Reasearch On Mechanisms Of Circular RNAs Involved In Carcinogenesis Of N-nitrosamines Promoted By Microcystins In Esophagus

Backgroud: As a kind of environmental cancer with high morbidity and mortality,esophageal squamous cell carcinoma（ESCC）seriously threatens people’s health.Lacking of effective targeted therapies and early diagnosis biomarkers determines poor outcome of ESCC.Exploring pathogenesis and looking for novel effective therapies are becoming challenging problems for reducing burdens caused by ESCC.We previously discovered high concentration of nitrosamines and microcystins in drinking water may be responsible for high morbidity of ESCC in Huaian City,Jiangsu Province.Still now,how nitrosamines and microcystins induced ESCC were largely unknown.As a class of endogenousRNA,circularRNAs（circRNA）are widely expressed in tissues and organs in human.The closed circular structure without 5’ end cap and 3’ end poly（A）tail make circRNAs not easy to be degraded by RNase R,which promoted the stabilization of circRNAs and make circRNAs to be excellent potential molecular biomarkers.Chapter 1 Screening and analysis of circRNAs related with ESCC1 Objects: This section aimed to identify differentially expressed circRNAs in ESCC and illuminate functions and signal pathways may these circRNAs involved.2 Methods: 2.1 Screening differentially expressed circRNAs in ESCC 100 cases pathological diagnosed ESCC patients were recruited,3 pair of tumor and adjacent non-tumor tissues were selected for circRNA Sequencing.2.2 Analysis of functions and pathways regulated by differently expressed circRNAs Source genes of differentially expressed circRNAs were analysed using GO and KEGG pathway assays for exploring functions and signal pathways regulated by differentially expressed circRNAs.2.3 Verification of differentially expressed circRNAs in ESCC patients 45 differentially expressed circRNAs identified byRNA Sequencing were selected for verification using Sanger sequencing,then RT-QPCR were performed for detecting expression of circRNAs verified by Sanger sequencing in 100 cases of ESCC patients.3 Results: 3.1 Screening differentially expressed circRNAs in ESCC A total of 571 differentially expressed circRNAs were identified in ESSC tissuses,were down-regulated and 236 were up-regulated.circRNA₁2733 was most significantly downregulated with the fold change of 77.04,Hsa_circ₀063865 was most significantly up-regulated with the fold change of 43.63.3.2 Analysis of functions and pathways regulated by differently expressed circRNAs GO analysis showed differentially expressed circRNAs were mainly enriched in Xenobiotic metabolic process and Epoxygenase P450 pathway.KEGG analysis revealed differentially expressed circRNAs were mainly enriched in Central carbon metabolism in cancer and Chemical carcinogenesis pathways.3.3 Verification of differentially expressed circRNAs in ESCC patients Hsa_circ₀011821,Hsa_circ₀012152,Hsa_circ₀008590,Hsa_circ₀005243,Hsa_c irc₀064388,Hsa_circ₀003429,circRNA₀6349,circRNA₁9252,Hsa_circ₀063865,c ircRNA₁9586 and Hsa_circ₀079227 were significantly up-regulated in tumor tissues（P<0.05）;Hsa_circ₀002490,Hsa_circ₀009043,Hsa_circ₀000915,Hsa_circ₀008832,Hsa_circ₀071106,Hsa_circ₀005941,Hsa_circ₀065214,Hsa_circ₀006867,circRNA₁9586,circRNA₃2026,Hsa_circ₀060927,Hsa_circ₀008865,Hsa_circ₀000745 and Hsa_circ₀002874 were significantly down-regulated in tumor tissues（P<0.05）.4 Conclusions: Differentially expressed circRNAs in ESCC may regulated chemical carcinogenesis of esophageal epithelial cells.Chapter 2 Analysis of circRNAs participated in malignant transformation induced by NMBz A and MC-LR1 Objects: Identify circRNAs involved in malignant transformation of esophageal epithelial cells induced by exposuring of nitrosamine and microcystin and assess effects of candidate circRNAs on malignant transformation.2 Methods: 2.1 Screening circRNAs participated in malignant transformation of Het-1A Malignant transformated Het-1A（Het-1A-T）were induced by exposuring of NMBz A and MC-LR.Expression of circRNAs differentially expressed among ESCC patients were then detected in Het-1A-T and Het-1A using RT-QPCR.2.2 Validation of candidate circRNAs participated in malignant transformation in cell Cells stably over-expressing or under-expressing candidate circRNAs（Hsa_circ₀063865,Hsa_circ₀006867 and Hsa_circ₀006867）were established using transfection of lentivirus.The established cell lines were then cultured in medium containing NMBz A and MC-LR for inducing malignant transformation.Plate cloning experiment,soft agar cloning experiment were conducted for analysing degrees of malignant transformation when cultured for 15,25 and 35 generations.2.3 Validation of circRNAs participated in malignant transformation in nude mice Tumorigenesis of subcutaneous inoculation and caudal vein injection in nude mice were performed for assessing tumorigenic ability of cells over-expressing or under-expressing candidate circRNAs which cultured for 40 generations with NMBz A and MC-LR.3 Results: 3.1 Screening circRNAs participated in malignant transformation of Het-1A In Het-1A-T,Hsa_circ₀006867 was significantly down-regulated,Hsa_circ₀063865 and Hsa_circ₀071106 were significantly up-regulated.3.2 Validation of candidate circRNAs participated in malignant transformation in cell Over-expressing of Hsa_circ₀063865 promoted but under-expressing of Hsa_circ₀063865 inhibited malignant transformation of Het-1A,on the contrary,overexpressing of Hsa_circ₀006867 inhibited but under-expressing of Hsa_circ₀006867 promoted malignant transformation of Het-1A.No effect on malignant transformation caused by under-expressing of Hsa_circ₀071106 were discovered.3.3 Validation of circRNAs participated in malignant transformation in nude mice Over-expression of Hsa_circ₀063865 promoted but under-expression of Hsa_circ₀063865 inhibited formations of subcutaneous transplanted and hepatic metastasized tumors grew from cells exposed with NMBz A and MC-LR.Under-expression of Hsa_circ₀063865 inhibited inhibited tumor formation rate and sizes of subcutaneous transplanted and hepatic metastasized tumors.4 Conclusions: Hsa_circ₀063865 and Hsa_circ₀006867 regulated malignant transformation of esophageal epithelial cells induced by expouring of NMBz A and MC-LR.In malignant transformation,Hsa_circ₀063865 played an oncogene role and Hsa_circ₀006867 played an anti-oncogene role.Chapter 3 Functional analysis of circRNAs participated in malignant transformation1 Objects: Identify effects of over-expression and under-expression of Hsa_circ₀006867 and Hsa_circ₀063865 on proliferation,migration,invasion and apoptosis in malignant transformed cells and recognize how these two circRNAs participated in malignant transformation of esophageal epithelial cells.2 Methods: 2.1 Analysis of characterization of Hsa_circ₀006867 and Hsa_circ₀063865 RNase R and Actinomycin D were used for analysing stabilities of Hsa_circ₀006867 and Hsa_circ₀063865 in Het-1A.Nucleocytoplasmic separation assay and Fish in situ hybridization assay were performed for analysing locations of Hsa_circ₀006867 and Hsa_circ₀063865 in cell.2.2 Analysis of functions of Hsa_circ₀006867 and Hsa_circ₀063865 in Het-1A-T Malignant Het-1A-T stably over-expressing or under-expressing Hsa_circ₀006867 or Hsa_circ₀063865 were established by transfecting lentivirus.Ttranswell and Ttranswell+Martial assays were performed for detecting cell migration and invasion.Ed U was used for detecting cell proliferation,Annexin V-APC/7-AAD assay was performed for detecting cell apoptosis.2.3 Functional analysis of circRNAs in tansplanted tumors Immunohistochemistry were performed for detecting expression of PCNA and E-cadherin in transplanted tumors located in lung,liver and subcutaneous of nude mice.3 Results: 3.1 Analysis of characterization of candidate circRNAs Hsa_circ₀006867 and Hsa_circ₀063865 mainly expressed in cytoplasm in Het-1A.Endogenous Hsa_circ₀006867 and Hsa_circ₀063865 were resistant to RNase R and were more stable than linerRNAs.3.2 Analysis of functions of Hsa_circ₀006867 and Hsa_circ₀063865 in Het-1A-T Over-expression of Hsa_circ₀006867 inhibited but under-expression of Hsa_circ₀006867 promoted cell migration,invasion and proliferation.Over-expression of Hsa_circ₀063865 promoted but under-expression of Hsa_circ₀063865 inhibited cell migration,invasion and proliferation,on the contrary,over-expression of Hsa_circ₀063865 inhibited but under-expression of Hsa_circ₀063865 promoted apoptosis of Het-1A-T.3.3 Functional analysis of Hsa_circ₀006867 and Hsa_circ₀063865 in tansplanted tumors Hsa_circ₀063865 promoted expression of PCNA but inhibited expression of E-cadherin in subcutaneous transplanted,hepatic metastasized and lung metastasized tumors,Hsa_circ₀006867 inhibited expression of PCNA but promoted expression of E-cadherin in subcutaneous transplanted,hepatic metastasized and lung metastasized tumors.4 Conclusions: Hsa_circ₀063865 promoted cell migration,invasion and proliferation but inhibited apoptosis.Hsa_circ₀006867 inhibited cell migration,invasion and proliferation.Chapter 4 Analysis of mechanisms on how candidate circRNAs participated in malignant transformation1 Objects: Identify how Hsa_circ₀063865 and Hsa_circ₀006867 participated in malignant transformation through regulating binding miRNAs and binding proteins.2 Methods: 2.1 Analysis of binding proteins of Hsa_circ₀006867 and Hsa_circ₀063865 Chromatin Isolation byRNA Purification assay was performed for separating binding proteins of candidate circRNAs,the separated proteins were then detected using Mass spectrometry,combinations of identified proteins and circRNAs were then verified using RIP assay.2.2 Analysis of competitive binding miRNAs of Hsa_circ₀006867 and Hsa_circ₀063865 Dual luciferase reporter assays were performed for identifying competitive binding miRNAs of Hsa_circ₀063865 and Hsa_circ₀006867.2.3 Hsa_circ₀063865 promoted cell migration and invasion through regulating e EF1A2/MYH9 Co-IP-MS assay was performed for identifying binding proteins of e EF1A2,then Co-IPWestern blot assay was performed for verifying the combination of identified MYH9 and e EF1A2.RIP assay was performed for identifying the combination of MYH9 and Hsa_circ₀063865.Western blot and RT-QPCR were performed for detecting effects of overexpressing or under-expressing Hsa_circ₀063865 on expression of e EF1A2 and MYH9.SiMYH9 and Si-e EF1A2 were used for detecting mutual regulation between MYH9 and e EF1A2.Phalloidin staining assay and Transwell assay were performed for detecting effects on cytoskeleton,migration and invasion of Hsa_circ₀063865 through regulating MYH9.Immunohistochemistry assay was performed for detecting MYH9 in subcutaneous transplanted,liver metastasized and lung metastasized tumors grown from cells over-expressing or under-expressing Hsa_circ₀063865,In addition,expressions of MYH9 and F-actin in Het-1A-T and Het-1A were detected using Phalloidin staining and Western blot for analysing how Hsa_circ₀063865-e EF1A2-MYH9 played in malignant transformation induced by Nnitrosamine and microcystin.2.4 Hsa_circ₀063865 inhibited cell apoptosis through regulating hsa-miR-450b-3p/RCN1RNA sequencing was performed for detecting potential targets of Hsa-miR-450b-3p,Ago2 RIP assay and Dual luciferase reporter assay were performed for verifying the combination of Hsa-miR-450b-3p and identified RCN1.RT-QPCR and Western blot were performed for detecting expressions of RCN1 in cells over-exressing and under-expressing Hsa-miR-450b-3p,mRNA and protein expressions of RCN1 were then detected in cells treated with Hsa_circ₀063865High+mimic NC,Hsa_circ₀063865High+ Hsa-miR-450b-3p mimic and circRNA NC+ mimic NC for analysing if Hsa-miR-450b-3p participated in regulating RCN1 regulated by Hsa_circ₀063865.Annexin V-APC/7-AAD assay were performed for analysing effects on apoptosis regulated by Hsa_circ₀063865/Hsa-miR-450b-3p/RCN1 axis.Western blot and JC-1 staining assays were performed for detecting effects on endoplasmic reticulum stress pathway（p PERK,p EIF2α,ATF4 and CHOP）and mitochondrial membrane potential regulated by Hsa_circ₀063865/Hsa-miR-450b-3p/RCN1.Immunohistochemistry assay was performed for detecting effects of over-expression and under-expression of Hsa_circ₀063865 on expressions of RCN1,p EIF2α,ATF4 and CHOP in subcutaneous transplanted,liver metastasized and lung metastasized tumors.In addition,expression of RCN1,p PERK,p EIF2α,ATF4,CHOP and mitochondrial membrane potential were detected in Het-1A and Het-1A-T for analysing how Hsa_circ₀063865/Hsa-miR-450b-3p/RCN1 played in malignant transformation of Het-1A induced by N-nitrosamine and microcystin.2.5 Hsa_circ₀006867 inhibited cell migration and invasion through regulating hsa-miR-499a-3p/MEF2 C Western blot was performed for detecting expression of MEF2 C in Het-1A and Het-1AT,Anti-Ago2 RIP assay was performed for verifying the combination of Hsa-miR-499a-3p and MEF2 C.RT-QPCR and Western blot were performed for detecting effects of Hsa_circ₀006867/Hsa-miR-499a-3p on expression of MEF2 C.Transwell assay was used for detecting effects of Hsa_circ₀006867/Hsa-miR-499a-3p on cell invasion and migration.Immunohistochemistry assay was performed for detecting effects of over-expression and under-expression of Hsa_circ₀006867 on expression of MEF2 C in subcutaneous transplanted,liver metastasized and lung metastasized tumors.3 Results: 3.1 Analysis of binding proteins of Hsa_circ₀063865 and Hsa_circ₀006867 Hsa_circ₀063865 can specifically bind with e EF1A2.3.2 Analysis of competitive binding miRNAs of Hsa_circ₀063865 and Hsa_circ₀006867 Hsa_circ₀006867 can competitively bind with hsa-miR-499a-3p,Hsa_circ₀063865 can competitively bind with hsa-miR-450b-3p.3.3 Hsa_circ₀063865 promoted cell migration and invasion through regulating e EF1A2/MYH9 Co-IP-MS assay revealed the combination of MYH9 and e EF1A2,RIP assay revealed the combination of MYH9 and Hsa_circ₀063865.Hsa_circ₀063865,e EF1A2 and MYH9 may formed as a complex in Het-1A-T.No effects on expression of e EF1A2 induced by over-expressing or under-expressing Hsa_circ₀063865 was discovered.In cells over-expressing Hsa_circ₀063865,expression of MYH9 was promoted,in cells with lower expression of Hsa_circ₀063865,expression of MYH9 was inhibited.No effects of over-expressing or under-expressing Hsa_circ₀063865 on expressions of e EF1A2（protein and mRNA）and MYH9 mRNA were discovered.Overexpression of Hsa_circ₀063865 promoted cytoskeleton reorganization,when expression of MYH9 was interfered,cytoskeleton reorganization was inhibited,the cytoskeleton changed to be neatly arranged,cell migration and invasion were also inhibited.In subcutaneous transplanted,liver metastasized and lung metastasized tumors grown from cells over-expressing Hsa_circ₀063865,expression of MYH9 was up-regulated.In tumors grown from cells under-expressing Hsa_circ₀063865,expression of MEF2 C was inhibited.In contrast with normal Het-1A,MYH9 were upregulated in Het-1A-T,which destroyed intermediate filaments and disordered arranged cytoskeleton.3.4 Hsa_circ₀063865 inhibited cell apoptosis through regulating hsa-miR-450b-3p/RCN1 Over-expression of Hsa_circ₀063865 promoted but under-expression of Hsa_circ₀063865 inhibited expression of RCN1 through regulating Hsa-miR-450b-3p.In cells over-expressing Hsa_circ₀063865,cell apoptosis was inhibited,when over-expressing Hsa-miR-450b-3p,the inhibition on apoptosis induced by over-expression of Hsa_circ₀063865 was eliminated.In cells over-expressing Hsa_circ₀063865,p PERK,p EIF2α,ATF4 and CHOP were down-regulated,endoplasmic reticulum reaction was inhibited,which in turn increased mitochondrial membrane potential.When over-expressing Hsa-miR-450b-3p in cells over-expressing Hsa_circ₀063865,inhibition on endoplasmic reticulum reaction induced by over-expression of Hsa_circ₀063865 was eliminated,mitochondrial membrane potential was down-regulated.In subcutaneous transplanted,liver metastasized and lung metastasized tumors overexpression of Hsa_circ₀063865 promote expression of RCN1 but inhibited expression of p EIF2α,ATF4 and CHOP.In contrast with normal Het-1A,RCN1 was up-regulated in Het-1A-T,the downstream proteins of p PERK,p EIF2α,ATF4 and CHOP were downregulated,which increased mitochondrial membrane potential in turn.3.5 Hsa_circ₀006867 inhibited cell migration and invasion through regulating hsa-miR-499a-3p/MEF2 C Over-expression of Hsa_circ₀006867 promoted but under-expression of Hsa_circ₀006867 inhibited expression of MEF2 C through regulating Hsa-miR-499a-3p.Over-expression of Hsa-miR-499a-3p promoted but under-expression of Hsa-miR-499a-3p inhibited cell migration and invasion through regulating MEF2 C.In cells over-expressing Hsa_circ₀006867,cell migration and invasion was inhibited,when Hsa-miR-499a-3p was inhibited,over-expression no longer inhibited migration and invasion.In subcutaneous transplanted,liver metastasized and lung metastasized tumors grew from cells under-expressing Hsa_circ₀006867,expression of MEF2 C was promoted.In tumors grown from cells over-expressing Hsa_circ₀006867,MEF2 C was inhibited.4 Conclusions: 1.Hsa_circ₀063865 promoted translation of MYH9 through promoting combination of e EF1A2 and MYH9,which promoted reorganization of cytoskeleton and malignant transformation.2.Hsa_circ₀063865 can inhibit cell apoptosis through regulating hsa-miR-450b-3p/RCN1 /PERK/CHOP signal pathway,which inhibited depolarization of mitochondrial membrane potential and apoptosis.3.Hsa_circ₀006867 can inhibit cell migration and invasion through regulating hsa-miR-499a-3p/MEF2 C,which inhibited malignant transformation of esophageal epithelial cells.