Study On The Mechanism Of Hsa?circ0006332 And Hsa?circ0000069 Regulating The Development Of Cervical Cancer

Background and objectiveCervical cancer(CC)is a common gynecological malignant tumor caused by human papillomavirus(HPV)infection.China is one of the countries with the largest number of CC cases,making the prevention and treatment of CC an arduous task.Although the application of HPV vaccine and cervical smear screening are effective in preventing CC,the prognosis of advanced CC is usually poor.Circular RNAs(circRNAs)play significant roles in tumour genesis and development,including cervical cancer(CC).In addition,due to their high stability and specific expression,circRNAs are promising in the treatment and diagnosis of tumors.However,the expression and function of a large number of circRNAs are currently unknown.This study aims to investigate the role of Hsa?circ0006332 and Hsa?circ0000069in the development of CC and the mechanism of their upregulation.Methods1.Verification the nature and expression of Hsa?circ0006332 and Hsa?circ0000069 in cervical cancer cells and tissuesThe structure of Hsa?circ00063322 and Hsa?circ0000069 were verified by RNase R digestion and genomic DNA amplification assays.The high stability and cell distribution of Hsa?circ00063322 and Hsa?circ0000069 were verified by actinomycin D and distribution assays.Real-time quantitative polymerase chain reaction(qPCR)was used to detect the expression of Hsa?circ00063322 and Hsa?circ0000069 in CC tissues and cells,as well as in normal tissues and cells,to determine the changes in the expression of Hsa?circ00063322 and Hsa?circ0000069 after cervical tissue carcinogenesis.2.Mechanism of Hsa?circ00063322 and Hsa?circ0000069 upregulationThe database predicted that Hsa?circ00063322 expression might be regulated by transcription factor FOXA1,while Hsa?circ0000069 expression was regulated by M6A methylation modification.FOXA1 binding to Hsa?circ00063322 promoter was detected by chromatin immunoprecipitation assay.The regulation of FOXA1 on Hsa?circ00063322expression was detected by qPCR.The binding site of FOXA1 to the promoter of Hsa?circ00063322 was confirmed by luciferase reporting assay.The m6A level of Hsa?circ0000069 in CC and normal cells,as well as the m6A level of Hsa?circ0000069 in METTL3 knockdown cells was detected by Me-RIP.The effection of METTL3 knockdown on Hsa?circ0000069 expression was detected by qPCR.Actinomycin D assay was used to detect the effect of METTL3 knockdown on Hsa?circ0000069 stability,as well as the effect of M6A modification at two M6A sites on Hsa?circ0000069 stability.Single and double mutants at two Hsa?circ0000069 M6A sites were prepared,and the relationship between the two M6A modifications of Hsa?circ0000069 was detected by ME-RIP and qPCR assay3.The function of Hsa?circ00063322 and Hsa?circ0000069Hsa?circ00063322 and Hsa?circ0000069 stable knockdown cells were prepared and verified by qPCR.Hsa?circ00063322 and Hsa?circ0000069 transient overexpressed cells were prepared.Cell Counting Kit-8(CCK8)and 5-ethyl-2-deoxyuridine(EDU)were used to detect the proliferation of Hsa?circ00063322 and Hsa?circ0000069 knockdown and overexpression cells.Hsa?circ00063322 and Hsa?circ0000069 knockdown and overexpression cells were treated with cisplatin and fluorouracil,and then the cell apoptosis was detected by flow cytometry.The migration ability of Hsa?circ00063322 and Hsa?circ0000069 knockdown and overexpression cells was detected by scratch and Transwell assay.4.Hsa?circ00063322 and Hsa?circ0000069 respectively targeted miR-548q and miR-4426,and miR-548q targeted L2The regulatory relationship of Hsa?circ00063322 and Hsa?circ0000069 to miR-548q and miR-4426,respectively,and the regulatory relationship of miR-548q to HPV16 microcapsid protein L2 were verified by qPCR.The binding sites between Hsa?circ00063322and miR-548q,Hsa?circ0000069 and miR-4426,as well as miR-548q and L2 were verified by luciferase assay.The expressions of miR-548q,miR-4426 and L2 in tumor and normal tissues were detected by qPCR.5.Validation of Hsa?circ00063322/miR-548q/L2 and Hsa?circ0000069/miR-4426 signaling pathwaysThe changes of L2 expression after Hsa?circ00063322 overexpression,Hsa?circ00063322 and miR-548q overexpression or Hsa?circ00063322 knockdown and Hsa?circ00063322 and miR-548q knockdown were detected by qPCR.Immunofluorescence(IF),EDU,flow cytometry and Transwell assay were used to detect the functional phenotypic changes of cells after miRNA overexpression,circRNA and miRNA overexpression or miRNA knockdown,and circRNA and miRNA knockdown.Results1.The expression of circRNA Hsa?circ00063322 was significantly up-regulated in CC tissues and cells,and could accurately distinguish tumor from normal tissues(AUC=0.8597).Hsa?circ00063322 is highly stable and mainly distributed in the cytoplasm.FOXA1 is a transcription factor for Hsa?circ00063322 and binds to the promoter region of MYBL2(the parent gene of Hsa?circ00063322)at two sites.2.Hsa?circ00063322 stable knockdown cell lines were prepared.Hsa?circ00063322knockdown significantly inhibited the proliferation of CC cells and might increased their drug sensitivity.The transient overexpression of Hsa?circ00063322 promoted its proliferation ability and weakened its drug sensitivity.3.Hsa?circ00063322 binds to miR-548q and regulates the level of miR-548q.The expression of miR-548q was significantly decreased in CC tissues,and the expression levels of miR-548q and Hsa?circ00063322 were negatively correlated.4.MiR-548q binds to the L2 transcript at two sites and mediates the regulation of L2 expression by Hsa?circ00063322.Overexpression of Hsa?circ00063322 prevented the tumor suppressing activity of miR-548q,and knockdown of Hsa?circ00063322 prevented the tumor promoting activity of miR-548q inhibitor.5.The expression of Hsa?circ0000069 is significantly up-regulated in CC,and it could accurately distinguish tumor from normal tissue(AUC=0.7901).Hsa?circ0000069 is highly stable and mainly distributed in the cytoplasm.6.Compared with normal cervical cells,the M6A modification level of Hsa?circ0000069 in CC cells was significantly increased.M6A modifies the stability of Hsa?circ0000069.There were two M6 a modifications in the Hsa?circ0000069 transcript,which acted synergistically with each other.7.Two stable knockdown cell line of Hsa?circ0000069 was prepared.The knockdown of Hsa?circ0000069 significantly inhibited the proliferation and migration of CC,while the transient overexpression of Hsa?circ0000069 promoted the proliferation and migration of CC.8.Hsa?circ0000069 binds to miR-4426 and regulates the level of miR-4426.Overexpression of Hsa?circ0000069 prevented the tumor suppressing activity of miR-4426,and knockdown of Hsa?circ0000069 prevented the tumor promoting activity of miR-4426 inhibitor.Conclusion1.In CC,the transcription factor FOXA1 stimulates the expression of Hsa?circ00063322,which promotes the expression of L2 by adsorbing miR-548,promotes the proliferation ability of CC,and weaken its drug sensitivity2.In CC,enhanced M6A modification of Hsa?circ0000069 improves its stability,and Hsa?circ0000069 promotes the proliferation and migration ability of CC by adsorbing miR-4426...