HPV16 CircE7 Promote Proliferation,migration And Inhibit Ferroptosis Of Cervical Cancer Cells

Background ＆ Objective: Cervical cancer（CC）is one of the most dangerous diseases for women’s health,and its morbidity and mortality are still at a high level in the word.High-risk human papillomavirus（HPV）infection is the main cause of cervical cancer.Meanwhile,non-coding RNAs produced by cells,including micro RNAs（miRNAs）,circular RNA（circRNA）and other molecules,can also play a role in the progression of cervical cancer through epigenetic regulation.HPV16 circE7 is a circRNA encoded by HPV16 virus.Circ E7 is modified by N6-methyladenosine（m6A）,preferentially localized in the cytoplasm,but its mechanism in cervical cancer cells is still unclear.This study mainly focused on the expression of HPV16 circE7 in cervical cancer,and the effect and potential mechanism of circE7 on proliferation and apoptosis of HPV16 positive cervical cancer cells through miR-214-3p.Meanwhile,the effect of circE7,miR-214-3p and it’s target gene GPx4 on ferroptosis in cervical cancer cells and the regulatory mechanism were investigated.Methods:1.HPV-negative C33 A cells and HPV16-positive cervical cancer cell Caski were cultured,and the expression levels of HPV16 E7,HPV16 circE7 and miR-214-3p in these two cell lines were detected by real-time fluorescence quantitative PCR.2.In order to explore the effects of HPV16 circE7 and miR-214-3p on proliferation,migration and apoptosis of the two cell lines,si RNA targeting HPV16 circE7,plasmid expressing HPV16 circE7,miR-214-3p mimics and mimics NC were transfected into C33 A and Caski cells,respectively.CCK8 assay was used to detect changes in cell proliferation,wound healing and transwell assay was used to detect the effects of HPV16 circE7 and miR-214-3p on cell migration and invasion,and cell cycle and apoptosis were detected by flow cytometry.3.Target gene of miR-214-3p was analyzed by starbase v3.0,then Real-time fluorescence quantitative PCR and dual luciferase assay were used to veriy whether miR-214-3p inhibited the target gene and whether there were binding sites between them.4.RSL3 was used to inhibit the target gene of miR-214-3p in HPV16-positive Caski cells,changes of ferroptosis were detected by GSH,GPx,MDA,ROS,JC-1 staining and Western blotting experiments.Meanwhile,si RNA targeting HPV16 circE7,miR-214-3p mimics and mimics NC were transfected into Caski cells,respectively.After transfection of HPV16 circE7 plasmid and pc DNA3.1（+）vector in C33 A cells,the changes of ferroptosis were detected by the above experiments.Results:1.Real-time fluorescence quantitative PCR results showed that compared with HPV negative C33 A cells,HPV16 E7 and HPV16 circE7 were significantly higher in HPV16 positive Caski cells（P < 0.01）,and miR-214-3p was significantly lower in HPV16 positive Caski cells（P < 0.01）.2.Results of CC8 assay,wound healing assay,transwell assay,cell cycle and apoptosis assay showed that HPV16 circE7 knockdown and miR-214-3p overexpression in HPV16 positive Caski cells inhibited cell proliferation,migration and invasion（P < 0.05）.The cell cycle in S phase was significantly decreased（P <0.05）,and the apoptosis level was significantly increased（P < 0.05）.After HPV16 circE7 was overexpressed in HPV-negative C33 A cells,the proliferation,migration and invasion abilities were significantly up-regulated（P < 0.01）,and the cell cycle in S phase was increased（P < 0.05）,the level of apoptosis decreased（P < 0.05）.3.Target gene of miR-214-3p were predicted by starbase v3.0.After analysis and screening,it was found that GPx4 had multiple binding sites with miR-214-3p.Then it was verified by real-time quantitative PCR and dual luciferase assay.After transfection of miR-214-3p mimics and mimics NC,q PCR results showed that GPx4 m RNA level decreased after overexpression of miR-214-3p（P < 0.01）.The dual luciferase report showed that the fluorescence signal with mut-GPX4 3’UTR fragment decreased significantly（P < 0.05）,indicating that miR-214-3p has a binding site with GPx4.4.GPx4 was inhibited by RSL3,a direct inhibitor of GPx4,and si RNA,miR-214-3p mimics and mimics NC targeting HPV16 circE7 were transfected into Caski cells,respectively,then,GSH,GPx,MDA,ROS and JC-1 staining experiments were performed.The results showed that the expression levels of GSH and GPx in Caski cells decreased（P < 0.05）,MDA and ROS level increased（P < 0.05）,and the red fluorescence/green fluorescence values of mitochondrial membrane potential decreased.After transfection of HPV16 circE7 plasmid and pc DNA3.1（+）vector in C33 A cells,GSH,GPx,MDA,ROS and JC-1 staining experiments were performed.The results showed that the expression levels of GSH and GPx in C33 A cells increased（P < 0.05）.The levels of MDA and ROS decreased（P < 0.05）,and the red fluorescence/green fluorescence values of mitochondrial membrane potential increased.Conclusion: Inhibition of HPV16 circE7 or overexpression of miR-214-3p can inhibit the occurrence and development of HPV16 positive cervical cancer Caski cells,and there is a mutual inhibitory relationship between HPV16 circE7 and miR-214-3p.miR-214-3p can bind to GPx4,thus affecting the occurrence and development of ferroptosis in HP16-positive cervical cancer cells.