Screening Of Key Pathways In Triple-Negative Breast Cancer And Inhibitory Effect Of Sericin On The Growth Of MDA-MB-468 Cells

Breast cancer is a malignancy,with the highest morbidity and mortality in women,according to The World Cancer Report 2020,2.1 million cases newly-diagnosed.Triple-negative breast cancer（TNBC）,as an aggressive and metastatic subtype,accounts for about 20% of all breast cancers and is characterized by absent expression of estrogen receptor,progesterone receptor,and human epidermal growth factor receptor 2.To date,no endocrine therapies or targeted therapies have been recommended for the treatment of TNBC,while chemotherapy remains the primary treatment modality.Patients with TNBC tend to have a high risk of recurrence and poor prognosis.Using bioinformatics methods,gene expression profiles,biological processes and signaling pathways in the tumorigenesis and progression of TNBC are analyzed to predict the potential pathogenic mechanisms.Further,based on this,the search for highly efficient and less-toxic drugs and new therapeutic strategies is warranted.Sericin,a natural protein,is produced in silkworm complete metamorphosis,accounting for 20%～30% of the total silk protein.It has various biological functions,such as anti?inflammatory,antibacterial,anti-oxidant.Our previous study reported that sericin could prevent diabetes-induced renal damage,reproductive damage,and hippocampal neuronal apoptosis.Furthermore,several studies have reported that sericin could exert antitumor effects in various tumor types,including gastric carcinoma,colon carcinoma,skin carcinoma,breast adenocarcinoma,and tongue carcinoma.In the present study,differentially expressed genes（DEGs）between normal mammary tissues and TNBC tissues were identified,and key pathways and key genes were screened by bioinformatics analysis.Based on this,the effects of sericin on the TNBC MDA-MB-468 cell growth and its underlying mechanism were explored.Part 1 Screening key pathways and key genes in triple-negative breast cancer by bioinformatics analysisObjective:To identify key pathways and key genes associated with tumorigenesis and progression of TNBC by bioinformatics analysis.Methods:1.Two gene expression profile datasets containing normal mammary tissue（GSE112825）and TNBC tissue（GSE76124）samples and their platform data were obtained from Gene Expression Omnibus（GEO）database.2.The quality of GSE112825 and GSE76124 datasets were evaluated by the R software.3.The GSE112825 and GSE76124 datasets were normalized.The probe names in the gene expression profiles were converted into gene names.The two datasets were merged according to the common probes and the batch effect was removed.4.The gene expression matrixes were analyzed and the DEGs were identified using the limma package in R software.5.The gene ontology（GO）function annotation and the kyoto encyclopedia of genes and genomes（KEGG）pathway analysis of the DEGs were performed using the Database for Annotation,Visualization and Integrated Discovery（DAVID）online tool.6.The KEGG analysis was performed using the gene set enrichment analysis（GSEA）software v3.0.7.The most key pathway was identified and the DEGs in the key pathway were extracted.8.A diagram,showing the expression levels of the DEGs in the key pathway in GSE112825 and GSE76124 datasets,were plotted using the ggplot2 package in R software.9.The GO function annotation and the KEGG pathway analysis of the DEGs in the key pathway were performed using the DAVID online tool.10.The relationships between the expression levels of the DEGs in the key pathway and prognosis in breast cancer patients were analyzed.The overall survival curves were constructed using the Kaplan-Meier Plotter database.11.The protein-protein interaction（PPI）network of DGEs was constructed using the STRING database and the Cytoscape software v3.6.2.12.The hub genes were identified using the Cytoscape plugin Cytohubba.The functional modules were extracted and the seed genes were obtained using plugin MCODE.13.A diagram,showing the expression levels of the key genes（hub genes and seed genes）in GSE112825 and GSE76124 datasets,were plotted using the ggplot2 package in R software.14.The GO function annotations and the KEGG pathway analysis of the key genes were performed using the DAVID online tool.Results:1.Grayscale plots,weight plots,residuals plots,residuals sign plots,RLE,NUSE,and RNA degradation curves indicated that the quality of normal mammary tissue（GSE112825）and TNBC tissue（GSE76124）samples were reliable.2.The GSE112825 and GSE76124 datasets were normalized and the sample distributions of each dataset were identical.The two datasets were merged and the batch effect was removed.3.A total of 1091 DEGs were screened out,including 764 upregulated genes and 327 downregulated genes.4.The GO analysis of the 1091 DEGs revealed that biological process terms were mainly enriched in ‘aromatic compound biosynthetic process’ and ‘heterocycle biosynthetic process’.The cell component terms were mainly related to ‘extracellular region’ and ‘extracellular region part’.The molecular function terms mainly involved ‘carbohydrate derivative binding’and ‘small molecule binding’.The KEGG analysis indicated that the most significant pathways included ‘pathways in cancer’ and the ‘PI3K/Akt signaling pathway’.5.Validation performed by the GSEA software v3.0 demonstrated that DEGs were mainly enriched in PI3K/Akt signaling pathway which was considered as the key pathway in TNBC（NES=1.947,NOM p-value=0.008,FDR q-value=0.037）.6.The 30 DEGs in the key pathway were extracted.7.The expression levels of 30 DEGs in the key pathway in GSE112825 and GSE76124 datasets were showed in the diagram（P<0.05）.8.The GO analysis of 30 DEGs in the key pathway revealed that biological process terms were mainly enriched in ‘positive regulation of cellular metabolic process’ and ‘positive regulation of macromolecule metabolic process’.‘Extracellular region’ and ‘receptor binding’ were the top?ranked cell component and molecular function,respectively.The KEGG analysis indicated that the most significant pathways included the ‘PI3K/Akt signaling pathway’ and ‘pathways in cancer’.9.Survival analysis indicted that 27 DEGs,among 30 DEGs in the key pathway,were associated with the overall survival of breast cancer patients（P<0.05）.10.The 1091 DEGs were used to construct the PPI network which was composed of 821 nodes.11.A total of 10 hub genes were identified based on PPI network data,including BUB1 B,CENPF,TOP2 A,NDC80,CCNB1,DLGAP5,CCNA2,CDK1,CCNB2,and UBE2 C.A total of 21 functional modules were extracted.And,19 seed genes were obtained including MAD2L1,GPSM2,MID1,LY6 E,KALRN,COL6A6,KRT86,MMRN1,SPTAN1,MCM5,SRD5A1,FABP4,WHSC1,NUP210,ITGA5,ATP11 B,PBRM1,RPS11,and HIST1H2 BG.12.The expression levels of 29 key genes（10 hub genes and 19 seed genes）in GSE112825 and GSE76124 datasets were showed in the diagram（P<0.05）.13.The GO analysis of 29 key genes revealed that biological process terms were mainly enriched in ‘mitotic cell cycle process’ and ‘mitotic cell cycle’.‘Nucleoplasm’ and ‘macromolecular complex binding’ were the top?ranked cell component and molecular function,respectively.The KEGG analysis indicated that the most significant pathways included the ‘Cell cycle’ and ‘Progesterone-mediated oocyte maturation’.Conclusion:1.The DEGs between TNBC tissues and normal mammary tissues mainly enriched in the PI3K/Akt signaling pathway,predicting that PI3K/Akt signaling pathway might be a key pathway in the mechanism of TNBC.2.The key genes of TNBC majorly enriched in cell cycle biological process and signaling pathway,indicating that cell cycle procedure involved in the tumorigenesis and progression of TNBC.Part 2 Sericin inhibits MDA?MB?468 cell growth via the PI3K/Akt signaling pathway in triple?negative breast cancerObjective:To investigate the effects of sericin on the TNBC cell proliferation,cell cycle and cellular apoptosis and to explore whether its mechanism is related to regulating PI3K/Akt signaling pathway.Methods:1.MTT assay was performed to assess viability of human TNBC MDA-MB-468 cell line and MDA-MB-453 cell line,non-TNBC MCF-7 cell line,and normal breast epithelial MCF?10A cell line after 24 h treatment with gradient concentrations of sericin.The optimal concentrations of sericin were determined.2.Colony formation assay was performed to assess the clone-forming capacity of MDA-MB-468 cells after 24 h treatment with indicated concentrations of sericin（0 mg/ml,2 mg/ml,4 mg/ml,and 8 mg/ml）.3.Immunocytochemistry was performed to assess the expression levels of Ki67 protein in MDA-MB-468 cells after 24 h treatment with indicated concentrations of sericin（0 mg/ml,2 mg/ml,4 mg/ml,and 8 mg/ml）.4.Flow cytometry was used to assess cell cycle progression of MDA-MB-468 cells after 24 h treatment with indicated concentrations of sericin（0 mg/ml,2 mg/ml,4 mg/ml,and 8 mg/ml）.5.Immunoblot assay was performed to assess the expression levels of Cyclin D1,Cdk4,E2F3,P21,and P27 proteins in MDA-MB-468 cells after 24 h treatment with indicated concentrations of sericin（0 mg/ml,2 mg/ml,4 mg/ml,and 8 mg/ml）.6.Immunocytochemistry was performed to assess the subcellular localization and expression levels of P21 protein in MDA-MB-468 cells after 24 h treatment with 0 mg/ml and 8 mg/ml concentrations of sericin.7.Flow cytometry was used to assess apoptosis status of MDA-MB-468 cells after 24 h treatment with indicated concentrations of sericin（0 mg/ml,4 mg/ml,and 8 mg/ml）.8.Immunoblot assay was performed to assess the expression levels of Bax,Bcl-2,and Cyto-C proteins in MDA-MB-468 cells after 24 h treatment with indicated concentrations of sericin（0 mg/ml,2 mg/ml,4 mg/ml,and 8 mg/ml）.9.Immunoblot assay was performed to assess the expression levels of PI3 K,Akt,p-Akt,HSP90,and Bcl-2 proteins in MDA-MB-468 cells after 24 h treatment with indicated concentrations of sericin（0 mg/ml,2 mg/ml,4 mg/ml,and 8 mg/ml）.10.The AKT agonist SC79 was used to treat MDA-MB-468 cells for 2 h,following sericin treatment for 22 h.Five groups were set as follows:DMSO group,0 mg/ml group,0 mg/ml+SC79 group,8 mg/ml group,and 8 mg/ml+SC79 group.MTT assay was performed to assess cell viability.11.The AKT agonist SC79 was used to treat MDA-MB-468 cells for 2 h,following sericin treatment for 22 h.Five groups were set as follows: DMSO group,0 mg/ml group,0 mg/ml+SC79 group,8 mg/ml group,and 8 mg/ml+SC79 group.Colony formation assay was performed to assess clone-forming capacity.12.The AKT agonist SC79 was used to treat MDA-MB-468 cells for 2 h,following sericin treatment for 22 h.Five groups were set as follows: DMSO group,0 mg/ml group,0 mg/ml+SC79 group,8 mg/ml group,and 8 mg/ml+SC79 group.Immunocytochemistry was performed to assess the expression levels of Ki67 protein.Results:1.Sericin inhibited viability of TNBC cells:The MTT assay showed that the viability of both MDA?MB?468 cells（F=74.516,P<0.001）and MDA-MB-453 cells（F=23.150,P<0.001）were decreased gradually with increasing sericin concentrations.2.Sericin inhibited the ability of MDA-MB-468 cell clone formation:Colony formation assay showed that the clone-forming capacity of MDA-MB-468 cells（F=81.602,P<0.001）were decreased gradually with increasing sericin concentrations.3.Downregulation P21 in MDA-MB-468 cells by sericin:Immunocytochemistry showed that the expression levels of Ki67 protein in MDA-MB-468 cells（F=76.470,P<0.001）were decreased gradually with increasing sericin concentrations.4.Sericin induced cell cycle arrest at the G0/G1 phase in MDA-MB-468 cells:Flow cytometry demonstrated that the sericin treatment led to an increased portion of cells at the G0/G1 phase（F=32.131,P<0.001）and a reduction at the S phase（F=38.818,P<0.001）,in a dose?dependent manner.5.Downregulation of Cyclin D1,Cdk4,E2F3 and upregulation P21, P27 in MDA-MB-468 cells by sericin:Immunoblot assay showed that the expression levels of Cyclin D1（F=35.866,P<0.001）,Cdk4（F=163.359,P<0.001）and E2F3（F=257.478,P<0.001）proteins in MDA-MB-468 cells were downregulated,while the expression levels of P21（F=80.107,P<0.001）and P27（F=471.443,P<0.001）proteins were upregulated gradually with increasing sericin concentrations.6.The nuclear signals for P21 protein were elevated in MDA-MB-468 cells by sericin:Immunocytochemistry showed that the nuclear signals（t=?10.918,P<0.001）and total expression levels（t=?7.948,P=0.001）of P21 protein were increased,the cytoplasmic signals（t=2.914,P=0.044）were decreased in MDA-MB-468 cells treated with 8 mg/ml sericin.7.Sericin induced early and late apoptosis in MDA?MB?468 cells:Flow cytometry demonstrated that early apoptosis rates（F=123.132,P<0.001）,late apoptosis rates（F=150.238,P<0.001）and total apoptosis rates（F=233.551,P<0.001）in MDA-MB-468 cells were increased with sericin treatment,in a dose?dependent manner.8.Upregulation of Bax,Cyto-C and downregulation Bcl-2 in MDA-MB-468 cells by sericin:Immunoblot assay showed that the expression levels of Bax（F=222.130,P<0.001）and Cyto-C（F=55.296,P<0.001）proteins in MDA-MB-468 cells were upregulated,while the expression levels of Bcl-2（F=92.422,P<0.001）protein were downregulated gradually with increasing sericin concentrations.9.Downregulation PI3 K,Akt,p-Akt,HSP90,Bcl-2 in MDA-MB-468 cells by sericin:Immunoblot assay showed that the expression levels of PI3K（F=35.494,P<0.001）,Akt（F=11.175,P=0.003）,p-Akt（F=260.064,P<0.001）,HSP90（F=398.631,P<0.001）,and Bcl2（F=92.422,P<0.001）proteins were downregulated gradually with increasing sericin concentrations.10.Sericin combined with SC79 treatment restored the viability of MDA?MB?468 cells:MTT assay showed that 8 mg/ml sericin significantly decreased the cell viability（P<0.001）,while 8 mg/ml sericin combined with SC79 treatment partially restored the cell viability（P<0.001）of MDA?MB?468 cells.11.Sericin combined with SC79 treatment restored the clone-forming capacity of MDA-MB-468 cells:Colony formation assay showed that 8 mg/ml sericin significantly decreased the clone-forming capacity（P<0.001）,while 8 mg/ml sericin combined with SC79 treatment partially restored the clone-forming capacity（P=0.020）of MDA-MB-468 cells.12.Sericin combined with SC79 treatment restored the expression levels of Ki67 protein in MDA-MB-468 cells:Immunocytochemistry showed that 8 mg/ml sericin significantly decreased the expression levels of Ki67 protein（P<0.001）,while 8 mg/ml sericin combined with SC79 treatment partially restored the expression levels of Ki67 protein（P=0.011）in MDA-MB-468 cells.Conclusion:1.Sericin significantly suppressed MDA-MB-468 cells proliferation,induced G₀/G₁ cell cycle arrest and promoted cell apoptosis.2.Sericin might inhibit MDA?MB?468 cell growth by regulating the PI3K/Akt signaling pathway.