The Impact On Biological Behavior In Esophageal Squamous Cell Carcinoma Of Expression And Methylation Of SEPT9 And Its Clinical Application

Objective:Esophageal cancer is one of the most common malignancies worldwide.It ranks sixth in leading cause of cancer related mortality.Esophageal cancer is subdivided into two main histological types:esophageal squamous cell carcinoma（ESCC）and esophageal adenocarcinoma（EAC）,and about 90%of the patients are ESCC.ESCC is characterized by aggressive local growth,early lymphatic metastasis and vascular invasion.Despite the improvement of surgery and chemoradiotherapy,the prognosis of ESCC is poor due to asymptomatic progression in early stage and diagnosis in advanced stage.The overall 5-year survival is merely about 15-25%.It is high time to develop sensitive,specific and none-invasive biomarkers for early diagnosis to improve the overall prognosis for ESCC.It is demonstrated that aberrant genetic and epigenetic changes lead to the pathogenesis and progression of human ESCC.There are three kinds of familiar epigenetic modifications:histone modification,DNA methylation and non-coding RNA.DNA methylation is a common epigenetic modification and plays a crucial role in tumor genesis by regulating gene expression without changing the DNA sequence.Aberrant DNA methylation usually occurs on cytosin-phosphate-guanosine islands（CpG islands）of gene promoter region which involved in the occurrence and development of ESCC.With the in-depth research in oncology and epigenetics,we find promotor methylation of tumor suppressor gene silence the expression of the protein which promotes the development of malignant tumors.Gene microarray technology is a high-tech in molecular biology that has developed rapidly since the mid-1990s.DNA methylation detection have experienced from the detection of a single candidate gene by methylation specific PCR to the detection of the whole genome DNA methylation level by methylation chip.The Illumina Human Methylation EPIC Bead Chip（850k）is widely used in researches nowadays which is an important means for whole epigenome association research.The chip covers more than 850,000 CpG sites and quantitatively detects the methylation sites of the whole genome at the level of single nucleotide.Septin belongs to the filamentous protein family.The gene was first found in Saccharomyces cerevisiae.The septin gene family includes 13 members in humans.Septin 9（SEPT9）gene is located at chromosome 17q25.3 and ubiquitous in eukaryotes.It encodes SEPT9 protein which plays important physiological role in cytoskeletal remodeling,actin dynamics,microtubule regulation,vesicle trafficking and exocytosis.It is abnormally expressed in various kinds of cancer with different biological effects.Peripheral cell free DNA detection is used for non-invasive diagnosis of tumors.At present,it is mainly used for the detection of mutations in oncogene and tumor suppressor genes.EGFR,APC,MET,TP53,KRAS and BARF are common detected genes in clinics.Previous studies have confirmed that the abnormal methylation of cell free DNA in peripheral blood can be used as a new biomarker for early and noninvasive screening of malignant tumors.Many studies have reported the hypermethylation expression of many genes in ESCC tissues.However,there are few reports on the methylation of cell free DNA in plasm of ESCC.In our study,we intend to screen the differentially methylated sites and regions of ESCC tissues by using Illumina Human Methylation EPIC Bead Chip （850k）.Then we try to verify the methylation and expression of target gene in ESCC according to the screening results.Furthermore,we try to uncover the mechanism of how the target gene participating in the development and occurrence of ESCC.Materials and Methods:1.Materials（1）Fresh frozen esophageal squamous cell carcinoma tissue and paracancerous tissue.（2）Blood specimens from esophageal squamous cell carcinoma patients and control cases.（3）Esophageal squamous cell carcinoma cell line KYSE-150 and TE-1.（4）Real-time PCR experiment reagents and instruments.（5）Western blot experiment reagents and instruments.（6）Cell culture related reagents and instruments.（7）SEPT9 over-expression vector.（8）Immunohistochemical related reagents.2.Methods:（1）Illumina human methylation epic beadchip（850k）chip was used to scan the differential methylation sites and regions of 4 pairs of ESCC and adjacent tissues.Combined the 850k database with UALCAN database of tumor methylation,the expression and methylation level of SEPT9 in ESCC were verified.（2）Real-time PCR to conform the methylation level in ESCC patients and control cases.Analyze the clinical information of ESCC cases to explore the relationship between clinical characters and methylation.（3）Western blot and immunohistochemistry experiments to confirm the expression of SEPT9 in ESCC tissue and para-cancer tissue.Real-time PCR to conform the methylation level in ESCC tissue and para-cancer tissue.（4）Build SEPT9 over-expression plasmid and transfect ESCC cell lines KYSE-150and TE-1.（5）Wound healing,transwell,CCK-8 and apoptosis experiments to confirm the impact of SEPT9 on ESCC cell lines.（6）Western blot experiment to compare the expression of phosphorylated GSK-3βin SEPT9 over-expression group and vector group.（7）Western blot experiment to compare the expression ofβ-catenin in SEPT9 over-expression group and vector group in nuclear protein.Then confirm whether GSK-3βinhibitor TWS119 could revert the expression ofβ-catenin.Results:1.4 pairs of ESCC tissues and para-cancer tissues were scanned with Illumina Human Methylation EPIC Bead Chip（850k）.We found 189489 differential methylation sites.There were 53307 hypermethylation sites and 136182 hypomethylation sites.Furthermore,we found 261 differential methylation regions.We chose top 30 differential methylation regions and found 34 genes located in these regions.The methylation chip information from UALCAN database were integrated with our screening database.We found 6 genes including ZNF132,SEPT9,ZIC1,ZIC4,USP4and ZNF154 possessed hypermethylation in both databases.Found the probe site which had the highest hypermethylation level among all probe sites of the gene to be the representative site.Sort the results in descending order among all 189489 differential methylation sites.The representative site cg20309069 of SEPT9 ranked18^th among all sites which was the first among the six genes.Therefore,we choose to confirm the expression and methylation of SEPT9 in ESCC.2.The methylation of SEPT9 was increased in ESCC tissue（1.20±1.92）than para-cancer tissue（3.64±1.92）.3.81%（51/63）of esophageal squamous cell carcinoma patients and 6%（4/71）of control cases had SEPT9 hypermethylation in plasm cell free DNA.Plasm cell free DNA from esophageal squamous cell carcinoma patients（10.42±4.36）had an obviously higher methylation level than control cases（16.72±1.68）.4.The methylation of SEPT9 was correlated with TNM stage and lymphatic metastasis.Hypermethylation occurred early in ESCC.5.The expression of SEPT9 was decreased in ESCC tissue than para-cancer tissue.6.Over-expression of SEPT9 inhibited migration,invasion,proliferation abilities of ESCC cell while promotes apoptosis abilities.7.Over-expression of SEPT9 inhibited the expression of phosphorylated GSK-3βwhich promoted the kinase activity of GSK-3β.8.Over-expression of SEPT9 inhibited the expression ofβ-catenin in nuclear while the expression was reverted when TWS119 was added.Conclusion:Our study suggested the expression of SEPT9 was decreased in ESCC which may due to the hypermethylation of the gene.Plasm from esophageal squamous cell carcinoma patients had an distinctly higher methylation status of SEPT9 than control cases which is parallel with ESCC tissue.The methylation of SEPT9 was correlated with TNM stage and lymphatic metastasis.Hypermethylation occurred early in ESCC.Plasm SEPT9 hypermethylation could be a potential biomarker for none-invasive and early screening of ESCC.SEPT9 may acts as a tumor suppressor gene in ESCC by adjusting the activity of GSK-3βto inhibit Wnt/β-catenin signaling pathway.