Mechanism Of FOXP3 Transcriptional Regulation Of RSPO3 In Radiation Resistance Of Non-small Cell Lung Cancer

Lung cancer is the most common malignancy and the leading cause of death in our country.Non-small cell lung cancer(NSCLC)accounts for 80 to 85 percent of all lung cancer cases.Radiotherapy is an indispensable part of comprehensive treatment of NSCLC.However,in clinical treatment,some patients may show radiation resistance,leading to local recurrence of lung cancer and even the failure of radiotherapy.Therefore,exploring the radiation resistance mechanism of NSCLC and improving the radiotherapy sensitivity of lung cancer will have an important impact on the clinical treatment of lung cancer.Our research group established X-ray resistant lung cancer cell lines in the early stage and screened differentially expressed genes by transcriptome sequencing.A total of 207 upregulated genes and 137 down-regulated genes were identified.Among them,RSPO3,a member of the RSPOs family,was significantly down-regulated in X-ray resistant strains,which attracted our research attention.Members of the RSPOs family share a similar structure and are widely involved in regulating a variety of biological functions,including angiogenesis,bone growth,and the development of the reproductive,respiratory,and digestive systems.In addition,RSPO3 has been found to be involved in the development of various malignant tumors including lung cancer through the Wnt/β-catenin signaling pathway.The Wnt pathway is involved in the regulation of radiation resistance by regulating cell cycle,DNA damage repair,cell apoptosis and other processes.At present,the mechanism of RSPO3’s involvement in the regulation of cellular radiation resistance is still poorly understood.Previous studies have confirmed that the expression of RSPO3 is down-regulated in Xray-resistant lung cancer cell lines.In order to explore the molecular mechanism of its abnormal expression,we analyzed its regulatory mechanism at the transcriptional level.Firstly,bioinformatics was used to predict the upstream transcription factors that can regulate RSPO3.Then the expression correlation,q-PCR and dual luciferase reporter assay confirmed that the transcription factor FOXP3 can regulate the expression of RSPO3.FOXP3,a member of the Forks family of transcription factors,is a major regulator of regulatory T cell(Treg)development and function.Abnormal expression of FOXP3 may lead to autoimmune diseases in humans.FOXP3 is expressed in a variety of malignant tumors,but its expression and function are different.FOXP3 plays two distinct roles in promoting and suppressing cancer in different tumors.As a transcription factor,FOXP3’s regulatory effect on tumorigenesis has been intensively studied,while the mechanism of radiation resistance in tumor cells has been less studied.Taking RSPO3 and the radiation resistance of NSCLC as the starting point,this study explored the upstream transcriptional regulation mechanism,and studied the mechanism related to the predicted transcription factor FOXP3 and the radiation resistance of NSCLC,so as to provide certain theoretical basis for radiotherapy of NSCLC.The main research results of this paper are as follows:(1)Bioinformatics was used to analyze the upstream transcriptional regulation mechanism of RSPO3.The biological function and clinical correlation of candidate transcription factors were analyzed.(2)mRNA and protein levels verified the expression correlation between FOXP3 and RSPO3.According to the predicted binding sites,site mutations were designed,and the transcriptional regulation and binding sites of FOXP3 and RSPO3 were verified by double luciferase reporter assay.(3)Cell functional experiments confirmed FOXP3’s involvement in the regulation of radiation resistance in NSCLC through RSPO3.