| BackgroundEpigenetics is usually defined as DNA sequence invariable,but gene expression has changed,and the change is heritable.It can be regulated by histone modification,noncoding RNA,chromatin remodeling and DNA methylation,so that network-based regulation of important DNA events in the nucleus can be realized at protein,chromatin,DNA and RNA levels.Abnormal epigenetic modification may lead to incorrect gene expression,metabolic disorder,disease and even tumor.Therefore,it is of great significance for clinical diagnosis,treatment and prevention to explore the epigenetic network related to tumor metastasis and understand its role in the occurrence and development of tumor.Ubiquitination of histone is one of the important contents of epigenetic modification.It can regulate gene transcription,DNA damage repair and cell cycle.Ubiquitination is a process in which the lysine site of a protein binds to the carboxylic terminus of a ubiquitin molecule.The ubiquitination of histone mainly occurs on H2A and H2B.Mysml is a histone H2A specific ubiquitinase,also known as 2a-dub,which was first discovered and identified by Zhu P in 2007.The common domain of its metalloenzyme family is the jamm domain,located at the N-terminal,with the hydrolysis activity of metalloproteinase,which can remove ubiquitin molecules.The molecular structure of mysml also determines that it can remove the ubiquitination of histone in the nucleus.In early studies,H2A deubiquitination was associated with prostate cancer.MYSM1 regulates androgen receptor-dependent gene activation by coordinating histone acetylation and deubiquitination,and destabilizes the association between connectome histone h1 and nucleosomes.When mysm1 was knocked down in colorectal cancer cells,the migration and invasion ability of cancer cells decreased significantly.The research shows that mysml can maintain the stability of genome and participate in the process of cell proliferation,which suggests that mysml may play an important role in tumor development.Mysml may play a role in tumor formation,but its mechanism in tumor metastasis has not been clearly reported.In order to find out the role and mechanism of melanoma with pulmonary metastasis,this project intends to verify the correlation between mysml and the lung metastasis of melanoma,to clarify its role in the lung metastasis of melanoma,to find and determine the target gene of mysm1,to explore the molecular mechanism involved in the regulation of mysml,and to provide potential targets and markers for the diagnosis,treatment and prognosis of melanoma lung metastasis.ObjectiveTo explore the biological role and mechanism of mysm1 in pulmonary metastasis of melanoma.Methods1.Pan-cancer analyses for mean expression levels of MYSM1 in different types of cancers;2.The expression of mysml in lung cancer cells was cut down by RNAi technology,and the expression level of mysml was identified by qRT-PCR and Western blot;3.Detection of the effect of down-regulation of mysml on the formation of melanoma cell clone by plate cloning experiment;4.The effect of down-regulation of mysml on the cell cycle and apoptosis of melanoma cells was analyzed by flow cytometry;5.The effect of down-regulation of mysm1 on the migration of melanoma cells was detected by scratch test and transwell;6.The animal model of C57BL/6 mice was established by injecting B16F10 cells into tail vein,so as to detect the effect of down regulating mysml on lung tumor metastasis 7.Western blot was used to detect the effect of down-regulated mysml on MMPs protein,and its effect on key molecules of related signaling pathway 8.Immunohistochemistry was used to detect the expression of MMP9 in the lung of mice after mysml knockout 9.Co IP method was used to confirm the interaction between mysml and p38.Results1.In order to explore the expression of mysm1 in human tumor,we use gepia bioinformatics to mine the data of TCGA based on Web.We found that in ACC(adrenocortical carcinoma),Cesc(cervical squamous cell carcinoma and cervical adenocarcinoma),coad(colon carcinoma),luad(lung adenocarcinoma),lusc(lung squamous cell carcinoma),OV(ovarian serous cystadenocarcin oma),read(rectal adenocarcinoma),the mRNA level of mysml was down regulated.In addition,the expression of mysml in dlbc and thymus was higher than that in normal tissues.The expression of mysm1 in melanoma cells was higher than that in normal tissues.2.Establishment of stable and low expression cell line and control cell line of mysml.Mysm1 interference lentivirus was used to infect melanoma cells.Puromycin was used to screen the cells for one week.RNA and protein were extracted from the cells after screening.The mRNA level of mysml was detected by Q RT-PCR,and the protein expression level of mysm1 was detected by Western blot.The results showed.that compared with the control group,the interference lentivirus could significantly down regulate the mRNA level of mysm1 and protein expression level.3.Mysml can significantly inhibit the migration and invasion of melanoma cells.A series of in vitro and in vivo functional experiments were carried out by using interference lentivirus to down regulate the expression of mysml in melanoma cells.The results showed that:plate cloning experiment confirmed that down regulating mysml can significantly inhibit the formation of melanoma cells;scratch experiment,Transwell,cell invasion experiment confirmed that down regulating mysml It can significantly inhibit the cell migration and invasion of melanoma cells;the lung metastasis experiment of melanoma shows that down regulating mysml can significantly inhibit the lung metastasis of melanoma cells;the flow cytometry detection of cell apoptosis and cycle experiment shows that down regulating mysml has no significant effect on the apoptosis and cell cycle of melanoma cells4.After the drop of mysml,the invasion and migration of cells were inhibited.We detected the MMPs family related to migration and found that the expression of MMP9 decreased.We also detected the related proteins in MMP9 signaling pathway,and found that p38 phosphorylation was activated and ERK phosphorylation was inhibited.5.Immunoprecipitation(co-IP)experiment confirmed that there was interaction between mysml and p38.We speculated that down regulating the phosphorylation of p38 activated by mysm1 could down regulate the expression of MMP9 through ERK dependent pathway,so as to inhibit the migration and invasion of melanoma cells.ConclusionAs an important epigenetic molecule,mysml plays an important role in the process of lung metastasis of melanoma.After knockdown mysml,the invasion and metastasis of melanoma cells are inhibited,and the lung metastasis of mouse melanoma is significantly reduced.After the removal of mysml,the expression of MMP9 decreased,the phosphorylation of p38 was activated,and the phosphorylation of ERK was inhibited.We speculate that the role of mysm1 in melanoma may depend on its down-regulation,which can activate p38 phosphorylation and down regulate the expression of MMP9 through ERK dependent pathway,so as to inhibit the migration and invasion of melanoma cells. |
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