Preliminary Study Of Construction And Key Molecules Of Non-coding RNA Regulatory Networks During Breast Cancer Progression

Breast cancer is one of the most common malignant tumors in women.It seriously threatens the physical and mental health of women.The incidence of breast cancer is more than twice in China than the global,and the incidence rate in young women is increasing,seriously affecting the lives and work of young women and women of childbearing age.With the improvement of medical,the treatment of breast cancer has become more abundant,the therapeutic effect has been improved.However,the prognosis of these patients with advanced and metastatic breast cancer is not good.In recent years,more and more non-coding RNA(ncRNAs)have been studied in human cancer,and their important regulatory role has been gradually recognized.In this study,stage-specific microRNAs and lncRNA were obtained from TCGA database,and the differentially expressed genes were analyzed by GO and pathway analysis.Then,the non-coding RNA regulatory network was constructed by expression trend analysis,bioinformatics analysis and negative correlation analysis.These key non-coding RNA may provide potential targets for early screening,disease progression monitoring and prognosis of breast cancer.Part 1 Identification of the stage-specific noncoding RNA and the lncRNA-miRNA-mRNA regulatory network construction in breast cancer progressionObjectives:We aimed to obtain differentially expressed miRNAs and lncRNAs in different stages and identify key noncoding RNA in the lncRNA-miRNA-mRNA interactions network based on The Cancer Genome Atlas(TCGA)data.Methods:Data were downloaded and integrated from TCGA database,and the patients were grouped according to clinical information(A.normal group,B.BRCA early group,C.BRCA middle group,D.BRCA late group).The data were extracted from miRNA-seq and RNA-seq.Compared with the adjacent normal tissues(n=84),differentially expressed microRNAs,lncRNA and mRNA of the early stage(n=104),middle stage(n=404)and late stage(n=192)were screened.Furthermore,non-coding RNA and its possible target genes were obtained by Series Test of Cluster analysis,bioinformatics analysis and negative correlation analysis to construct the lncRNA-microRNA-mRNA regulatory network.The key non-coding RNA and target genes in the network were further analyzed for overall survival,and the possible regulatory relationship was verified by transient transfection and real-time quantitative PCR.Results:Based on the above analysis,we found 148 differentially expressed miRNAs and 193 differentially expressed lncRNAs in early stage,166 differentially expressed miRNAs and 208 differentially expressed lncRNAs in middle stage,163 differentially expressed miRNAs and 207 differentially expressed lncRNAs in late stage.122 1ncRNA,67 miRNAs and 119 mRNAs were used to construct the lncRNA-microRNA-mRNA regulatory network.The results suggested that the miR-20b/93/106a/106b family was at the center of the regulatory network.The expression of miR-20b/93/106a/106b in breast cancer was higher than adjacent tissues in GSE57897,GSE44124,and GSE58606,which was consistent with our analysis results.Furthermore,4 key lncRNAs(MESTIT1,LOC100128164,LINC00667,and DNMBP-AS1)and 2 target genes(ATP1A2 and NR4A3)were positively correlated with overall survival(OS,log-rank p<0.05).The regulatory relationship between miR-93 and target gene ATP1A2 were validated by transient transfection and Quantitative real-time PCR in breast cancer cell line.Conclusions:Non-coding RNA is differentially expressed in early,middle and late stages of breast cancer.With the development of breast cancer,it shows a certain expression trend in different stages.The construction of a regulatory network of lncRNA-microNA-mRNA reveals that the miR-20b/93/106a/106b family may play an important role in the progression of breast cancer.Targeting these key non-coding RNAs may provide effective control of the progression of breast cancer and provide new therapeutic options.Part 2 Studies on the function of miR-93-5p in breast cancerObjective:To detect the effect of miR-93 on the biological behavior of breast cancer cells by overexpressing or knocking down miRNAs-93 through transfection.Methods:After overexpressing or knocking down microarray-93 in breast cancer MCF-7 cell line by liposome transfection,the proliferation of breast cancer MCF-7 cell was detected by CCK-8 analysis,the migration and invasion of breast cancer cell line MCF-7 were detected by Transwell analysis,and the apoptosis of MCF-7 cells were analyzed by flow cytometry.Results:MiR-93 could significantly promote the proliferation of MCF-7 cells,but not affect the apoptosis of MCF-7 cells.MiR-93 could significantly promote the invasion of breast cancer,but not affect the migration of MCF-7 cells.Conclusion:MiR-93 can significantly promote the biological malignant behaviors proliferation and invasive of breast cancer MCF-7 cells,and may participate in regulating the progression of breast cancer.