Modification Of M~6A Enhances The Effect And Mechanism Of CircTANK On Promoting Malignant Behavior Of Cervical Cancer Cells

[Objective]Cervical cancer is a common malignant tumor of women,with high morbidity and mortality.N-adenosine(m6A)is a common,rich,and conservative modification among eukaryotic messengers(mRNA),microRNA(miRNA),and long noncoding RNA.Some studies have shown that m6A methylation plays an important role in the development of cancer.CircRNA also affects the occurrence and development of cancers.Our study aims to explore the effect of m6 A modification on the expression of circRNA,malignant behavior of cervical cancer cells and the specific molecular mechanism.So as to clarify its role in cervical cancer,and may provide new ideas and methods for the diagnosis and treatment of cervical cancer.[Methods]First,we analyzed the deep sequencing results of three pairs of cervical cancer tissues and adjacent tissues,and screened out 12 circRNAs that were highly expressed in cervical cancer tissues.RT-qPCR and the treatment of RNase R were used to verify the existence expression level of circRNAs in cervical cancer tissues and cells,MeRIP-qPCR expriment detected the level of m6 A modification in 12 circRNAs,and determined the circTANK with the highest level of m6A modification as a further research object.The stability and cell localization of circTANK were detected by RT-qPCR.MTT assay,clone formation experiment,transwell migration and invasion experiment and tumor xenograft experiment to detect the effect of circTANK on the malignant behavior of cervical cancer.Then,RT-qPCR and MeRIP-qPCR were used to examine the effect of METTL3 on the expression and methylation level of circTANK.The effect of m6 A modification level on the biological function of circTANK were examined by cytofunctional experiments.We used RegRNA software to predict the miRNAs that circTANK can adsorb,and verified the targeting and regulation relationship between circTANK and miR-2392 by RT-qPCR and Western blot exprements.Then through cell biology experiments and Western blot experiments to detect the effect of miR-2392 on cervical cancer cell malignant behavior.Bioinformatics prediction,RT-qPCR and Western blot experiments were used to determined the targeting and regulation relationship between miR-2392 and TAF7,YTHDF3.At the same time,the effects of TAF7 on the malignant behavior of cervical cancer cells were detected by cell biology experiments and Western blot.Rescue experiments proved that miR-2392 mediates the functional effect of circTANK on cervical cancer cells.The effects of YTHDF3 on circTANK expression level,modification level and stability were determined by RT-qPCR and MeRIP-qPCR assay.[Results]The RT-qPCR experiment proved that 12 circular RNAs were resistant to RNase R digestion,and the expression levels were consistent with the results of deep sequencing.MeRIP-qPCR showed that circTANK had the highest m6A enrichment.RT-qPCR assay proved that circTANK expression has high stability,and mainly distributed in the cytoplasm.Cell biology,Western blot and animal experiments have showen that circTANK plays oncogene role in cervical cacner.RT-qPCR showed that METTL3 promoted the expression and stability of circTANK.MeRIP-qPCR indicated that METTL3 mediates m6A methylation of circTANK,and cell biology experiments proved that m6A modification can enhance the cancer-promoting effect of circTANK.Western blot and RT-qPCR confirmed the targeting and regulation relationship between circTANK and miR-2392.Cell biology and Western blot experiments showed that miR-2392 plays a role in suppressing cancer in cervical cancer.The results of the rescue experiment showed that miR-2392 can mediate the biological function of circTANK in cervical cancer cells.RT-qPCR showed that YTHDF3 promoted the expression and stability of cricTANK.MeRIP-qPCR experiment showed that YTHDF3 promoted the m6A modification of circTANK.[Conclusion]We found that METTL3 as the main m6A methylase of circTANK promoted the expression of circTANK,and circTANK can promote the malignant behavior of cervical cancer cells as an oncogene in vitro and in vivo.CircTANK promoted the expression of downstream target genes TAF7 and YTHDF3 by adsorbing miR-2392,thereby promoting the malignant behavior of cervical cancer cells,and the stability of circTANK.Thus,METTLE/m6A-circTANK/miR-2392/YTHDF3-TAF7 axis promoted the malignant behavior of cervical cancer cells.