The Mechanism And Its Inner Mechanism Of The Role Which Epb4113 Plays In Esophageal Squamous Cell Carcinoma And Its Effect On Prognosis

BackgroundUntil 2013, Lancet magazine pointed out that death from esophagus cancer occupies the sixth place in the etiology of cancer, affecting more than 450,000 people globally. This data is constantly increasing. The total five-year survival rate of esophageal cancer is about 15%-25%.The difficulty of early detection and diagnosis of esophagus cancer and distant metastasis are the main causes for the low five-year survival. Distribution of esophageal cancer in the world exist the so-called "Asian belt". It includes the Asian population of Iran, kazakhstan, northern and central China, where is a region of high incidence of esophageal squamous cell carcinomas. The incidence is 100 cases of esophageal squamous carcinoma each year in every 100000 people. The treatments of esophageal cancer include surgery, radiotherapy, chemical therapy and comprehensive therapy. And most of the middle to late patients have lost the operation chance, who can only adopt chemotherapy combined with radiotherapy. However, the effect of chemotherapy in late stage patients is not ideal. Patients who are diagnosed early with conventional treatments can get the highest five-year survival rate which is more than 90%. Exploring new esophageal tumor suppressor genes and analyzing the mechanism may provide therapy target for the early diagnosis and treatment of esophageal cancer. Currently, squamous cell carcinoma (SCC) is the major histological type of advanced esophageal cancer worldwide. However, in Australia, the United Kingdom, the United States and some European countries, the incidence rates of adenocarcinoma exceed squamous cell carcinoma.Like other tumors, the morbidity of esophageal squamous cell carcinoma is also a multi-stage process of many steps. It includes the activation of oncogenes and inactivation of tumor suppressor genes. Tumor suppressor genes refer to a large class of genes which can inhibit cancer cell growth and carcinogenesis potentially. There have been three characteristics: ①It is normally expressed in certain tumors corresponding normal tissues; ② There should be changes happened to this gene in certain tumors, such as point mutations, deletions of DNA fragments or whole gene or expression defection; ③ After overexpressing this gene, the expression of the gene should be upregulated. Some or all of the relevant tumor phenotype whose expression was of deficiency would be inhibited. Tumor suppressor genes have played an important role in the regulation of the cell cycle, angiogenesis, signal transduction or even chemotherapy drug resistance. The concept of a tumor suppressor gene was originally developed with functional complementation, which inhibits transformed phenotypes through whole cell-cell fusion of normal and cancer cells; the two-hit theory that led to discovery of loss of heterozygosity (LOH) was also important. Inactivation of TSGs is because that genes and epigenetic level work jointly. Gene expressing deletion and point mutations directly affects tumor suppressor gene function. The epigenetic mechanisms including promoter CpG island region methylation and those modification processes of histone methylation, acetylation, phosphorylation, polyadenylation, ubiquitination are often key factors in tumorigenesis and progression, resulting in the loss of function for tumor suppressor genes.The nature of DNA methylation is a covalent chemical modification. S-adenosylmethionine is the methyl donor. It is a response catalyzed by DNA methylation enzymes which converts cytosine to 5-methylcytosine. In the mammalian genome, the main site of DNA methylation is CpG dinucleotides which were unevenly distributed in the genome. When methyl groups connect to the gene’s DNA, gene transcription terminates and gene silences. CpG oligonucleotides concentrated in certain regions and the density of CpG sequences is about 10 to 20 times higher than the average density. G+C content are more than 50% and its length is greater than 200 bases. Those regions were named as CpG islands. CpG islands play a regulatory role in gene expression which has special meanings. CPG islands usually located in the promoter region of the gene which can also be extended to the exons, ranging from 500bp to 5000bp. DNA methylation is an epigenetic modification. In mammalian genomes, the main site of DNA methylation is CpG dinucleotide. It was unevenly distributed in the genome. In tumor, low methylation usually occurs on the DNA repetitive sequence, and methylation mainly involves the CpG island. Approximately,50% of human genes contain CpG islands which often located in the promoter of upstream regulatory region of the gene. These genes are housekeeping genes or tissue-specific expression gene. Aberrant methylation of multiple genes often occurs in tumors, including tumor suppressor genes. Besides, abnormal methylation often occurs in the DNA repair related genes, tumor invasion related genes and DNA metabolism and repair genes. The aberrant methylation of CpG islands leads to gene silencing, resulting in the inactivating of TSG, which can fasten tumor formation through disabling multiple normal cellular processes, such as apoptosis and cell cycle. In general, there are two kinds of DNA tumor abnormal methylation, including wide hypomethylation and localized hypermethylation. The former allows the proto-oncogene activation, the formation of mutation hot spots, increased chromosomal instability, so transposon abnormal expression; the latter is directly or indirectly affecting gene transcription, which also leads to cytosine-thymine mutation.EPB41L3 is also called 4.1B, Dal-1, is an important part of the membrane skeleton protein 4.1 family which are widely expressed in various human tissues and organs. A growing number of studies have found EPB41L3 is abnormally expressed in a variety of tumors. As expressed in many tumor tissues of esophageal squamous cell carcinoma, non-small cell lung cancer, breast cancer, glioma, meningioma, renal cell carcinoma, etc. down. Yajie Zhang found that more than 71.4% of the reduction in non-small cell lung cancer cells and clinical tissues; Kittiniyom Kearly reported in breast tumor tissue also appears its expression is missing, and about two-thirds of ductal carcinoma in situ and invasive ductal carcinoma in this situation have emerged; Bernkopf research results EPB41L3 missing expression in prostate cancer findings and prompt regulation if its expression can be a therapeutic strategy; EPB41L3 Daisuke Yamada et al found that often occurs in renal cell carcinoma methylated, it can become an independent prognostic factor for recurrence and metastasis after surgery. Generally speaking, there are reports proving EPB41L3 may play the role of tumor suppressor genes in a wide variety of tumors. Its methylation is associated with cancer and poor prognosis. However, at present, there have been no similar studies reporting EPB41L3 and esophageal cancer. Revealing EPB41L3 in esophageal cancer cannot play the role of tumor suppressor genes, and whether its because the methylation and what impact the process of tumor cancer, and what molecular biological mechanism are involved is our concern.Methods1 Cell culture and treatmentHetla, TE1, KYSE150, KYSE450, KYSE510 cells using RPMI-1640, Eca109, Caes17 cells using high glucose DMEM, supplemented with 10% fetal bovine serum were respectively, at 37 ℃,5% CO2 concentration, relative humidity of 50% of the cells within the incubator, the cell growth rate according to replace the medium in due course, with 0.25%-0.02% EDTA trypsin digestion dispersion fluid cells were subcultured or seeded culture.2 Invasion AssaySelect the cells grew well passaged to 24 or 12-well culture plate, cultured normal overnight until the cells in logarithmic growth phase, cell fusion density of about 80%, the company’s use invitorgen Lipofectamine 2000 transfection reagent transfection, concrete operation reference manual,48 hours after transfection, cell invasion and migration of cells to detect or extract the total protein. When the migration test were resuspended in serum-free medium 1.0× 105 cells were seeded in a small indoor BD; invasive test with serum-free culture medium were resuspended when 2.0 x 105 cells were seeded in Matrigel-coated BD closet.37 ℃ incubator, after 24h culture chamber removed, gently rinsed with PBS, fixed with 95% methanol at room temperature for 20 min; 0.25% crystal violet (prepared in methanol) staining, dyeing in room temperature for 30min, rinse with water, wipe with a cotton swab on the interior endoscopic camera and count cell numbers.3 Scratch TestFirst, with a marker pen, we drew a line about 0.5～1cm together across the every hole in 6-well plates behind the uniform underlined. Each hole went through at least five lines. Then we added about 5 x 105 cells into each well, the next day with a ruler than the tip, as far as possible perpendicular to the horizontal scratches behind the tip to the vertical, preferably using the same can not be tilted between (different hole only the tips). PBS cells were washed three times, draw the place cells, serum-free medium. And finally into the 37 ℃ 5% CO2 incubator. We chose 0,24h, 48h time point to collect samples and take photos.4 ImmunoblottingCells were lysed on ice to extract total protein; SDS-PAGE electrophoresis and transferred PVDF membrane protein; 5% skim milk for 1 hour; 4 ℃ incubated with primary antibody overnight; secondary antibody was incubated at room temperature for an hour; chemiluminescence film exposure and scanning.5 Ordinary PCRCompanies using Omega Omega Total RNA Kit I (Omega, GA, USA) Total RNA was extracted esophageal squamous cell carcinoma; spectrophotometer and agarose gel electrophoresis of RNA concentration and purity, quality control testing; use PrimeScript TM RT Reagent Kit with a gDNA Eraser (Takara Bio Inc, Japan) reverse transcription; cDNA encodes reverse finished mixed with other agents, to configure the PCR reaction solution, and placed in a thermal cycler, PCR reaction according to the corresponding heating; finally heating the product subjected to agarose gel electrophoresis.6 Quantitative PCRApplication of the company Omega Total RNA Kit I (Omega, GA, USA) Total RNA was extracted esophageal squamous cell carcinoma; spectrophotometer and agarose gel electrophoresis of RNA concentration and purity, quality control testing; use PrimeScript TM RT Reagent Kit with a gDNA Eraser (Takara Bio Inc, Japan) was reverse transcribed, followed by SYBR Premix Ex Taq (Takara Bio Inc, Japan) for real-time quantitative, GAPDH as an internal control in this experiment. Specific experimental process should refer to the corresponding kit instructions.7 CCK8Nippon colleagues in the company CCK8 kit to detect cell proliferation, Specific experimental process should refer to the corresponding kit instructions.8 Flow cytometry detection cycle and apoptosisKGI use China’s kit to detect the cell cycle and apoptosis state. Specific experimental process should refer to the corresponding kit instructions.9 ImmunohistochemistryConventional dewaxing hydration; heating; cooling to room temperature; 3% H2O2 at room temperature processing 10min; goat serum closed at room temperature; antibody 4 ℃ overnight; secondary antibodies were incubated at room temperature; DAB color; hematoxylin; 1% hydrochloric acid alcohol differentiation; anti-washing blue; dry mounted.10 Methylation and methylated PCRUsing the US Omega company’s Tissue DNA Kit, we extracted DNA from seven cells. Then DNA concentration was measured by spectrophotometer, the application Epigentek’s BisulFlash DNA Modification Kit methylation modification, using China’s Takara TaKaRa Taq Hot Start Version dosing and treatment, agarose gel electrophoresis EPB41L3 methylation status in the cells. Protocols of each step should refer to the corresponding kit instructions.11 PyrosequencingExtraction, purification and modification of seven cellular DNA, methylation PCR, followed by reaction in the United States QIAGEN’s Pyrosequencing detector, with Pyro Q-CpG software automatically analyzing the methylation status of each site.12 ZymographyUse Chinese Rydell’s kit MMP Zymography Assay Kit to detect cells mmp2 and mmp9 activity. Detailed protocol should refer to the corresponding kit instructions.13 TumorigenesisUsing commercially available 4-5 week old male nude mice from Southern Medical University, four as a group, were inoculated subcutaneously in the back of stable overexpression lentivirus KYSE150 or interfere EPB41L3 of the shRNA KYSE450 esophageal squamous cell suspension per week measured once the tumor volume is calculated as 1/2** long diameter short diameter 2.14 StatisticsDifferences between the two groups compare with independent samples t test (Independent-samples t test), and above three groups were compared using single factor analysis of variance (One-Way ANOVA). The relationship between EPB41L3 and clinical pathological features of esophageal squamous cell cancer applies X2 test. Survival curves use the Kaplan-Meier method and log-rank. Univariate and multivariate analysis using the Cox proportional hazards regression model (Cox proportional hazards regression). Statistical significance was set at p<0.05. All experiments were performed at least three times. All data are represented as the mean ± SD. Statistical analysis was performed using SPSS version 13.0 software.Results1 EPB41L3 in esophageal squamous cell carcinoma cells and low expression and play a tumor suppressor role in esophageal squamous cell carcinoma(1) The expression of EPB41L3 in ESCC tissues and cells was lower than that in normal esophageal tissue and esophageal epithelial cells. Besides, the depth of tumor invasion, TNM stage and survival were negatively correlated with the expression of EPB41L3.In order to detect the expression of EPB41L3 in esophageal cancer, we first started from the esophageal tissue level. After performing immunohistochemistry on 97 pairs of paraffin-embedded esophageal squamous cell carcinoma and adjacent normal esophageal epithelium tissue microarray, we found in the cell membrane and cytoplasm in 91 cases of esophageal squamous cell carcinoma, EPB41L3 showed high expression. To the opposite, EPB41L3 showed low or no expression at 72 cases of esophageal carcinoma. Then it is detected at the cellular level. We found six strains of esophageal squamous cell carcinoma, the expression EPB41L3 were lower than normal esophageal epithelium. Then we performed IHC in the tissue microarray and analyzed pathological and survival data, finding that the expression level of EPB41L3 in esophageal squamous cell carcinoma patients wasn’t related with age, sex, type of tumor or lymph node status. However, it is significantly correlated with the depth of invasion and TNM stage which were negatively correlated (P=0.016). Moreover, survival time of patients is also negatively correlated with the expression of EPB41L3(P=0.002).(2) EPB41L3 plays a tumor suppressor role in esophageal squamous cell carcinomaOur experiments from proliferation, apoptosis, migration and invasion and cell cycle four aspects EPB41L3 ESCC tumor suppressor function. First, we make EPB41L3 by transfection in esophageal squamous cell KYSE150, KYSE510 and KYSE450 overexpression were CCK8 and colony formation assay, the results confirmed esophageal squamous carcinoma cell growth after transfection and colony formation was significantly inhibited. After in vitro tumor bearing by subcutaneous tumor-bearing mice, the results support our previous in vivo proliferation experiments. Then we use the method of flow cytometry to detect apoptosis were related research. The results showed that apoptosis rate EEPB41L3 over expression group was significantly higher. To detect cell migration and invasion capabilities, we scratches and Transwell experiment..Confirmed EPB41L3 inhibit esophageal squamous cell migration and invasion. Finally, we use the method of flow cytometry to detect found EPB41L3 can cause G2/M arrest.Mechanism 2 EPB41L3 low expression in esophageal squamous cell carcinoma tissues and cells and play a tumor suppressor functionThe downregulation (1) EPB41L3 methylation in esophageal squamous cell carcinomaFor normal esophageal epithelium Het-la and six esophageal squamous cell carcinoma (Eca109, Tel, KYSE150, KYSE450, KYSE510), we methylation PCR, this qualitative experiment EPB41L3 found in normal esophageal Het-la cells did not occur methylation, and in esophageal squamous cell carcinoma have occurred partially methylated. Then we conducted this quantitative Pyrosequencing. Detect CpG island located-141551,-141494,-141439,-33 methylation status sites 141379 bp at the results in six lines of Five EPB41L3 esophageal squamous cell carcinoma cell lines was shown as hypermethylation, while in normal esophageal cells unmethylated. And when the demethylating drugs after 5-Aza-2-deoxygcitidine (5-aza-2’-deoxycytidine) treatment of esophageal squamous cell re-PCR, RT-PCR and Western blot to detect the expression of EPB41L3 the results are shown EPB41L3 expression in esophageal squamous cell recovery. Comprehensive above experiments illustrate EPB41L3 occur in esophageal squamous cell methylation.(2) Epb4113 by PI3K/MAPK, Caspase3/8/9, and Cdc2/Cyclin B1 pathway regulates tumor suppressor role to playTo further investigate the role of tumor suppressor EPB41L3 play in ESCC mechanism, we conducted a series of western blot. After EPB41L3 treated transfected expression in esophageal squamous cell carcinoma, we found that MAPK pathway in proliferation-related classic two important nodes, P-erk expression and up-regulation of P-p38, no significant change; subsequently withered Caspase pathway in death, caspase8, expression caspase9 and caspase3 upregulated happen; we also examined the expression of cyclin five found expression Cdc2 (cdkl) and by inhibiting cyclin B1 expression CyclinA, CyclinD and CyclinE did not change; Finally, we examined the expression of MMP2 and MMP9, and the results of its expression was suppressed. The above experiments concluded:Epb4113 by PI3K/MAPK, Caspase3/8/9 and Cdc2/Cyclin B1 pathway regulates tumor suppressor role to play.Our findingsAlthough the above-mentioned findings can occur with the progression of esophageal squamous cell carcinoma EPB41L3 linked, but the specific situation EPB41L3 expression in esophageal squamous cell carcinoma, and whether to play a tumor suppressor role and the corresponding mechanism is not clear, the need for further the study. This study aims to explore the methylation and inhibition effect in which EPB41L3 plays and the mechanism about tumorigenesis of esophageal squamous cell carcinoma.Local progression and distant metastases are the main cause of death in patients with esophageal squamous cell carcinoma. Surgical and chemotherapy treatment, the five-year survival rate of patients with esophageal squamous cell carcinoma remains low. Studies have shown that a variety of molecular markers used to evaluate the prognosis of patients with esophageal squamous cell carcinomaConclusionsIn this study, we first revealed the appearance and mechanism that EPB41L3 is methylated in squamous cell carcinoma and plays as a tumor suppressor. We found the expression of EPB41L3 was decreased in esophageal squamous cell carcinoma, which was lower than the adjacent normal tissues. In esophageal squamous carcinoma cell, EPB41L3 was down-regulated by methylation, thereby inhibiting proliferation and invasion of esophageal squamous cell carcinoma cells, inducing apoptosis and G2 /M arrest. Further analysis also proved that MAPK, Caspase3/8/9, Cdc2/Cyclin B1 pathway involved in how EPB41L3 suppresses esophageal squamous cell carcinoma tumor. In addition, the analysis esophageal squamous cell carcinoma tissue microarray and its pathological parameters and prognosis also found out that the expression of EPB41L3 was correlated with tumor invasion, TNM staging and survival. These findings reveal the mechanism that EPB41L3 was downregulated in ESCC, and provides a theoretical basis for EPB41L3 playing a tumor suppressor role in ESCC. Inactivation of tumor suppressor genes is an important mechanism involved in tumorigenesis, and methylation is one of the reasons of tumor suppressor gene inactivation. The study may be providing the basis for squamous cell carcinoma tumor early diagnosis and monitoring, also providing a reference for individual diagnosis and treatment.