Fund programs: National Natural Science Foundation of China (No. 82300944); Tianjin Medical Key Discipline Construction Fund (No. TJYXZDXK-3-003A); Tianjin Medical University Institute & Hospital Scientific Research Fund (No. Y1905)
Authors:Bai Lili 1,2,3,4, Zhang Jin5, Fan Yatong 1,2,3,4, Zhang Pengyu 1,2,3,4
Keywords:cervical cancer; TNF-α; circWBSCR22; miR-512-5p; FAM60A; migration; invasion
DOI:专辑:医药卫生科技
〔Abstract〕 Objective To explore the effect and mechanism of tumor necrosis factor alpha (TNF-α)-regulated circular RNA Williams Beuren syndrome chromosomal region 22 (circWBSCR22) on the malignant behaviors of cervical cancer cells. Methods Human cervical cancer HeLa cells were treated with TNF-α . Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression level of circWBSCR22. Nuclear-cytoplasmic RNA separation experiments were conducted to determine the localization of circWBSCR22 in cells. Cell migration, invasion, viability, and proliferation were assessed through Transwell assay, tetrazolium salt (MTT) assay, and colony formation assay, respectively. Dual-luciferase reporter gene assays were performed to verify the targeting relationship between microRNA-512-5p (miR-512-5p) and circWBSCR22 or family with sequence similarity 60A (FAM60A). RT-qPCR was used to detect the RNA levels of miR-512-5p and FAM60A. Western blot was used to detect the protein level ofFAM60A. Results Compared with the Control group,the expression level of circWBSCR22 in HeLa cells treated with TNF-α increased (P<0.05). About 66% of circWBSCR22 was found in the cytoplasm. Compared with the control group, the migration (P<0.01) and invasion (P<0.01) of HeLa cells transfected with circWBSCR22 were enhanced, but the cell activity and cell proliferation were not significantly affected (P>0.05). The double luciferase reporter gene assay showed that the relative luciferase activity of the co transfected circWBSCR22-WT and miR-512-5p groups was lower than that of the co transfected circWBSCR22-WT and miR-NC groups (P<0.01). Compared with the control group, the expression level of miR-512-5p in HeLa cells transfected with circWBSCR22 decreased (P<0.01). Compared with the control group, the migration (P<0.01) and invasion (P<0.05) ability of HeLa cells transfected with miR-512-5p decreased. Double luciferase reporter gene assay showed that compared with the co transfected FAM60A 3´-UTR-WT and miR-NC groups, the fluorescence activity of the co transfected FAM60A 3´-UTR-WT and miR-512-5p groups decreased (P<0.01).Compared with the control group, the expression levels of FAM60A mRNA (P<0.05) and protein (P<0.01) in HeLa cells transfected with miR-512-5p decreased. Compared with the control group, the migration (P<0.01) and invasion (P<0.01) of cervical cancer HeLa cells transfected with FAM60A were enhanced. Compared with the control group, the expression levels of FAM60A mRNA (P<0.01) and protein (P<0.05) in the cells transfected with circWBSCR22 increased. The results of rescue experiment showed that compared with the pcDNA+miR-NC group, the expression levels of FAM60A RNA (P<0.01) and protein (P<0.001) in the pcDNA+miR-512-5p group decreased, and the ability of migration (P<0.01) and invasion (P<0.05) decreased; Compared with pcDNA+miR-512-5p group, the expression levels of FAM60A RNA (P<0.01) and protein (P<0.001) in circWBSCR22+miR-512-5p group increased, and the ability of migration (P<0.01) and invasion (P<0.05) was enhanced. Conclusion TNF-α promotes the migration and invasion of cervical cancer cells through the circWBSCR22/miR-512-5p/FAM60A regulatory axis.