| Objective: Breast cancer is one of the most common malignant tumors in the world,and also the "number one killer" of women’s health.According to the level of hormone receptor expression,breast cancer is clinically divided into four subtypes: lumen type A,lumen type B,human epidermal growth factor receptor 2(HER2)overexpression type and triple negative breast cancer(TNBC).Among them,TNBC accounts for 12-18% of all breast cancer patients.Its molecular characteristics are estrogen receptor(ER),progesterone receptor(PR)and HER2 receptor are negative.Compared with other types of breast cancer,TNBC is more aggressive in biological behavior,and has attracted much attention because of its rapid progress,easy recurrence and metastasis,and poor prognosis.At present,TNBC still lacks effective treatment methods,and problems such as chemotherapy resistance still need to be solved.Therefore,it is necessary to explore the pathogenesis of TNBC and provide new treatment strategies.With the rapid development of biotechnology in recent years,multiple evidence shows that long non coding RNA(lnc RNA)has a key role in the occurrence and development of breast cancer and other tumors.Lnc RNA is a class of transcriptional fragments with a length of > 200 nucleotides,usually located in the nucleus or cytoplasm,and has no protein-coding function.However,like the protein-coding genes,it plays the role of carcinogenic or tumor suppressor genes,regulating tumor metastasis and invasion,cell apoptosis and drug resistance.Lnc RNA affects gene function through epigenetic regulation and post-translational protein activity regulation,and has higher specificity,indicating that it is more suitable to be a molecular target for cancer diagnosis and treatment.Therefore,based on the RNA expression profile of the cancer genome atlas(TCGA)database,this study screened and identified the new lnc RNA related gene regulatory network of TNBC through multiple bioinformatics analysis such as weighted gene co-expression network analysis(WGCNA),and revealed its complex molecular mechanism in the process of occurrence,development and poor prognosis,providing basis for finding the best treatment strategy and prevention of TNBC.Methods: Part I:(1)Edge R package was used to analyze the difference between the RNA-seq data of clinical early and advanced TNBC cancer tissues compared with normal breast tissues in TCGA database;WGCNA analysis on differential gene expression profile was applied to obtained significant gene aggregation module.(2)The GO of DAVID software was used to conduct functional annotation and the KEGG of Cytoscape was used to perform pathway enrichment analysis on the genes in the gene coexpress modules,and to explore the biological process in which genes significantly participate.(3)Further survival analysis was carried out on the co-expression gene cluster in the most significant module,and the lnc RNA LINC00466 that affected the prognosis of TNBC was screened out.(4)The survival and nomogram packages of R language were used to analyze the prognosis and construct the nomogram model identifying the ability of LINC00466 predicting the prognosis of TNBC.(5)Gene set enrichment analysis(GSEA)was used to analyze the function enrichment according to the differential expression level of LINC00466.(6)The expression of LINC00466 in TNBC patient’s blood was verified by q PCR.(7)The LINC00466 stable knockdown cell line was constructed using the lentivirus plasmids.Cell proliferation(plate cloning,CCK-8 and Ed U experiments)and cell cycle(flow cytometry)were detected,and tumor growth was observed by nude mice tumorigenesis experiment.Part II:(1)Using bioinformatics methods such as transcription factor prediction and gene-protein interaction,LINC00466 was predicted to play biological roles by interacting with SRSF3 protein.(2)The lentivirus plasmids were used to construct LINC00466 stable over-expression TNBC cell line.The expression levels of FOXM1,which was the SRSF3 targeting protein,and its downstream molecules were detected by q PCR and Western blot.(3)On the basis of LINC00466 overexpression,SRSF3 was knocked down.And the expression of FOXM1 and its downstream target genes was detected by q PCR and Western blot.(4)Then the binding of LINC00466 and SRSF3 protein was verified by RNA immunoprecipitation(RIP)and RNA pull down experiments.(5)SRSF3 was knocked down in LINC00466 overexpression cells.And cell proliferation(plate cloning,CCK-8 and Ed U experiments)and cell cycle(flow cytometry)were detected,and tumor growth was observed by nude mice tumorigenesis experiment.(6)Micro RNAs(mi RNAs)that had potential binding sites with SRSF3 and LINC00466 were predicted by bioinformatics,and the dual luciferase reporter gene detection system was performed to verify the interaction.(7)The lentivirus plasmids were used to construct LINC00466 stable knockdown cell line,and mi RNA inhibitors were applied to observe the changes of cell proliferation,cell cycle,and tumor growth through the above methods.Results: Part I:(1)The data of 79 human TNBC tissues(63 early and 16 late)and 139 normal breast tissues in the TCGA database were selected for cross comparison.The results showed that there were 2136 differential genes between the early TNBC group and the control group,and 2141 differential genes between the late TNBC group and the control group.(2)For the differential genes in the early group and the late group,7 and10 co-expression modules were identified by WGCNA package analysis to be related to the clinical characteristics of TNBC,and the early blue and late blue-green modules were significantly related to the progression of TNBC.(3)The enrichment analysis of GO function and KEGG pathway showed that the biological process,biological function,cytological components and signal pathway of co-expression gene cluster in the significant module were enriched in cell cycle and microtubule movement.(4)The key node analysis of the first 50 genes in the prominent module was carried out using Cytoscape software.It was found that the most critical genes in the early blue module were KIF4 A,KIF20A,NUSAP1,BUB1,CCNB1 and CDCA8,and the most critical genes in the late blue-green module were TPX2,BUBIB,CDCA5,KIF2 C and DLGAP5.(5)The single-factor Cox proportional risk and Kaplan-Meier survival analysis of genes in the late significant module showed that there were 141 highly expressed hub genes significantly associated with TNBC poor prognosis,among which LINC00466 was the only lnc RNA that was up-regulated in TNBC and associated with poor prognosis.(6)The nomogram of the risk score of clinical stage,age,sex and LINC00466 expression level shows that LINC00466 expression has good independent prediction ability for the survival rate of patients with TNBC,indicating that the high expression of LINC00466 can be used as an independent predictor of poor prognosis of TNBC.(7)GSEA analysis showed that the high expression of LINC00466 was significantly correlated with cell cycle function.(8)The high expression of LINC00466 in TNBC tissue was detected by q PCR,and the knockdown of LINC00466 inhibited the proliferation of TNBC cells and tumor growth,resulting in cell cycle arrest in G2/M phase.Part II:(1)Using the correlation of gene expression in tissues and the prediction of the target binding sites of transcription factors,the LINC00466 related gene regulatory network was constructed with FOXM1 as the transcription factor and CCNB1,CDCA5 and CDCA8 as the target genes.(2)Cat RAPID results showed that the upstream regulatory factor SRSF3 of FOXM1 has a high interaction value with LINC00466 and contains recognition motifs.(3)Overexpression of LINC00466 increased the expression of FOXM1 and its downstream CCNB1,CDCA5 and CDCA8 genes.(4)Knockdown of SRSF3 could inhibit the expression of FOXM1 and its downstream genes.(5)RIP and RNA pull down results showed that LINC00466 could bind to SRSF3 protein.(6)Knockdown of SRSF3 could inhibit the proliferation and tumor growth of TNBC cells with overexpressed LINC00466,and led to cell cycle arrest in G2/M phase.(7)Micro RNAs that bind to LINC00466 and SRSF3 m RNA were screened by bioinformatics methods: mi R-29c-3p,mi R-29b-3p and mi R-802.(8)The administration of mi R-29c-3p,mi R-29b-3p and mi R-802 inhibitors can rescue the inhibition of LINC00466 on the proliferation and tumor growth of TNBC cells and the arrest of G2/M phase of cell cycle.Conclusion: The expression of long-chain non-coding RNA LINC00466 can be an independent predictor of the poor prognosis of TNBC.It is highly expressed in TNBC tissue and promotes the proliferation of TNBC cells and tumor growth.Abnormal expression of LINC00466 is associated with cell cycle disorder.LINC00466 up-regulates the expression of FOXM1-CCNB1/CDCA5/CDCA8 by directly binding with SRSF3 or competitive inhibition mechanism to disrupt the cell cycle of TNBC and promote the malignant progression of TNBC. |
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