Effects Of TP53 Mutations At Different Sites On The Biological Characteristics Of Breast Cancer

Objective:Breast cancer has become the most prevalent cancer among women in recent years,and the number of deaths is increasing year by year,posing a serious threat to women’s health.TP53 is a widely studied tumor suppressor gene,and its encoded protein TP53 plays an important role in cells.TP53 gene mutations are one of the key factors leading to the development and progression of different types of tumors,including breast cancer.In this paper,we compare the effects of TP53 mutations at different sites on the important functions of breast cancer and analyze their mechanisms in order to improve the understanding of TP53 mutations at different sites and to provide potential novel clinical treatment strategies for TP53 mutated breast cancer.Methods:1)Bioinformatics analysis of genomic and transcriptome characteristics of TP53 mutations of different breast cancer subtypes:Download TCGA(the The Cancer Genome Atlas)breast cancer genome and expression profile data,identify differential genes and signaling pathways associated with TP53 mutations of different breast cancer subtypes by differential expression analysis and functional enrichment,and calculate immunity and m~6A scores.2)Construction of TP53 stable cell lines with different mutation sites:TP53 plasmids with different mutation sites were constructed by designing primers,PCR amplification,enzymatic cleavage,ligation and transformation process,using 293T cells packed with lentivirus and infected with breast cancer cell line MCF-7.3)The effect of TP53 mutated at different sites on the function of breast cancer cells: for the TP53 stably transformed cell lines with different mutations sites,the cell proliferation ability was investigated by CCK-8 assay and clone formation assay, the cell cycle and apoptosis level were detected by flow cytometry,the two- dimensional and three-dimensional migration ability was detected by cell scoring assay and Transwell assay,respectively,Western blot assay was used to detect the expression level of related proteins,and q PCR assay was used to detect the m RNA transcription level of related genes.4)The effect of TP53 mutation on m~6A modification in breast cancer:The TP53 mutation-related m~6A analysis of breast cancer samples was performed by ss GSEA method,and the changes of m~6A modification in cells were detected by dot blot assay,and the changes of related gene expression were detected by Western blot and q PCR assay.5)The effect of TP53 mutated at different sites on the tumorigenic ability of breast cancer:4-5 weeks immunodeficient nude mice were used for nude mice tumor loading experiments,and the weight status and tumor size of the mice were recorded to observe the difference in tumorigenic ability of nude mice caused by mutated TP53 at different sites,and tumor volume histograms were plotted.Results1)The results showed that TP53 mutation had an important effect on breast cancer: TCGA breast cancer samples were grouped into TP53 wild type and mutant according to TP53 mutation status,and differential expression analysis and KEGG signaling pathway analysis were used,and the results showed that TP53 mutation group was significantly enriched in immune signaling pathways such as antigen presentation and natural killer cytotoxicity.In addition,by grouping different subtypes and TP53 mutations,it was shown that the m6A score in the Basal-like subtype was significantly increased compared to other molecular subtypes.In addition,the expression levels of m6A methyltransferase molecules METTL3 and METTL14 decreased after TP53 mutation,while the expression levels of RBM15 and WTAP increased.2)Successful construction of TP53 stable-transformed cell lines with different mutation sites:firstly,PCR amplification was performed after extracting cell genomic DNA,and PCR products were sequenced,thus wild-type TP53 breast cancer cell line MCF-7 was selected as the experimental cell line.Then five breast cancer TP53 hotspot mutations in TCGA database were screened:R175H,R248Q, R248W,R273C and R273H,and TP53 hotspot mutations MCF-7 stable cell lines were constructed and experimented.3)TP53 mutated at different sites was found to exert oncogenic effects in breast cancer cells in different ways:R248Q,R248W,R273C and R273H TP53 mutations significantly promoted breast cancer cell proliferation and cycle progression;R248W and R273C TP53 mutations significantly inhibited breast cancer cell apoptosis;R175H TP53 mutation significantly promoted breast cancer cell migration.TP53 mutations at different sites affect the immune microenvironment of breast cancer,enhance cellular immunosuppression and promote immune escape of tumor cells.4)TP53 mutation affects m~6A modification in breast cancer cells:TP53 mutation increases the level of m~6A modification in breast cancer cells and affects the expression of m~6A modification-related genes.The expression levels of m~6A methyltransferase molecules METTL3 and METTL14 decreased and the expression levels of RBM15 and WTAP increased in breast cancer cells after TP53 mutation.5)TP53 mutation promoted breast cancer tumorigenesis in vivo:the tumorigenic ability of breast cancer in vivo was enhanced and the tumor size became larger after TP53 mutations.Conclusions:TP53 mutated at different sites exerted oncogenic effects in breast cancer in different ways,promoting proliferation and migration of breast cancer cells,inhibiting apoptosis and immune level of breast cancer cells,and promoting tumorigenic ability of breast cancer in vivo.TP53 mutation increased the level of m~6A modification in breast cancer cells and affected the expression level of m~6A-related genes.