Screening And Interaction Of MAGEA6 Expression Regulatory Proteins In Esophageal Cancer

The melanoma antigen gene A family（MAGIA）is highly expressed in A variety of tumors and in male testis and is low or not expressed in normal cells.In recent years,this protein family has become a popular antigen in the research of antitumor drugs and immunotherapy.In the early stage,my research group found that MAGEA6 was generally highly expressed in esophageal cancer,while not expressed in normal tissues.Other studies have also confirmed that increased expression of MAGEA6 has been found in many tumors.At present,there are relatively few studies on the function and mechanism of MAGEA6 in the MAGEA family.In this study,the function of MAGEA6 and the proteins interacting with it were studied,and the gene MSMO1 closely related to it was screened out,and the interaction mechanism between it and MAGEA6 was further explored.This study will provide more data support for the prevention and control of tumors with high expression of MAGEA6.The research is carried out through the following parts:A stable Eca109 cell line with high expression of MAGEA6 has been constructed in the laboratory at the early stage,and transcriptome sequencing of this cell line has been performed to obtain a high-throughput sequencing data,in order to obtain genes closely related to the function of MAGEA6 when it is highly expressed.First of all,bioinformatics analysis was performed on the data.Sequencing data before and after high expression of MAGEA6 were evaluated and sorted,and low-quality data were filtered out.Clean reads with high quality were compared with the reference genome,and sequencing data that could not be directly aligned with the reference gene were taken as differential gene data.After screening,the data were input into the DAVID database to obtain GO enrichment analysis and KEGG pathway enrichment analysis data.The molecular functions and biological processes of these enrichment genes are closely related to RNA polymerase II.In the experiment,it was found that through high-throughput sequencing data,5 genes with significantly up-regulated expression and 8 genes with significantly down-regulated expression were obtained,which were significantly different from the expression of MAGEA6 upregulated expression.The data of MAGEA6 in STRING was downloaded and imported into Cytoscape（V3.5.1）for the construction of PPI protein network,and 13 core regulatory genes related to MAGEA6 were screened out.Through differential gene screening and the construction of protein interaction network,it was found that MSMO1 was both a significant differential gene and a core regulatory gene after the upregulation of MAGEA6,and MSMO1 was also related to the regulation of RNA polymerase II,so we speculated that the two were closely related.In order to clarify the interaction between MAGEA6 and MSMO1,Eca109 cells with high expression of MAGEA6（Eca109-MAGEA6-3.1）,control Eca109 cells（Eca109-3.1）and Eca109 cells with low expression of MAGEA6（Eca109-MAGEA6-si RNA）were compared,respectively.q RT-PCR,Western blot,cell migration,invasion and proliferation of CCK8 cells were used to analyze and study the results showed that the high expression of MAGEA6 in esophageal cancer cells could accelerate the growth rate,enhance the cell viability and enhance the invasion ability.The high expression of Eca109 cells（Eca109-MAGEA6-3.1）and knockdown of MSMO1 Eca109 cells（Eca109-MAGEA6-si MSMO1）were compared,and q RT-PCR,Western blot,cell migration,invasion and proliferation of CCK8 cells were found.Altering the levels of MSMO1 did not lead to significant differences in these levels.However,the expression of MSMO1 increased and decreased when MAGEA6 was overexpressed or knocked down,suggesting that MAGEA6 positively regulates the expression of MSMO1,and the two may synergically promote the canceration process of cells.In order to explore the functional complex proteins related to the regulation of MAGEA6 expression,Eca109 cells with high expression of MAGEA6 gene（carrying the V5 fusion gene tag）were constructed and screened.MAGEA6 interacting proteins were dragged by magnetic beads specifically attached to V5.By immunoprecipitation combined with mass spectrometry analysis,it was found that MSMO1 and MAGEA6 were indirectly interacting.In addition,three proteins L10 K,RS10L and CHTOP that may form functional complexes with MAGEA6 were found.In this study,the main functions of MAGEA6 were identified,and proteins related to the expression and regulation of MAGEA6 were screened and verified,and the gene MSMO1 that was most closely related to its function was found.Subsequently,it was proved that the function of MAGEA6 was positively regulated by MSMO1.Then it was determined that the main components of the complex formed when MAGEA6 functions were L10 K,RS10L and CHTOP,and MAGEA6 indirectly interacts with MSMO1 to mutually regulate its functions.Therefore,we believe that MAGEA6 may exert its functions by forming a complex with L10 K,RS10L and CHTOP,and indirectly regulate MSMO1 to affect RNA polymerase II and thus affect cell transcription.This study provides a new idea for cancer research on the basis of further exploring the function of MAGEA6.