analysed the differential circRNA epression profiles of CC and normal colonic mucosa(NCM).We have investigated the relationships between circRNA＿100859 and clinicopathologic features,explored molecular mechanism.Our findings will provide new insights into understanding the mechanisms of colon carcinogenesis.It will provide new evidences for the diagnosis and treatment of CC.Objective:1.Differential expression analysis of circRNAs between CC and NCM.2.Analyze the correlation of circRNA＿100859 and clinical clinicopathologic characters,or prognosis in CC patients.3.Explore the effects of circRNA＿100859 in the regulation of proliferation,apoptosis,migration and invasion abilities of CC cells,as well as epithelial-mesenchymal transformation(EMT)and autophagy.4.Investigate the molecular mechanism of circRNA＿100859function as a miRNA sponge.Methods:1.circRNAs array was performed to screen for differentially expressed circRNAs in CC and NCM tissues.2.Divergent primers and convergent primers for circRNAs and linear transcripts were designed to verify the property of circular RNA.3.The expression of circRNA＿100859 was measured by quantitative PCR(qPCR)in CC cell lines and 50 pairs of CC and NCM samples.4.The clinicopathological and prognosis data of these 50 patients were collected.The correlation between expression of circRNA＿100859and clinicopathologic characters was analysed.Furthermore,the 3-years progression-free-survival(PFS)were estimated by using the Kaplan-Meier method.Receiver operating characteristic(ROC)curve was used to shown the diagnostic performance of circRNA＿100859 in distinguishing CC from NCM tissue.5.Lentivirus transfection method was used to construct the circRNA＿100859 overexpressed HCT116 cell lines and silenced Lovo cell lines.MTT assay,clone formation assay,flow cytometry assay,scratch healing and transwell experiment,western blot were applied to detect the proliferation,apoptosis,EMT,migration and invasion abilities of CC cells in HCT116 or Lovo cells,after overexpression or silencing circRNA＿100859.6.Autophagy flux was assessed by western blot and analysed using tandem m RFP-GFP-LC3 fluorescence microscopy.7.Based on miRanda,Target Scan and miRbase databases,we predicted the downstream target miRNAs and mRNAs.8.qPCR assay was applied to detect the miR-217 and HIF-1αexpression in 50 pairs of CC and NCM samples,analyzing the expression correlation of miR-217 and circRNA＿100859,as well as miR-217 and HIF-1α.Meanwhile,qPCR assay to detect the alteration expression of miR-217 or HIF-1α after changing the epression level of circRNA＿100859 or miR-217.9.The dual-luciferase reporter assay system was utilized to analyze the relationship between circRNA＿100859 and miR-217,as well as miR-217 and HIF-1α.10.qPCR and western blot were used to anlyze whether miR-217 can rescue HIF-1α epression brought by overexpression or silencing expression of circRNA＿100859.11.Using a series of assays,such as MTT,clone formation,flow cytometry assay,scratch healing,transwell and western blot,we evaluated whether miR-217 mimic could alleviate the tumorigenesis capacities which were brought by circRNA＿100859 overexpression.12.Lentivirus-m RFP-GFP-LC3 and western blot assays were applied to detect whether both miR-217 and siHIF-1α could reduce the activation of autophagy which was brought by circRNA＿100859overexpression.Results:1.287 differentially expressed circRNAs were discovered,including84 up-regulated and 203 down-regulated circRNAs.2.Agarose gel electrophoresis showed the products when using the divergent primers to amplify the c DNA,verifing the circular character of circRNA＿100859,circRNA＿000320 and circRNA＿101736.3.The expression level of circRNA＿100859 in CC tissues and cell lines were higher than normal control respectly.4.The higher expression of circRNA＿100859 was related with more lymph vessel tumor emboli,lower tumor differentiation,poorer clinical staging.5.In the group with high expression of circRNA＿100859,the median PFS time was 25.1 months,shorter than 31.2 months in low expression group.ROC analysis showed the area under the curve of tissue circRNA＿100859 was 0.974.6.In HCT116 cells,overexpressing circRNA＿100859 enhanced proliferation,invasion,migration ability and colony forming efficiency,while inhibited apoptosis.Contrarily,in Lovo cell,circRNA＿100859silencing prevented apoptosis,promoted proliferation,invasion,migration and colony forming abilities.7.The expression of E-cad was downregulated,while N-cad,Slug and Vimentin were upregulated in circRNA＿100859 overexpressing HCT116 cell.Adversely,silencing circRNA＿100859 group exhibited increased epression of E-cad and reduced epression of N-cad,Slug and Vimentin.8.The number of both autophagosomes and autolysosomes in circRNA＿100859 overexpressing HCT116 cell were increased,especially for autolysosomes.Consistently,the levels of autophagy-related protein LC3 II:I was upregulated and p62 was downregulated.9.In Lovo cell,silencing circRNA＿100859 exhibited lower number of autophagosomes and autolysosomes,especially for autolysosomes.Meanwhile,expression of LC3 II:I was lower and p62 was elevated.10.miR-217 significantly inhibited luciferase activity when co-transfected with wildtype circRNA＿100859;However,when binding site of circRNA＿100859 is mutant,miR-217 could not reduce luciferase activity.The expression level of miR-217 in CC was lower than in NCM.Meanwhile,there was a negative correlation between the expression of miR-217 and circRNA＿100859.11.miR-217 significantly downregulated luciferase activity when co-transfected with wildtype HIF-1α 3′-UTR;However,when binding site of HIF-1α 3′-UTR is mutant,miR-217 could not alter luciferase activity.The CC tissues had a higher expression level of HIF-1α than NCM.Similarly,there was a negative correlation between the expression of miR-217 and HIF-1α.12.Overexpression of circRNA＿100859 promoted the expression of HIF-1α,both in mRNA and protein level.Cotransfection of miR-217 mimic could counteract the growth of HIF-1α in part.On the contrary,silencing circRNA＿100859 inhibited the expression of HIF-1α,and this effect was partly reversed by miR-217 inhibitor.13.miR-217 mimic could alleviate the tumorigenesis capacities which were brought by circRNA＿100859 overexpression,such as proliferation,apoptosis,migration,invasion and EMT.14.miR-217 mimic and siHIF-1α could partly rescue the activation of autophagy brought by circRNA＿100859 overexpression.Conclusion:1.The higher expression of circRNA＿100859 could related with more lymph vessel tumor emboli,lower tumor differentiation,later clinical staging and poorer prognosis.The expression level of circRNA＿100859 could be a useful classifier for diagnosis of CC.2.circRNA＿100859 could promote the ability of proliferation,migration,invasion,EMT and autophagy,while inhibit apoptosis.3.circRNA＿100859 could competitively bind miR-217 in CC cells,and relieve the inhibition effect of miR-217 targeting at HIF-1α,thereby promoting proliferation,migration,invasion,EMT and autophagy,while inhibiting apoptosis. |
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