Epigenetic Study Of Early Breast Cancer(EBC) Based On DNA Methylation And Gene Integration Analysis

Breast cancer has become the most common malignant tumor worldwide and is the second leading cause of cancer death in women,after lung cancer.The molecular mechanism of breast carcinogenesis is complex,which is accompanied by changes in mRNA,lncRNA and DNA methylation.In this study,transcriptome sequencing of cancer tissues and adjacent tissues of breast cancer patients was carried out,the information was obtained from the database and analyzed,and molecular biological verification was carried out in early breast cancer patients.The aim was to identify key molecular markers related to the diagnosis and prognosis of early breast cancer（EBC）.Part One Bioinformatics analysis of DNA methylation and gene data-base integration in early breast cancer and its epigenetic characteristicsObjective:Through high-throughput sequencing technology,high-throughput data of early breast cancer in TCGA database were mined to identify key molecular markers related to diagnosis and prognosis of early breast cancer（EBC）.Methods:Breast cancer-related mRNA,lncRNA,and DNA methylation data were downloaded from the Cancer Genome Atlas（TCGA）database for identification of differentially expressed mRNAs（DEmRNAs）,differentially expressed lncRNAs（DelnRNAs）,and DNA methylation analysis.The biological functions of differentially expressed mRNA and the signaling pathways involved were identified by GO enrichment analysis and KEGG pathway analysis.Correlation analysis was performed between DNA methylation and differentially expressed mRNAs,and diagnostic and prognostic analyses were performed for identified differentially expressed mRNA and differentially expressed lncRNAs.Finally,the methylation status of identified differentially expressed mRNAs was verified using the GSE32393 dataset.Results:1633 differentially expressed mRNAs,750 differentially expressed lncRNAs,and 8042 differentially methylated sites were obtained by bioassay.The biological functions of differentially expressed mRNA were identified by GO enrichment and KEGG pathway analysis,and most DEmRNA were found to be involved in cell activity,cell cycle,transcription,etc.In the Venn analysis,we identified 11 key genes（ALDH1L1,SPTBN1,MRGPRF,CAV2,HSPB6,PITX1,WDR86,PENK,CACNA1H,ALDH1A2and MME）.The ROC curve was used to analyze the diagnostic value of 11differentially expressed mRNA.ALDH1A2（hypermethylated）,ALDH1L1（hypermethylated）,HSPB6（hypermethylated）,MME（hypermethylated）,MRGPRF（hypermethylated）,PENK（hypermethylated）,PITX1（hypmethylated）,SPTBN1（hypermethylated）were found.WDR86（hypermethylation）and CAV2（hypermethylation）may be considered as potential diagnostic gene biomarkers for early breast cancer.Summary:1.1633 differentially expressed mRNAs,750 differentially expressed lncRNA and 8042 differentially methylated sites were obtained by bioinformatics studies,and most DEmRNA were found to be involved in cell activity,cell cycle,transcription,etc.2.In the Venn analysis,we identified 11 key genes（ALDH1L1,SPTBN1,MRGPRF,CAV2,HSPB6,PITX1,WDR86,PENK,CACNA1H,ALDH1A2and MME）.Besides CACNA1H,it was found that the remaining 10 genes may be considered as potential diagnostic gene biomarkers for early breast cancer.Part Two Validation of differential genes obtained by DNA methylation and gene integration analysis in clinical samplesObjective:To detect and verify differentially expressed genes in clinical samples using RT-PCR techniques.Methods:Six tissue samples of early breast cancer（disease group）and adjacent tissue samples（control group）were obtained for RT-PCR.TotalRNA was extracted using the TRIzol kit,mRNA was reverse transcribed using the Fast King c DNA first-strand synthesis kit;then the Super Real Pre Mix Plus kit was used for real-time quantitative fluorescent PCR to determine the differential expression changes of the genes,and glyceraldehyde-3-phosphate dehydrogenase（GAPDH）and actin beta（ACTB）were used as internal references.Relative gene expression levels were calculated using the 2^-ΔCtmethod.Results:Compared with the adjacent tissues,the expression of CAV2,MME,FHL1,CAV1,LINC01537 and TRHDE-AS1 were down-regulated,the mRNA expression of CACNA1H was down-regulated to the 0.27 of the adjacent tissues,the mRNA expression of CAV2 was down-regulated to the0.18 of the adjacent tissues,and the mRNA expression of FHL1 was down-regulated to the 0.09 of the adjacent tissues.The expression of CAV1mRNA was down-regulated to the 0.57 of the adjacent tissues,the expression of LINC01537 was down-regulated to the 0.37 of the adjacent tissues,the expression of TRHDE-AS1 mRNA was down-regulated to the 0.40 of the adjacent tissues,the expression of LINC01614 and FOXD-3-AS1 was up-regulated to the 13.51 of the adjacent tissues,and the expression of FOXD-3-AS1 mRNA was down-regulated to the 3.59 of the adjacent tissues.Significant differences were found in the expression of all genes except CAV1and TRHDE-AS1（P<0.05）Summary:The expression of CAV2,MME,FHL1,CAV1,LINC01537and TRHDE-AS1 was down-regulated in early breast cancer tissues compared with adjacent tissues,and LINC01614 and FOXD-3-AS1 were expressed in early breast cancer tissues,which are consistent with the results of bioinformatics analysis,while CACNA1H and ENSG0000261294 were contrary to the results of bioinformatics analysis.Part Three Verification of the mechanism of AC093110.1 in early breast cancer at cytological levelObjective:To investigate the potential biological function of AC0931-10.1 in breast cancer at the cellular level and the effect of overexpression of AC093110.1 on the expression of SPTBN1 protein.Methods:AC093110.1 was overexpressed in human early breast cancer cell line MCF-7.The expression level of AC093110.1 was determined by RT-PCR,and then the effect of AC093110.1 on cell proliferation was analyzed by MTT assay,and the effect of AC093110.1 on cell migration was analyzed by cell scratch assay.The effect of AC093110.1 on SPTBN1 protein expression was determined by Western Blot assay.Results:1.Compared with normal cells,the gene expression of AC093110.1overexpressing cells increased,indicating that AC093110.1 overexpression was successful.The proliferation and migration of MCF-7 cells were detected by MTT assay and scratch test,and the proliferation rate of overexpressing cells was significantly reduced compared with normal cells（P<0.05）.The migration of AC093110.1 overexpressing cells decreased significantly at 24h and 48h（P<0.05）,indicating that overexpression of AC093110.1 inhibited cell migration.2.Western Blot results showed that the expression level of SPTBN1protein increased significantly after overexpression of AC093110.1,which indicated that AC093110.1 may play an important role in the proliferation and migration of early breast cancer cells by regulating SPTBN1.Summary:Overexpression of AC093110.1 inhibited the proliferation and migration of MCF-7 cell lines.AC093110.1 regulates SPTBN1 expression and plays an important role in tumor cell proliferation and migration.Conclusions:1.Through bioinformatics,potential biomarkers that can be used for early breast cancer diagnosis were identified,such as ALDH1A2,ALDH1L1,HSPB6,MME,MRGPRF,PENK,PITX1,SPTBN1,WDR86 and CAV2.2.RT-PCR techniques were used to verify the differential genes CACNA1H,CAV2,MME,FHL1,CAV1,LINC01537,TRHDE-AS1,LINC01614,FOXD3-AS1 and AC120498 in early breast cancer tissue samples and adjacent tissue samples.The results were basically consistent with the results of bioinformatics analysis.3.AC093110.1 can regulate the expression of SPTBN1 and play an important role in tumor cell proliferation and migration,providing clues for elucidating the regulatory mechanism of early breast cancer.