Construction Of LncRNA Regulation Network And Studies Of Functional Role And Regulatory Mechanism Of DGUOK-AS1 In Breast Cancer

BackgroundCancer is a major public health concern,which has brought huge and heavy social burden to mankind.Among female malignant tumors,the incidence of breast cancer is about 30%,ranking first,and the mortality rate also ranks second.Similarly,the incidence of breast cancer in China is as high as 17.07%,which also ranks the first and shows a younger trend Although the overall survival rate and prognosis of breast cancer patients have improved in recent years,progressive metastasis is still the most important cause of death for breast cancer patients,which can reduce the overall survival rate of breast cancer patients from more than 90%to less than 30%.Therefore,revealing the new mechanism of breast cancer metastasis and looking for new therapeutic targets play an important role in improving the prognosis of breast cancer patients.Studies have shown that protein-coding genes make up only 3%of the genome,and the remaining 97%are "dark regions" with molecular functions that can be transcribed into various RNAs.Most of the RNAs do not have the ability to code for proteins,which are called non-coding RNAs(ncRNAs).Among them,the long non-coding RNA(1ncRNA)is one of the transcripts,which has got much attention,could regulate the expression of oncogenes or tumor suppressor genes and functions through a variety of mechanisms to play an important role in the process of cancer development and progression.For example,lncRNAs could regulate the stability of proteins or mRNAs,modulate transcription,form nuclear scaffold or mediate the interaction between proteins and DNAs.LncRNA BCYRN1 can competitively bind to miR-619-5p to regulate the expression of CUEDC2,activate the PTEN/AKT/P21 pathway,and thus inhibit the occurrence and development of glioma.LINC00319 can be induced by the increased expression of CCL18,thereby promoting proliferation and metastasis of oral squamous cell carcinoma through miR-199A-5p/FZD4 pathway.LncRNA MALAT1 can bind with and inactivate TEAD,prevent TEAD from binding to YAP,and thus inhibit the progression and metastasis of breast cancer.LncRNA LINC-A could promote glycolysis and tumorigenesis in triple negative breast cancer by activating the HIF-1 signaling pathway.Therefore,from the perspective of lncRNA,new mechanisms mediated the progression and metastasis of breast cancer would be elucidated.The deeper research about lncRNAs would provide new directions and theoretical basis for the diagnosis and prognosis prediction of breast cancer,which has important clinical application significance and social value.Various studies have shown that lncRNAs could regulate the biological function or expression of miRNA as competitive endogenous RNAs(ceRNAs).For example,LINC00963 could act as a ceRNA of miR-324-3p to regulate tumor development and radiotherapy in breast cancer.NONHSAT101069 could function as a ceRNA of miR-129-5p to reduce the inhibition on Twistl expression,thus promoting the resistance to epirubicin,migration and invasion of breast cancer cells.Although recent studies have revealed various regulatory mechanisms of lncRNAs,the fact that one single lncRNA could absorb multiple miRNAs and one single miRNA could regulate multiple target genes,constructed a more complex regulatory network of lncRNAs.Therefore,further integrated analysis is needed to find the key regulatory pathway of lncRNAs.In recent years,bioinformatics related researches have developed rapidly.Massive data have been obtained through high-throughput sequencing technology.Analyzing and processing the data through different analysis softwares and databases,researchers could screen out the key regulatory factors from the numorous data.In this study,the expressions of lncRNAs,miRNAs and mRNAs obtained from GEO and TCGA databases were further analyzed.Combining with bioinformatics analysis results,database prediction,and expression detection in cells and tissues,we constructed a breast cancer-related IncRNA regulatory network and DGUOK-ASl(lncRNA deoxyguanosine kinase antisense RNA 1)was further selected out as the key lncRNA.Then,in vitro experiments and in vivo experiments were used to further investigate the specific function and molecular regulatory mechanism of DGUOK-AS1 in the progression and metastasis of breast cancer.Part ⅠConstruction of lncRNA regulation network in breast cancerObjective1.Explore the differential expression of lncRNAs,miRNAs and mRNAs in normal breast tissues and breast cancer tissues.2.Construct the lncRNA regulation network and screen the key lncRNA associated with breast cancer progression and metastasis.3.Investigate the expression of key lncRNA(DGUOK-AS1)in breast cancer tissues and breast cancer cells.4.Reveal the association between the expression of key lncRNA(DGUOK-AS1)and the prognosis of patients with breast cancer.5.Construct a prognostic model for breast cancer and evaluate its clinical application value.MethodsThe expression profiles of lncRNAs,miRNAs and mRNAs in normal breast tissues and breast cancer tissues were got from GEO and TCGA public databases.The differentially expressed lncRNAs,miRNAs and mRNAs were screened using limma data packet or edgeR data packet of R software.WGCNA analysis was used to further explore the correlation between differential expressed lncRNAs and breast cancer.The regulatory network of lncRNA-miRNA-mRNA was constructed based on Diana-lncbase V2,miRTarBase,Starbase,TargetScan and miRDB databases,and Cytoscape software was used for the visualization of the lncRNA regulatory network.Then we used STRING database to analyse the interaction between the DEmRNAs,and a rotein-protein interaction(PPI)network was thus constructed.Cytoscape software was used for the visualization of the network.Key genes in PPI were then extracted using cytoHubba module of Cytoscape software.GO analysis and KEGG analysis were used to investigate the putative molecular functions of these key genes and the putative signaling pathways they might be involved in.Based on key genes,the regulatory sub-network was constructed and the key lncRNA related with breast cancer was then screened out.We used qRT-PCR assay to investigate the expression of key lncRNAs(DGUOK-AS1)in breast cancer cells and breast cancer tissues.The qRT-PCR assay was performed to further detect the expression of the key lncRNA(DGUOK-AS1)in 182 breast cancer tissues,and the breast cancer patients were divided into the DGUOK-AS1-high expression group and the DGUOK-AS1-low expression group based on the above results.The Kaplan-Meier method was used to plotted the survival curve for patients with breast cancer,and log-rank analysis was performed to analyse the difference of the survival curves between DGUOK-AS1-high expression group and the DGUOK-AS1-low expression group.Finally,Cox single/multiple regression analysis and Lasso analysis were used to analyse the relationship between the expression of key lncRNA and the prognosis of patients with breast cancer.ResultsIn this study,6 of differentially expressed lncRNAs(DDElncRNAs),29 of differentially expressed miRNAs(DEmiRNAs)and 253 of differentially expressed mRNAs(DEmRNAs)were selected after evaluating the expression profiles in breast cancer tissues and normal tissues.WGCNA results showed that up-regulated lncRNAs were positively correlated with breast cancer,while down-regulated lncRNAs were negatively correlated with breast cancer.Combined with the prediction results of public database,a breast cancer related regulatory network,including 6 of lncRNAs,29 of miRNAs and 253 of mRNAs,was preliminarily constructed.By analyzing the correlation among 253 mRNAs,the protein-protein interaction network was further established.Seven key genes were extracted using the cytoHubba module of Cytoscape sofeware to construct a key subnetwork,containing 2 of DElncRNA,5 of DEmiRNA,and 7 of key genes.The results of GO and KEGG analysis showed that the key genes mainly participate in cell mitosis,cell cycle,p53 pathway,and metabolism.By analyzing the expression of molecules in the subnetwork and based on the mechanism of competitive endogenous RNA,DGUOK-AS1,which was up-regulated in breast cancer tissues,was finally screened out as a key breast cancer-related IncRNA.The DGUOK-AS1 expression was higher in breast cancer cells compared to normal breast cells.The DGUOK-AS1 expression was also higher in breast cancer tissues compared to normal breast tissues.The results of prognostic analysis showed that DGUOK-AS1 expression was significantly negatively correlated with the prognosis of breast cancer patients,and could be one of the independent risk factors affecting the prognosis of breast cancer patients.Multivariate regression analysis further revealed three independent prognostic factors,and constructed a prognostic model,including histological grade,lymph node status,and DGUOK-AS1 expression,which showed significant value in prognosis prediction of breast cancer patients.Conclusions 1.The expression patterns of lncRNA,miRNA and mRNA in breast cancer tissues were different from those in normal breast tissues.2.According to the results of differential analysis and prediction,a complex breast cancer-related lncRNA regulatory network was further constructed.3.Based on the analysis of the key regulatory network,DGUOK-AS1 was selected as a key breast cancer-related lncRNA.4.The DGUOK-AS1 expression was higher in breast cancer cells compared to normal breast cells.The DGUOK-AS1 expression was also higher in breast cancer tissues compared to normal breast tissues.These results indicated a tumor-promoting role of DGUOK-AS1 in breast cancer.5.DGUOK-AS1 expression was negatively correlated with the prognosis of patients with breast cancer and has important prognostic value.Part Ⅱ Long noncoding RNA DGUOK-AS1 promote progression through regulating miR-204-5p/IL11 axis in breast cancerObjective1.Analyze the function of DGUOK-AS1 on the malignant phenotypes of breast cancer cells in vitro.2.Investigate the effect of DGUOK-AS1 on the malignant phenotypes of breast cancer cells in vivo.3.Clarify the molecular regulatory mechanism of DGUOK-AS1 in regulating the malignant phenotypes of breast cancer.MethodsDGUOK-AS1 was knockdown or overexpressed in breast cancer cells(MDA-MB-231、MDA-MB-468、MCF-7)using liposome transfection,and the function of DGUOK-AS1 in breast cancer cells was further evaluated.The MTT assay,colony formation assay,and EdU assay were used to evaluate the functional role of DGUOK-AS1 in mediating breast cancer cell proliferation.The wound repair assay and transwell assay were performed to evaluate the functional role of DGUOK-AS1 in mediating breast cancer cell metastasis.Combined with the bioinformatics analysis and website prediction results,the molecular regulatory pathways of DGUOK-AS1 in breast cancer were searched.RIP,luciferase and qRT-PCR assays were used to verify the regulatory mechanism between DGUOK-AS1,miR-204-5p,and IL11.In addition,the results of in vivo nude mouse subcutaneous tumor formation experiment and lung metastasis model also showed that DGUOK-AS1 also played a role in promoting the malignant phenotypes of breast cancer in vivo.ResultsMTT,clonal formation,and EdU assays showed that DGUOK-AS1 knockdown could led to obviously decreased proliferative ability of breast cancer cells,while DGUOK-AS1 overexpression led to obviously increased proliferative ability of breast cancer cells.The wound repair assay and transwell assays showed that DGUOK-AS1 knockdown could led to obviously decreased metastatic ability of breast cancer cells,while DGUOK-AS1 overexpression led to obviously increased metastatic ability of breast cancer cells.Through site prediction and verification,we found that DGUOK-AS1 and miR-204-5p could inhibit each other,and miR-204-5p could significantly reverse the function of DGUOK-AS1.Further mechanism analysis showed that DGUOK-AS1 could affect the expression of IL11 by sponging miR-204-5p,thus playing a pro-oncogene role in breast cancer.The tumor formation experiment and caudal vein metastasis model in nude mice further confirmed that DGUOK-AS1 could promote the progression and metastasis of breast cancer in vivo.Conclusions1.In vitro experiments confirmed that DGUOK-AS1 can significantly improve the proliferation,migration and invasion abilities of breast cancer cells.2.In vivo experiments have confirmed that DGUOK-AS1 can significantly promote the progression and metastasis of breast cancer.3.DGUOK-AS1 can act as an oncogene in breast cancer by affecting miR-204-5p/IL11 pathway.