TNF?-induced CircDMD Promotes Cervical Cancer Development Through The R-loop Formation

[Objective] Inflammation is the basic innate immune response to damaged tissue,many tumorigenic stages are affected by chronic inflammation.As an important regulator of inflammatory response,TNF? plays an important role in the occurrence and development of tumors.Circular RNAs(circRNAs)is a type of covalently closed circular RNA,which is widely expressed in various human tissues and cells.Its formation process is regulated by alternative splicing.Some circRNAs acted as oncogenes or tumor suppressor genes to regulate the development of tumors.Therefore,our study aims to explore the mechanism of TNF? related inflammation pathways in regulating circRNA expression and affecting the malignant phenotype of tumor cells.[Methods] We first obtained the expression profile of circRNA that regulated by the inflammatory factor TNF? in cervical cancer by deep sequencing,combined with RNase R treatment and Sanger sequencing to verify the existence of circRNA,and determined the differentially expression level of circRNA by RT-qPCR experiments.The interaction between circRNA and p65,p50 were detected by RIP and S1-tag precipitation experiment,circDMD was selected as a further research object.RT-qPCR,Western blot,FISH,and S1-tag experiments were used to explore the effects of circDMD on the expression and nuclear distribution of p65 and p50,as well as the specific sites of interaction.We treated cervical cancer cells with actinomycin,and performed western blot to detect the effect of circDMD on the half-life of p65 and p50.Then,we treated cervical cancer cells with MG132,and performed ubiquitinated protein co-precipitation experiment to detect the effect of circDMD on the ubiquitination of p65.We performed bioinformatic analysis,RT-qPCR,fluorescence reporting system analysis experiment to explore the specific mechanism of circDMD on the ubiquitination of p65.Researchers have found that the promoter of VEGFR3 was regulated by NF-?B.So,we performed RT-qPCR and NF-?B fluorescence reporting system analysis to detect the expression level and promoter activity of VEGFR3 in circDMD overexpression cervical cancer cells.Next,bioinformatics analysis,FISH,Dot blot,DRIP,Western blot,dual luciferase reporting system analysis and co-immunoprecipitation experiments were used to analyze the effects of circDMD on VEGFR3.The reason for the high expression of circDMD in the inflammatory environment and its effect on the expression of downstream inflammation related factors were explored through RT-qPCR and Western blot.We conducted MTT assay,Western blot,Transwell assay,Immunofluorescence assay,Angiogenesis experiment,Tumor xenograft and immunohistochemical experiment explored the effects of circDMD in cervical cancer.[Results] We found a new circRNA affected by inflammatory factors by deep sequencing and RT-qPCR,named circDMD.circDMD not only promoted the expression of p65,p50 and IL-6,but also promoted and accompanied nuclear translocation of p65 and p50.According to the results,we verified that the promotion of circDMD on p65 expression is achieved by inhibiting its ubiquitination degradation.circDMD can be translocated into the nucleus and bind to the promoter of VEGFR3 to form a R-loop,and stabilize the binding of p65 and p50 on the promoter to promote the expression of VEGFR3.In addition,we found that the expression of DHX9 was inhibited in the inflammatory environment,the reduction level of DHX9 promoted the formation of circDMD and the expression of VEGFR3.The same results were obtained after the treatment of Poly I:C on cervical cancer cells.Finally,we confirmed that circDMD can promote cell proliferation,metastasis,autophagy and angiogenesis.Tumor xenograft experiments have also verified that high expression of circDMD promoted tumorgenicity and angiogenesis in nude mice.[Conclusion] We found that TNF? can promote the expression of circDMD,which can activate the canonical NF-?B signaling pathways.circDMD accompanied p65 and p50 translocation into the nucleus and binded to the promoter region of VEGFR3 to form a R-loop structure.R-loop helped improve the stability of p65 and p50 binding on the promoter of VEGFR3,and promoted the expression of VEGFR3.In addition,circDMD can promote cell metastasis,autophagy,cell proliferation and angiogenesis in vivo and vitro,and ultimately promote the occurrence and development of cervical cancer.