DEPDC1B Promotes The Metastasis And Proliferation Of Breast Cancer Via Binding CTNNB1 To Activate The Wnt/β-catenin Signaling Pathway

Part Ⅰ The Hub gene DEPDC1 B of breast cancer was screened via bioinformatic analysis Objective To predict and screen out the hub gene that can affect the occurrence,development,and prognosis of breast cancer(BRCA).Methods TCGA-BRCA database was downloaded,which contains the transcriptome data and clinical data.Differentially expressed genes(DEGs)were visualized by volcano and heatmap using ggplot2 and heatmap packages,respectively.STRING,MCODE,and cyto Hubba were utilized to construct the PPI network,module analysis,and calculate hub genes,respectively.KM-plotter was used to carry out survival analysis of hub genes to screen the critical DEG in BRCA.We used CCLE,EMBLEBI,Oncomine,and HPA to analyze the expression of this gene in BRCA cells and tissues.Then,GEPIA and Bc-Genex Miner were utilized to analyze the effect of clinical indicators and prognosis of BRCA with this gene.Finally,function enrichment analysis of this gene was conducted to clarify its possible role in the development of BRCA.Result 4190 DEGs were founded through the analysis of the difference expression of TCGA-BRCA.Then,the 364 DEGs were got by a higher screening condition,|logFC| >3,which includes 117 up-regulated genes and 247 down-regulated genes.Subsequently,hub genes were screened by constructing a protein interaction network and module analysis.KM-plotter was performed survival analysis of hub genes,and DEPDC1 B was identified as the critical hub gene in BRCA.The DEPDC1 B expression in BRCA was analyzed through multiple online tools,and the results indicated that DEPDC1 B was a high expression.Besides,the analysis of the prognostic indicators of BRCA patients showed that DEPDC1 B was positively correlated with lymph node metastasis and clinical stage,and negatively correlated with age.Meanwhile,DEPDC1 B could bring shorter DMFS,DFS,and OS to BRCA patients.Further functional analysis showed that DEPDC1 B promotes the proliferation,metastasis,and cell cycle transition of BRCA cells.Conclusion Based on bioinformatics analysis of TCGA-BRCA,DEPDC1 B was eventually screened as a hub gene that can affect the occurrence and development of BRCA.There is a research value that DEPDC1 B maybe a new therapeutic target for BRCA patients.Part Ⅱ Study the expression and clinical significance of DEPDC1 B in breast cancer tissuesObjective To study the effect of DEPDC1 B on the clinical characteristic and prognosis of BRCA,through detecting the DEPDC1 B expression in paracancerous and cancer tissues.Methods Immunohistochemical(IHC)was utilized to detect DEPDC1 B expression in paracancerous and cancer tissues microarray of BRCA,that is the staining intensity and expression of positive rate for DEPDC1 B.A Chi-square test was used to calculate the correlation between DEPDC1 B and clinical-pathological indicators in BRCA patients.KM-plotter was used to analyze the overall survival(OS)of BRCA patients with DEPDC1 B expression.Besides,univariate and multivariate COX regression was utilized to analyze whether DEPDC1 B was a risk factor for the BRCA.Result DEPDC1 B expression in BRCA tissues was higher than paracancerous,the results of paired cancer and paracancerous tissues analysis also showed that DEPDC1 B was significantly higher in cancer tissues than paracancerous(P < 0.001).The OS of DEPDC1 B high expression patients was significantly shorter than the low expression(P = 0.030).The results of the chi-square test indicated that DEPDC1 B correlated with the N stage(χ~2 = 10.105,P = 0.018)and the survival status of patients(χ~2 = 5.158,P = 0.023).Univariate COX regression analysis showed that DEPDC1 B expression(P = 0.030),tumor size(P = 0.011)and T stage(P = 0.001)influenced the prognosis of BRCA patients.Multivariate COX regression analysis showed that DEPDC1 B was an independent risk factor for the prognosis of BRCA patients(HR = 4.316,95% CI = 1.025-18.1701,P = 0.046).Conclusion We found that the DEPDC1 B expression in BRCA tissues was significantly higher than that in the normal breast tissue,and whose expression had a correlation with N stages,and the poor prognosis of patients.Multivariate analysis showed that DEPDC1 B expression was an independent prognostic marker for shorter OS,suggesting that the DEPDC1 B plays an important role in the BRCA tumorigenesis.Part Ⅲ Study the biological function of DEPDC1 B in breast cancer cellsObjective To detect the expression of DEPDC1 B in BRCA cells;to explore the function of DEPDC1 B in BRCA cells in vitro.Methods Western blot assay was utilized to explore the DEPDC1 B expression in BRCA and normal breast epithelial cells.Two cell lines with high and low expression levels of DEPDC1 B were screened out,respectively.Then,the expression of DEPDC1 B was silenced or increased by transfection of si RNA and DEPDC1 B overexpression plasmid,respectively.CCK-8 assay,clone formation assay,and immunofluorescence assay were used to detect the proliferation ability of BRCA cells with the DEPDC1 B silenced or overexpressed.Scratch assay and Transwell assay were used to explore the effect of DEPDC1 B on the migration and invasion of BRCA cells.Flow cytometry was utilized to explore the effect of DEPDC1 B on the cell cycle.EMTrelated markers were detected by western blot for exploring the influence of DEPDC1 B on EMT.Result DEPDC1 B was higher expressed in BRCA cells than that in normal breast epithelial cells.After silencing the DEPDC1 B expression in BRCA cells with DEPDC1 B high expression(Hs578T and MDA-MB-231),the proliferation ability,clone formation rate,invasion,and migration ability of the cells were all reduced,and the cell cycle was blocked in the G1 phase and decreased in S phase.Besides,the results of western blot combined with immunofluorescence showed that with the decrease of DEPDC1 B expression,E-cadherin was increased,meanwhile N-cadherin and vimentin was decreased.After the overexpression of DEPDC1 B in the BRCA cell lines with DEPDC1 B low expression(MDA-MB-157 and MDA-MB-468),the ability of proliferation,clone formation rate,invasion,and migration were enhanced,and the cell cycle was blocked in the S phase and decreased in the G1 phase.Also,the results of western blot combined with immunofluorescence showed that with the expression of DEPDC1 B increased,E-cadherin expression was decreased,accompanied by the increased expression of N-cadherin and vimentin.Conclusion DEPDC1 B is highly expressed in BRCA cells.DEPDC1 B expression promotes the cell cycle transition from G1 phase to S phase and the proliferation,invasion and migration of BRCA cells.Part Ⅳ Study the effect of the interfered expression of DEPDC1 B in breast cancer cells on the formation of lung metastases in vivoObjective To study the effect of silencing DEPDC1 B on invasion and migration of BRCA cells in vivo.Methods The stable cell lines were constructed by infecting with lentivirus,the DEPDC1 B expression in Hs578T-Lv-shDEPDC1 B and Hs578T-Lv-NC were detected by qRT-PCR,western blot,and CCK-8 to verify the successful construction of the stably transfected cell line.10 nude mice were randomly divided into 2 groups,and 2 kinds of stable transfection cells were inoculated by tail vein injection,respectively,to establish the model of lung metastatic tumor in nude mice.After 4-6 weeks,the lung tissue of the nude mice was scanned using a small animal imaging technique to detect the formation of lung metastases.Subsequently,the size of the metastatic tumor in the lung tissue sections was determined by HE staining.IHC method was used to detect the changes of EMT-related indexes in tissue sections.Result qRT-PCR and western blot results showed that DEPDC1 B expression in Hs578T-Lv-shDEPDC1 B cells was significantly lower than Hs578T-Lv-NC cells.CCK-8 results indicated that the proliferation ability of Hs578T-Lv-shDEPDC1 B was significantly lower than Hs578T-Lv-NC cells,indicating that the stable silenced DEPDC1 B cells were successfully constructed.In vivo imaging of small animals showed significantly lower lung metastases in mice with DEPDC1 B expression silenced compared to the control group.The results of HE indicated that compared with the Lv-NC,the number and size of cancer nests formed by lung metastases of mice in the DEPDC1 B silenced group was significantly reduced.IHC results suggested that with the silenced expression of DEPDC1 B,the expression of E-cadherin was increased and the expression of N-cadherin and vimentin was decreased in lung metastatic tumor tissues.Conclusion The formation of lung metastases of BRCA cells in nude mice is decreased via silencing the DEPDC1 B expression.DEPDC1 B can affect the ability of BRCA cells to metastasize in vivo.Part Ⅴ Study the molecular mechanism of DEPDC1 B in breast cancer cellsObjective To study the mechanism of DEPDC1 B in regulating the biological function of BRCA cells.Methods DEPDC1 B related proteins were identified by the protein profiling.Bioinformatics was utilized to explore the biological functions of proteins and the signal pathways involved.Western blot was used to identify the expression of key factors in the signal pathways.Then,the key protein interacting with DEPDC1 B were explored by co-immunoprecipitation(COIP)and immunofluorescence(IF)detection co-localization.Finally,the molecular mechanism of DEPDC1 B in BRCA was revealed.Result 414 proteins of DEPDC1 B interaction were identified by protein profiling,GO and KEGG enrichment analysis showed that these proteins were highly enriched in wnt signaling pathway,cell migration,adhesion function,DNA replication,and cell adhesion junctions.Western blot results showed that the expression of wnt3 a,β-catenin and p-GSK-3β(Ser9)were decreased after silencing DEPDC1 B expression,while GSK-3β increased.While the overexpression of DEPDC1 B produced the opposite result.IF analysis showed that the nuclear translocation of β-catenin was decreased when DEPDC1 B was interfered with,while overexpression of DEPDC1 B produced the opposite result.The results of GSEA analysis showed that the high DEPDC1 B group could be significantly enriched in RB1＿TARGETS,E2F＿TARGETS,CELL＿CYCLE,and cell＿cycle＿checkpoint.Western blot results showed that the overexpression of DEPDC1 B in BRCA cells could increase the expression of cyclin D1,CDK4,p-Rb(ser780),and E2F1,but did not affect Rb.After silencing DEPDC1 B expression,Rb expression did not change,and the results were opposite to the overexpression part.Besides,the cells treated with XAV-939 showed that XAV-939 restored the activation of the wnt/β-catenin signaling pathway induced by the DEPDC1 B overexpression.Finally,COIP and IF results showed that DEPDC1 B and CTNNB1 were co-localized in BRCA cells,and there was an interaction between them.Conclusion DEPDC1 B activates the wnt/β-catenin signaling pathway via interacting with CTNNB1,which promotes the EMT process of BRCA cells,promoting cell invasion and migration.Meanwhile,it promotes cyclin D1 expression,then activates the Rb-E2F1 pathway through binding with CDK4,thus promoting the transformation of cells from G1 phase to S phase,promotes cell proliferation.Part Ⅵ Study the upstream regulator of DEPDC1 B in breast cancer cellsObjective To explore miRNAs that can target and interfere with DEPDC1 B expression,in BRCA cells.Methods Starbase v3.0 database was used to predict the miRNAs that could interfere DEPDC1 B expression.The dual luciferase reporter gene assay was used to confirm the targeted correlation between miRNA and DEPDC1 B.qRT-PCR and western blot were utilized to detect DEPDC1 B expression,validating the effectiveness of targeted regulation.Then,the biological function of miRNA in BRCA cells was detected by CCK-8,Transwell,and western blot.The expression of key markers in the wnt/β-catenin signal pathway was detected by western blot.Finally,the effect of miRNA targeting interference with DEPDC1 B expression on cell proliferation ability was detected by Ed U assay.Result MiR-199a-5p was found as a targeted regulation miRNA for DEPDC1 B via starbase v3.0 and dual luciferase reporter genes assay.qRT-PCR and western blot results showed that the transcription and expression of DEPDC1 B were significantly inhibited after the overexpression of miR-199a-5p in BRCA cells.Moreover,the increased expression of miR-199a-5p can inhibit the proliferation,invasion,and migration of BRCA cells,otherwise,the E-cadherin expression was increased,with the decreasing of N-cadherin and vimentin.Besides,wnt3 a,β-catenin,and cyclin D1 were also significantly decreased.Noticeably,miR-199a-5p restored the promotion of cell proliferation caused by the overexpression of DEPDC1 B.Conclusion MiR-199a-5p is an upstream regulator of DEPDC1 B,which can inhibit the invasion,migration and proliferation of BRCA cells,and reduce the activity of Wnt /β-catenin pathway.