The Study On The Role And Mechanism Of MPDZ Gene In Human Cancer

Background:For a long time,the high morbidity and mortality of malignant tumors is a serious threat to human life and health.An increasing number of research has confirmed that genetic and epigenetic changes of tumor suppressor gene play an important role in the expression regulation and the occurrence of the tumor.It is closely related to the copy number variation(CNV)and(or)promoter CpG island methylation of tumor-suppressor gene.Therefore,exploring the mechanism of gene copy number variations and DNA methylation changes in the process of tumorigenesis is a new way for early clinical diagnosis and targeted therapy of malignant tumors.MPDZ,a new gene regulated by DNA methylation,was found in our early study of lung cancer.However,its role and mechanism in lung cancer and other tumors are not clear.Therefore,this study proposes to analyze the characteristic of MPDZ gene in human tumors,evaluate its preliminary clinical significance,and confirm the function and mechanism using experiment.This study will provide effective and reliable information for subsequent biological significance and effective treatment of the tumor in further study.Objective:1.To clarify the occurrence and major forms of the mutation of MPDZ in human tumors and analyze the influence of mutations on patients' survival and prognosis.2.To clarify the methylation status of MPDZ in lung cancer tumor tissues,sputum and blood plasma and evaluate the diagnosis value of MPDZ methylation in patients with lung cancer.3.To confirm the role and function of MPDZ on cell proliferation,cell cycle and apoptosis,invasion and metastasis,and clarify the signaling pathways of MPDZ in cancers.Methods:1.The COSMIC and cBioPortal online software were used to analyze the expression of MPDZ in normal tissue,tumor tissue and tumor cells,the occurrence and main form of MPDZ gene mutation in human cancers,and its influence on the prognosis of patients with tumors.2.The TCGA database were used to analysis the copy number variations and expression of MPDZ in hepatocellular carcinoma,renal clear cell carcinoma and lung cancer and the relationship between copy number variations and expression,then evaluate the impact of copy number variation and gene expression on the prognosis of patients with tumors.3.MSP was used to detect the methylation status of MPDZ lung cancer tissue sputum and plasma.TCGA database was to evaluate the potential biological value in lung cancer diagnosis by means of the detection of MPDZ methylation.4.The qRT-PCR and WB were used to detect the expression of MPDZ in lung cancer cell lines and the bronchial epithelial cells.At the same time,immunohistochemistry was used to analyze the protein expression of MPDZ in carcinoma tissue and adjacent tissue.The Kaplan Meier method was used to analyze the survival of patients with lung cancer in different groups of MPDZ protein expression.5.MTS were used to analyze cell proliferation.Flow cytometry instrument was used to analyze cell cycle and apoptosis.Scratched and transwell experiment were used to evaluate the invasion and metastasis.Nude mice were used to estimate the capability of tumor growth,invasion and metastasis in vivo.All above cell model and animal model were used to analyze the functions of MPDZ in tumors.6.The qRT-PCR,WB,gene transfection,RNAi technology and a variety of molecular biology technology such as immunofluorescence were used to study the molecular mechanism and determine the main signaling pathways that MPDZ had in human cancers.Results:1.The expression of MPDZ in normal tissues,tumor tissue and tumor cells are significantly different,which is low expression in blood system and high expression in the nervous system.Most of human cancer has mutations of MPDZ with the highest proportion of DLBC(16%),but the survival time was not significant difference between the patients with mutation and patients without mutation.2.Copy number variations of MPDZ were found in liver cancer and renal clear cell carcinoma,which the mainly form is deletion.Copy number variations of MPDZ association with its expression.Patients with MPDZ copy number deletion have significantly shorter survival time than that patients with normal copy number in renal clear cell carcinoma.3.Obviously,MPDZ is hypermethylated in lung cancer,which the detection rate of tumor tissue,sputum and plasma is 69.4%,46.5% and 76.7%,respectively.There are three CG sites in MPDZ gene promoter CpG island that is cg14655855,cg06202228,cg10899099.The methylation levels of three CG in lung cancer tissues are significantly higher than that in adjacent carcinoma tissues.ROC curves showed that the methylation level of three CG and their average is a sensitive and specific for clinical diagnosis of lung cancer with the AUC 0.80~ 0.84,95% CI 0.74 ~ 0.88.4.The expression of MPDZ in lung cancer tissues and most of lung cancer cell line is lower than that in bronchial epithelial cells,and is regulated by DNA methylation.Patients with high MPDZ expression have longer survival time than patients with low MPDZ expression.The MPDZ protein expression is an independent prognostic factor in lung cancer.5.High MPDZ expression can inhibit lung cancer cell invasion,metastasis and proliferation by promote cell apoptosis,but has no impact on cell cycle.Knockdown MPDZ expression can promote lung cancer cell proliferation,invasion and metastasis,the tumor growth in vivo and distant metastases to liver.6.MPDZ play a tumor suppressor role that is dependent on the Hippo-YAP pathway.MPDZ protein interacted with most proteins that in hippo pathway.High MPDZ expression inhibits the expression of YAP and its downstream target molecular CTGF and CYR61,while knockdown MPDZ expression can increase the expression of YAP and its downstream target molecular CTGF and CYR61.Moreover,knockdown MPDZ expression increases the amount protein of YAP.Conclusion:There were mutations of MPDZ in most human cancers,but the mutation rate was so low that it did not affect the prognosis of patients.In liver cancer and renal clear cell carcinoma,the expression of MPDZ associated with deletion,which had an influence on patient's prognosis.MPDZ was hypermethylated in lung cancer and the detection of methylation level is a potential biomarker for lung cancer diagnosis.MPDZ was downregulation in lung cancer tissue and cell line and associated with patients' survival.The expression of MPDZ is an independent prognostic factor in patients with lung cancer.MPDZ inhibited cell proliferation,invasion and metastasis,tumor growth in vivo and distance metastasis that depend on the Hippo-YAP pathway.