The Role Of Structure-specific Endonuclease FEN1 In Cervical Cancer Treatment And Its Mechanism Study

Objectives:Cervical cancer is one of the most common causes of cancer associated mortality among affected women in the world.At present,treatment with weekly cisplatin plus radiotherapy is the standard regimen for cervical cancer,especially for local advanced cervical cancer.However,patients who initially responded to cisplatin therapy often develop resistance to the drug during subsequent treatment.The potential nephrotoxicity,ototoxicity and highly emetic effects of cisplatin also limit its use to special populations.DNA flap endonuclease-1(FENI)is a nuclease which possesses flap endonuclease(FEN)activity,gap dependent endonuclease(GEN)activity and exonuclease(EXO)activity,and plays essential roles in Okazaki fragment maturation of DNA replication and DNA repair pathways.FEN1 overexpression has been found in many forms of cancer,and FEN1 inhibitor has been shown to sensitize DNA damage related chemotherapy drugs.However,whether FEN1 deficiency or activity inhibition is help for radiotherapy for cervical cancer is still unknown.Since FEN1 plays a central role in DNA metabolism,FEN1 knock out mice model is embryonic lethal at 9.5 days.It is hard to study the functions of FEN1 during cell proliferation and metabolism process by omics technology,which is powerful and effective for the comprehensive study of FEN1.In this study,we aimed to study the potential function of FEN1 during cervical cancer cell proliferation,the function of FEN1 inhibitor to suppress cervical cancer cell growth,and the sensitizing effects of FEN1 inhibitor to radiotherapy for killing the cervical cancer,which would helpful for clinical application.Besides,we also detected and analyzed the gene expression profiles of FEN1 knockdown cells,which would lay a foundation and define the research directions for comprehensive study of FEN1 functions.Methods:1.Analyze the expression of FEN1 and ?H2AX in the Cervical cancer samples and normal tissues by the Cancer Genome Atlas(TCGA)database,and enrich the?HZAX markers in FEN1high samples and FEN1Low samples through GSEA gene enrichment analysis.And detect the expression levels of FEN1 and ?H2AX in Hela cells which were treated with ionizing radiation(2 hours later).2.The effect of FEN 1 inhibitor SC 13 on iouizing radiation sensitivity of Hela cells was studied through the analysis of Hela cell vitality,colony forming ability and cell apoptosis.3.The FEN1 gene in 293T cells was knock out by CRISPR technology,observed the ability of cell colony formation and the effect on ionizing radiation sensitivity after knock outof FEN1 gene.4.Establish nude mouse tumor model to study the effect of FEN1 inhibitor SC 13 synergistic ionizing radiation on the tumorigenic ability of Hela cells in mice.5.Using 18F-FDG Micro-PET display technology to evaluate the sensitizing effects of FEN1 inhibitor with ionizing radiation treatment on the nude mouse tumor model.6.Construct FEN1 gene knock-down 293T cell line by transfected with FEN1-siRNA and validated by western blot assay.7.The effect of FEN1 gene knock-down on cell proliferation would be analyzed by CCK-8 cell proliferation assay.8.Extract the total RNA from FEN1 gene knock-down and normal cell lines and reversely transcribe into cDNA for RNA-seq analysis.9.The RNA-seq results would be statistically analyzed by KEGG enrichment analysis and GO analysis to screen out differentially expressed genes.10.Selected genes which showed differential expression level between normal and FEN1 knock-down cell strains would be detected the expression level by Real-time quantitative PCR technology.Results:1.FEN1 was overexpressed in cervical cancer and upregulated further by ionizing radiation induction.2.FEN1 inhibitor SC 13 can enhance the ionizing radiation sensitivity of the Hela,which was mainly caused by inducing apoptosis.3.The FEN1 gene in 293T cells was knock out by CRISPR technology,and it was found that knock out of FEN1 expression can enhance the ionizing radiation sensitivity of the 293T cells.4.The FEN1 inhibitor SC 13 had a radiation sensitization effect on tumor formation in mice with cervical cancer cells.5.It was found that the standardized uptake value(SUV)of each intervention group was lower than the control group,18F-FDG Micro-PET display technology was good strategy for the therapeutic efficacy evaluation of cervical cancer xenograft mice model.6.FEN1 played an important role in cell growth,and knock-down of FEN1 gene inhibited the proliferation of 293T cells.7.RNA-seq analyses were performed on 293T normal and FEN1 gene knock-down cells.From the analysis,5,730 differentially expression genes were identified bctween the two groups,3,117(54%)genes were upregulated and 2,613(46%)genes were downregulated in the FEN1 gene knock-down group.8.The differentially expressed genes were analyzed through DAVID databases,the data revealed that nucleic acids related metabolisms and cell cycle related metabolisms were significantly interrupted in the FENI gene knock-down group.For the up regulated gene clusters of the FEN1 gene knock-down group,most genes are related to morphogenesis or development of cellular organ in biological process category,and protein binding activity in molecular function category.4.KEGG enrichment analysis of the pathway revealed that FEN1 down-regulation was associated with RNA transport,ribosomal biogenesis and RNA degradation,as well as viral infection and tumor-related pathways5.Six up regulated genes and four down regulated genes were chosen for further analysis by qRT-PCR to confirm the expression profiles of FEN1 gene knock-down cells.The data showed that all the qRT-PCR results were in good agreement with the RNA-seq analysis.Conclusion:In this study,we determined if FEN1 inhibitor SC 13 could sensitize radiotherapy of cervical cancer cell.We demonstrated that FEN1 is overexpressed in Hela cell and upregulated further by ionizing radiation induction.We also showed that FEN1 inhibitor enhances ionizing radiation sensitivity of cervical cancer in vitro and in vivo,and this beneficial effect was largely due to the induction of apoptosis.Furthermore,the present study demonstrated that FEN1 deregulation will suppress cell proliferation,and interrupt nucleic acids related metabolisms and cell cycle related metabolisms.It was also found that FEN1 can be involved in non-coding RNA processing,ribosomal RNA processing,transfer RNA proeessing and ribosomal biogenesis,which laid some foundation to understand the role of FEN1 nucleases in regulating cell growth,DNA repair and cancer therapy.It also provides clues for understanding the role of other RAD2 family nucleases in cell growth and metabolism.