| Objective: Cervical cancer is one of the most serious gynecological malignant tumors with the highest morbidity and mortality in China.Although it has been clear that persistent infection of high-risk types of human papillomavirus(HPV)is the important pathogenesis of cervical cancer,it still cannot explain the cause of the onset of HPV-negative cervical cancer.Although the tertiary prevention of cervical cancer has been achieved,cervical cancer is still one of the killers that threaten women's health in developing countries.In particular,the breakthrough in the mechanism of cervical cancer in the post-HPV vaccine era will become the eradication of cervical cancer in the field of gynecological tumors.Circular RNA(circRNA)has a unique closed-loop structure,it is more stable than linear RNA and is not easily degraded.So it has the potential to become a biomarker.In recent years,studies have found that the abnormal expression of circRNA can cause a variety of malignant tumors,but the specific molecular mechanism urgently needs further in-depth study.Some circRNAs have been shown to inhibit the expression of mi RNAs by adsorbing mi RNAs,and further regulate the expression of downstream target genes,thereby promoting or inhibiting tumor cell proliferation,invasion and metastasis.In addition,some circRNAs have been shown to be able to regulate target gene expression by interacting with RNA binding protein(RBP).In the in-depth research on the mechanism of circRNA,the mechanism by which circRNA binds to protein to regulate complexes has just been paid attention in recent years,and there are not many reports.It is also a brand new field.Based on the abnormally high expression of circRNA in cervical cancer tissues screened in the early stage,this study conducted in-depth research on the molecular mechanism of circRNA regulating the occurrence and development of cervical cancer,aiming to find the core driving mechanism for the occurrence and development of cervical cancer.Methods: Firstly,combined with the results of the preliminary microarray screening,the candidate circRNA of this study was determined by bio-information analysis,and then the RT-q PCR experiment was used to detect the matched cervical cancer and normal tissues adjacent to the cancer to determine circZFR as the object of this study.In the in vitro cell experiment,firstly,the HeLa and SiHa stable transfected cell lines with overexpression and knockdown of circZFR were constructed through gene recombination technology.And the effect of circZFR on the proliferation ability of cervical cancer cells was tested by CCK8 experiment,Ed U experiment and plate cloning experiment.We detected the effect of circZFR on the invasion and metastasis of cervical cancer cells by scratch experiment and Transwell chamber experiment.We also detected the regulation effect of circZFR on cervical cancer cell cycle by flow cytometry.Bio-information analysis and RT-q PCR testing proved that E2F1 transcriptional activation is a key signaling pathway for cervical cancer.Western Blot experiments were used to detect E2F1 protein acetylation and Rb protein phosphorylation in cervical cancer cell lines overexpressing circZFR.These results were verified in matched cervical cancer and adjacent normal tissue.Through the RNA pull-down experiment and the subsequent mass spectrometry analysis,we found the protein that can directly bind to circZFR.At the same time,the circRIP experiment was used to reversely verify the binding effect between circZFR and RNA binding protein.Then the immunoprecipitation experiment was used to detect the regulatory effect of circZFR on the formation of SSBP1/CDK2/cyclin E1 protein complex.Results: 1.According to the microarray screening results of 3 pairs of cervical cancer and the matched adjacent normal tissues,combined with another set of microarray results of cervical cancer and adjacent normal tissues were found from GEO database,we found that6 circRNA are abnormally highly expressed in cervical cancer,but only circZFR is the circRNA derived from exons.The RT-q PCR experiment was used to detect 40 cases of cervical cancer tissues,30 cases of adjacent normal tissues and 7 cases of normal cervical epithelial tissues.The results showed that circZFR was abnormally overexpressed in cervical cancer tissues(P<0.001).In addition,the area under the receiver operating characteristic(ROC)curve of cervical cancer predicted by circZFR could reach 0.88(P<0.0001).Through the analysis of the clinical data of cervical cancer,we found that the high expression of circZFR is closely related to lymph node metastasis of the patients with cervical cancer(P=0.049),and is related to the Squamous Cell Carcinoma Antigen(SCC Ag)value and Ki67 value(P=0.049,P=0.003).2.We first constructed HeLa and SiHa stable transfection cell lines that successfully overexpressed and knocked down circZFR.The CCK-8 experiment,Ed U experiment,and plate cloning experiment were used to detect cell proliferation.The results suggested that the overexpression of circZFR could significantly increase the proliferation of HeLa and SiHa cells.In addition,the overexpression of circZFR could significantly increase the invasion and metastasis of HeLa and SiHa cells.The effect of circZFR on the cell cycle of HeLa and SiHa cells was detected by flow cytometry.The results suggested that the proportion of HeLa and SiHa cells in G0/G1 phase significantly decreased after overexpressing circZFR,while the proportion of cells in S phase increased.On the contrary,knocking down circZFR could significantly inhibit the proliferation,invasion and metastasis of HeLa and SiHa cells and inhibit the cells enter into S phase from G0/1 phase.3.Through the biometric analysis of the TCGA and GTEx databases,we found that the abnormally highly expressed genes in cervical cancer tissues were mainly concentrated in the three important signal pathways of DNA replication,cell proliferation and cell cycle progression.And 14 key genes were found by taking the intersection.RT-q PCR experiments verified that the expressions of the five genes CCNB1,CCNA2,CDC25 A,CDC6,and TFDP1 were all up-regulated by circZFR.The common upstream transcription factor E2F1 of these 5 genes was found through biosynthesis analysis.The next Western Blot test proved that overexpression of circZFR in cervical cancer cells could significantly increase the degree of acetylation at K117 and K125 of E2F1 protein,increase the degree of phosphorylation at S807 and S608 of Rb protein,and increase the expression of CDK2 and cyclin E1 protein.The increase of E2F1 protein acetylation,Rb protein phosphorylation,and CDK2 and cyclin E1 protein expression were verified in 30 matched cervical cancer and adjacent normal tissues.The correlation analysis results showed that the expression of circZFR and the phosphorylation degree of Rb protein S807 and S608 and the acetylation degree of E2F1 protein K117 and K125 were significantly positively correlated.Through immunoprecipitation experiments,we found that circZFR overexpression promoted the combination between CDK2 and Cyclin E1.On the contrary,circZFR knockdown inhibited the combination between CDK2 and Cyclin E1.Further through the RNA pull-down experiment and subsequent mass spectrometry analysis,we found that circZFR can directly bind to the SSBP1 protein.And the combination between circZFR and SSBP1 were verified by circRIP experiment.The further coimmunoprecipitation experiments proved that circZFR overexpression promoted the combination between SSBP1 and CDK2,and circZFR knockdown inhibited the combination between SSBP1 and CDK2.Conclusion: 1.CircZFR is abnormally highly expressed in cervical cancer tissues,and associated with the lymph node metastasis,SCC value and Ki67 expression in cervical cancer patients.2.CircZFR could promote the proliferation,invasion and metastasis of cervical cancer cells and promote the cells to progress from G0/1 into S phase.3.CircZFR could directly bind with SSBP1 protein,and promote the formation of SSBP1/CDK2/cyclin E1 protein complex,thereby promote the phosphorylation of Rb protein in cervical cancer cells. |
PDF Full Text Request