| Background and Objective: Cervical squamous cell carcinoma and endocervical adenocarcinoma(CESC),collectively called cervical cancer,is one common gynecological malignant tumor type.In China,there are 106000 new cases of CESC and 48000 CESC-related deaths each year.China and India account for one third of cervical cancer cases worldwide each year.The 5-year relative survival rate for localized CESC was 92%,whereas the 5-year relative survival rate for CESC patients with distant metastases was only 20%.Therefore,the search for effective and accurate CESC biomarkers and effective therapeutic targets is critical for early diagnosis,prognosis,and effective treatment of CESC.As a member of the E2 F transcription factor family,E2F1 is an important effector of retinoblastoma(RB)tumor suppressor protein.The hypo-phosphorylated RB protein inhibits transcriptional activity of E2F1 by binding to E2F1,thus maintaining the stability of cell cycle.About 99% of CESCs are caused by long-term infection with high-risk human papillomavirus(HPV).Integration of HPV DNA into the host(human)genome results in overexpression of HPV E6 and E7 oncogenes.The binding of HPV E7 oncoprotein to RB protein leads to functional inactivation of RB protein and its inability to suppress the transcriptional activity of E2F1.In turn,E2F1 activates the transcription of downstream target genes required for DNA replication in the S phase of the cell cycle,an event that is likely to accelerate the occurrence of CESC by promoting cellular proliferation.However,the biological role of E2F1 in the occurrence of CESC,especially its key downstream target genes,remains to be elucidated.In this study,we examined the m RNA and protein levels of E2F1 in non-tumor and CESC clinical samples,and explored the phenotypic changes related to proliferation,apoptosis and migration of CESC cells after stable knockdown of E2F1.Results showed that E2F1 promoted the proliferation of CESC cells but did not have significant impacts on their apoptosis or mirgration.In addition,bioinformatic analysis was used to screen and verify the key downstream target genes and downstream micro-RNAs(miRNAs)regulated by E2F1 in CESC.Methods: 1.Differential expression of E2 F family members in CESC from The Cancer Genome Atlas(TCGA)and normal cervical tissues from The Genotype-Tissue Expression(GTEx)was demonstrated through the GEPIA2 online analysis platform.2.Correlation between expression levels of E2 F family members and prognosis of CESC patients was analyzed by the TCGA online analysis tool UCSC XENA.3.Reverse transcription-quantitative polymerase chain reaction(RT-q PCR)was used to evaluate m RNA levels of E2F1 in 90 clinical samples(35 cases of non-tumor samples and 55 cases of CESC samples).4.Western blot(WB)and immunohistochemical staining(IHC)methods were used to evaluate the levels of E2F1 protein in clinical samples.5.E2F1 expression levels in Hela and Siha cells were evaluated by RT-q PCR and Western blot.6.sh-negative control(NC)He La,sh-E2F1 Hela,sh-NC Siha and sh-E2F1 Siha cell lines were established by stable lentivirus transfection.7.Cell count method and Methyl tetrazolium(MTT)colorimetry were used to detect the changes of cell number after E2F1 knockdown in above four groups of cells.8.The protein level of Ki-67 after E2F1 knockdown in the four groups was detected by Western blot.9.The apoptosis levels of sh-NC Hela,sh-E2F1 Hela,sh-NC Siha and sh-E2F1 Siha cells were assessed by terminal d UTP nick-end labeling(TUNEL)cell apoptosis assay.10.The migration ability of sh-NC Hela,sh-E2F1 Hela,sh-NC Siha and sh-E2F1 Siha cells was assessed by wound healing assay.11.Potential downstream target genes regulated by E2F1 in CESC were screened by bioinformatics analysis.12.The expression of potential downstream target genes in sh-NC Hela,sh-E2F1 Hela,sh-NC Siha and sh-E2F1 Siha cells was evaluated by RT-q PCR.Also differential expression of potential downstream target genes in clinical samples and their correlation with E2F1 expression detected by RT-q PCR.13.Potential downstream target miRNAs that can be regulated by E2F1 in CESC were screened by bioinformatic analysis.14.The differential expression of potential downstream target miRNAs in clinical samples and their correlation with E2F1 expression were assessed by RT-q PCR.15.Expression levels of potential downstream target miRNAs in aforementioned four groups of lentivirus-transfected cells were evaluated by RT-q PCR.Results: 1.Analysis of TCGA and GTEx data showed that expression of E2F1,E2F2,E2F3,E2F7 and E2F8 were increased in CESC than in normal tissues.2.Analysis of public databases showed that high expression of E2F1 was positively correlated with good prognosis(including overall survival)of CESC.3.Compared with non-tumor clinical samples,E2F1 was highly expressed in CESC samples.4.E2F1 knockdown inhibited the proliferation of Hela and Siha cells.5.E2F1 knockdown had no significant effect on the apoptosis of Hela and Siha cells.6.E2F1 knockdown had no significant effect on the migration of Hela and Siha cells.7.Bioinformatic analysis showed that ASF1 B,CENPM,CHAF1 B,GINS2,MCM3,PCNA,RNASEH2 A,FANCG,LIG1,MCM2,MYBL2 and TYMS were potential downstream target genes activated by E2F1 in CESC.8.E2F1 knockdown decreased CENPM expression in Hela cells and Siha cells.9.CENPM was highly expressed in CESC clinical samples as compared to non-tumor tissues,and there was a positive correlation between CENPM expression and E2F1 expression in CESC.10.Bioinformatic analysis showed that HsamiR-16-2-3p and Hsa-miR-20b-3p were potential downstream miRNAs upregulated by E2F1 in CESC.11.Compared with non-tumor samples,Hsa-miR-16-2-3p and HsamiR-20b-3p were highly expressed in CESC clinical samples,and their expression levels were positively correlated with those of E2F1 in CESC clinical samples.12.E2F1 knockdown decreased the expression of Hsa-miR-16-2-3p and Hsa-miR-20b-3p in Hela and Siha cells.Conclusion: 1.E2F1 is highly expressed in CESC and is positively associated with good prognosis in patients with CESC.2.E2F1 promotes the proliferation of CESC cells.3.In CESC cells,E2F1 up-regulates the expression of CENPM.4.In CESC cells,E2F1 up-regulates the expression of Hsa-miR-16-2-3p and Hsa-miR-20b-3p. |
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