Expression Of FoxM1Gene In Ovarian Cancer And The Preliminary Research Of Transcriptional Regulatory Mechanism

BackgroundOvarian cancer is one of the most life-thereat cancer in females worldwide. Theincidence of ovarian cancer is relatively low, while the mortality is very high. Ovariancancer can be symptomless in the early stage and progress rapidly. Ovarian cancer is notsensitive to chemotherapy and the five-year survival rates are around30%-40%. The currentstandard treatment of ovarian cancer is paclitaxel and platinum-based chemotherapy on thebasis of cytoreductive surgery. However, with the emerging of drug resistance the curativeeffect is unsatisfactory. Therefore, for early diagnosis and effective treatment, it is of greatsignificance of better exploring the molecular mechanisms of ovarian cancer carcinogenesisand shedding light on related target genes.As a member of the Forkhead transcription factor family, FoxM1is an importanttranscription factor closely related to the occurence and development of cancer. It iscomposed of10exons, locating on20KB of telomere position12p13-3. FoxM1have threespliced isomers, namely FoxM1A, B and C. FoxM1B and C are highly expressed in cancertissues while lowly expressed in normal tissues, playing the roles of transcription activation,promoting occurrence and development of cancer. FoxM1A has a low expression in cancertissue and plays transcriptional suppression actions. FoxM1performs the main function ofregulating proteins through the transcriptional activation of cell cycle, accelerating theprocess of tumor cell cycle; promoting the formation of tumor blood vessels by transcriptional activation of VEGF; promoting tumor invasion and metastasis bytranscriptional activation of MMP2and MMP9Furthermore, a large number of biochipresearch shows that FoxM1are closely associated with ovarian cancer. Therefore, FoxM1,as an important tumor marker, is of great significance for the diagnosis and treatment ofovarian cancer.Regulation of transcriptional level is the most important part in the gene expressionregulation of eukaryotes, Which could accomplish effective regulation of gene transcriptionthrough the combination of specific transcription factor and the regulatory sequencesas wellas promoter of the target gene. But so far there are no reports about the combination betweenspecific transcription factor and core promoter region of FoxM1effectively regulate itstranscriptional activity. Thus, it provides a theoretical basis for elucidating the relationshipbetween the biological function of FoxM1and the development of ovarian cancer throughthe cloning of FoxM1promoter gene and research of its expression regulation mechanism.Abnormal methylation of genomic DNA is closely associated with tumorigenesis,partial methylation of CpG island is a common phenomenon existing in the malignant tumor,which is apt to occur earlier than obvious malignant phenotype. So far, it is generallybelieved that DNA methylation can affect the transcription factor binding to the recognitionsequence containing CpG islands, with regulating gene expression.Therefore, it is of greatsignificance for understanding the molecular mechanism of the occurrence and developmentof ovarian cancer to explore the correlation between the methylation status of CpG island ofFoxM1promoter region and ovarian cancer, as well as to explore the influence oftranscriptional regulation relating DNA methylation to FoxM1, and provides a new ideas orearly diagnosis and targeted therapy for ovarian cancer.Objectives:1. To definite the FoxM1expression in cell lines and tissues of ovarian cancer;2. To definite the FoxM1core promoter regions;3. To explore the transcription regulation role relating Sp1transcription factor to FoxM1; 4. To determine relationship between the FoxM1expression and modification of DNAmethylation, evaluate the applicational feasibility of FoxM1in the early diagnosis andtreatment of ovarian cancer.The content and methods1.To detect the expression of FoxM1in ovarian cancer cell lines and ovarian cancer grouptissues.A.To detect the expression levels of FoxM1in ovarian cancer cell lines (HO-8910PM,HO-8910, A2780and CP70) respectively with quantitative real-time PCR and Western blot.B.Meanwhile, to detect the expression of35cases of epithelial ovarian cancer and50casesof normal ovarian tissue with quantitative real-time PCR and immunohistochemistry.2. To determine the FoxM1core promoter regionsWith the template of normal human whole blood genomic DNA which were amplifiedto build six different lengths but overlapping truncated promoter reporter FoxM1genevectors series by PCR technology, the core promoter region of the gene is determined bytransfecting293T cells transiently and using Dual-Luciferase FoxM1reporter gene system.3.With Sp1gene eukaryotic expressed plasmid and reported gene plasmid transfectingovarian cancer cells (cisplatin sensitive cell line A2780and cisplatin resistance cell lineCP70) respectively and transfecting vector pcDNA3.0in control group empty,determine theeffect on the transcriptional regulation ralting Sp1to FoxM1by using dual luciferasereporter gene system.4.The relationship between the modification of DNA methylation and the FoxM1expression.Firstly, with using Search software for CpG island named CpGPLOT,analyze FoxM1promoter sequences.By using sulfite cloning sequencing method, we detected the status ofmethylation in the core promoter FoxM1in HO-8910PM belonging to high transfered cellline, HO-8910belonging to low transfered cell line, A2780belonging to cisplatin andsensitive cell line, and CP70belonging to cisplatin resistance cell line of the ovarian cancer; Meanwhile, we detected the status of methylation in core promoter FoxM1region in15cases of epithelial ovarian cancer,14cases of normal ovarian tissue respectively by usingpyrophosphate sequencing technologies.Results:1. It is showed the high expression of FoxM1in ovarian cancer cell lines (HO-8910PM,HO-8910, A2780and CP70) in the quantitative real-time PCR and Western blot; Meanwhile,it is showed that FoxM1in epithelial ovarian cancer tissue level (30/35=86%) issignificantly higher than in normal ovarian tissue (6/50=12%, P <0.01) upon quantitativereal-time PCR and immunohistochemistry assay.2. It is showed that the core promoter FoxM1region located upstream of the transcriptionstart site on-95bp of the sequence according to double luciferase reporter gene detection.3. It is showed that Sp1has the transcriptional activation with combining into core promoterFoxM1region according to the luciferase reporter gene system experiment.4. According to the result of sulfite cloning sequencing method, it is showed that the levelsof methylation are very low in the core promoter FoxM1in HO-8910PM belonging to hightransfered cell line, HO-8910belonging to low transfered cell line, A2780belonging tocisplatin and sensitive cell line, and CP70belonging to cisplatin resistance cell line of theovarian cancer. In addition, according to the result of pyrophosphate sequencingtechnologies, it is showed that the low degree (P>0.05) of methylationthe in core promoterFoxM1region in15cases of epithelial ovarian cancer and14cases of normal ovarian tissuerespectively, with no statistical significance, and with degree of methylation in ovariancancer cell lines being basically identical, which indicates that the modification of DNAmethylation is not involved in regulating the expression of FoxM1.Conclusion:1. It is showed that FoxM1has low or no expression in normal ovarian tissue and has highexpression in the four strains and epithelial cell lines of ovarian cancer, with the consistence in transcription and translation level, which indicates that FoxM1gene is closely related tothe occurrence of ovarian cancer.2. Determine the core promoter FoxM1region in its upstream transcription start site-95bpof the sequence for the first time, laying a foundation for further study on FoxM1relationwith tumor molecular mechanism.3. Determine the transcriptional expression of upregulation relating Sp1genes to FoxM1,indicating to explain the mechanism of the high expression in the invasion and metastasis inovarian cancer.4. Determine the low levels of methylation in ovarian cancer cells, ovarian cancer tissue andnormal ovarian tissue, with basically identical results and no statistical significance,indicating that the modification of DNA methylation is not involved in regulating theexpression of FoxM1.