Construction and analysis of a survival-associated competing endogenous RNA network in breast cancer

BackgroundRecently, increasing studies have shown that non-coding RNAs are closely associated with the progression and metastasis of cancer by participating in competing endogenous RNA (ceRNA) networks. However, the role of survival-associated ceRNAs in breast cancer (BC) remains unknown.MethodsThe Gene Expression Omnibus database and The Cancer Genome Atlas BRCA_dataset were used to identify differentially expressed RNAs. Furthermore, circRNA-miRNA interactions were predicted based on CircInteractome, while miRNA-mRNA interactions were predicted based on TargetScan, miRDB, and miRTarBase. The survival-associated ceRNA networks were constructed based on the predicted circRNA-miRNA and miRNA-mRNA pairs. Finally, the mechanism of miRNA-mRNA pairs was determined. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of survival-related mRNAs were performed using the hypergeometric distribution formula in R software.The prognosis of hub genes was confirmed using gene set enrichment analysis.ResultsBased on the DE-circRNAs of the top 10 initial candidates, 162 DE-miRNAsand 34 DE-miRNAs associated with significant overall survival were obtained. The miRNA target genes were then identified using online tools and verified using the Cancer Genome Atlas (TCGA) database. Overall, 46 survival-associated DE-mRNAs were obtained. The results of GO and KEGG pathway enrichment analyses implied that up-regulated survival-related DE-mRNAs were mostly enriched in the “regulation of cell cycle” and “chromatin” pathways, while down-regulated survival-related DE-mRNAs were mostly enriched in “negative regulation of neurotrophin TRK receptor signaling” and “interleukin-6 receptor complex” pathways. Finally, the survival-associated circRNA-miRNA-mRNA ceRNA network was constructed using 34 miRNAs, 46 mRNAs, and 10 circRNAs. Based on the PPI network, two ceRNA axes were identified. These ceRNA axescould be considered biomarkers for BC.GSEA results revealed that the hub genes were correlated with “VANTVEER_BREAST_CANCER_POOR_PROGNOSIS”, and the hub genes were verified using BC patients' tissues.ConclusionsIn this study, we constructed a circRNA-mediated ceRNA network related to BC. This network provides new insight into discovering potential biomarkers for diagnosing and treating BC

Calcium-binding protein S100P promotes tumor progression but enhances chemosensitivity in breast cancer

Background: Chemoresistance remains one of the obstacles to overcome in the treatment of breast cancer. S100 calcium-binding protein P (S100P) has been observed to be overexpressed in several cancers and has been associated with drug resistance, metastasis, and prognosis. However, the role of S100P in chemoresistance in breast cancer has not been thoroughly determined. Methods: Immunohistochemistry was used to evaluate the expression level of S100P protein in 22 pairs (pre-chemo and post-chemo) of breast cancer tissue from patients who underwent neoadjuvant chemotherapy. The influence of S100P on the biological behavior and chemosensitivity of breast cancer cells was then investigated. Results: The protein level of S100P in breast cancer tissue was significantly higher than in benign fibroadenoma (p<0.001). The S100P expression level was shown to be decreased by 46.55% after neoadjuvant chemotherapy (p=0.015). Subgroup analysis revealed that S100P reduction (57.58%) was mainly observed in the HER2+ tumors (p=0.027). Our in-vitro experiments showed that the knockdown of S100P suppressed the proliferation, adhesion, migration and invasion abilities of T47D and SK-BR-3 breast cancer cells. We further demonstrated that this knockdown increased the chemoresistance to paclitaxel and cisplatin in SK-BR-3 cells. We found that S100P exerted its function by activating NF-κB, CCND1 and Vimentin, but downregulating E-cadherin. Conclusions: S100P promotes the aggressive properties of breast cancer cells and may be considered as a promising therapeutic target. Moreover, S100P can be used to predict the therapeutic effect of chemotherapy in HER2+ breast cancer patients

Tim-3 promotes cell aggressiveness and paclitaxel resistance through the NF-κB /STAT3 signalling pathway in breast cancer cells

Objective: Although T-cell immunoglobulin and mucin-domain containing molecule-3 (Tim-3) has been recognized as a promising target for cancer immunotherapy, its exact role in breast cancer has not been fully elucidated. Methods: Tim-3 gene expression in breast cancer and its prognostic significance were analyzed. Associated mechanisms were then explored in vitro by establishing Tim-3-overexpressing breast cancer cells. Results: In a pooled analysis of The Cancer Genome Atlas (TCGA) database, Tim-3 gene expression levels were significantly higher (P<0.001) in breast cancer tissue, compared with normal tissues. Tim-3 was a prognosis indicator in breast cancer patients [relapse-free survival (RFS), P=0.004; overall survival (OS), P=0.099]. Tim-3 overexpression in Tim-3low breast cancer cells promoted aggressiveness of breast cancer cells, as evidenced by enhanced proliferation, migration, invasion, tight junction deterioration and tumor-associated tubal formation. Tim-3 also enhanced cellular resistance to paclitaxel. Furthermore, Tim-3 exerted its function by activating the NF-κB/STAT3 signalling pathway and by regulating gene expression [cyclin D1 (CCND1), C-Myc, matrix metalloproteinase-1(MMP1), TWIST, vascular endothelial growth factor (VEGF) upregulation, concomitant with E-cadherin downregulation). Lastly, Tim-3 downregulated tight junction-associated molecules zona occludens (ZO)-2, ZO-1 and occludin, which may further facilitate tumor progression. Conclusions: Tim-3 plays an oncogenic role in breast cancer and may represent a potential target for antitumor therapy

Identification of DHX36 as a tumour suppressor through modulating the activities of the stress-associated proteins and cyclin-dependent kinases in breast cancer

The nucleic acid guanine-quadruplex structures (G4s) are involved in many aspects of cancer progression. The DEAH-box polypeptide 36 (DHX36) has been identified as a dominant nucleic acid helicase which targets and disrupts DNA and RNA G4s in an ATP-dependent manner. However, the actual role of DHX36 in breast cancer remains unknown. In this study, we observed that the gene expression of DHX36 was positively associated with patient survival in breast cancer. The abundance of DHX36 is also linked with pathologic conditions and the stage of breast cancer. By using the xenograft mouse model, we demonstrated that the stable knockdown of DHX36 via lentivirus in breast cancer cells significantly promoted tumour growth. We also found that, after the DHX36 knockdown (KD), the invasion of triple-negative breast cancer cells was enhanced. In addition, we found a significant increase in the number of cells in the S-phase and a reduction of apoptosis with the response to cisplatin. DHX36 KD also desensitized the cytotoxic cellular response to paclitaxel and cisplatin. Transcriptomic profiling analysis by RNA sequencing indicated that DHX36 altered gene expression profile through the upstream activation of TNF, IFNγ, NFκb and TGFβ1. High throughput signalling analysis showed that one cluster of stress-associated kinase proteins including p53, ROCK1 and JNK were suppressed, while the mitotic checkpoint protein-serine kinases CDK1 and CDK2 were activated, as a consequence of the DHX36 knockdown. Our study reveals that DHX36 functions as a tumour suppressor and may be considered as a potential therapeutic target in breast cancer

Identification of DHX36 as a tumour suppressor through modulating the activities of the stress-associated proteins and cyclin-dependent kinases in breast cancer

The nucleic acid guanine-quadruplex structures (G4s) are involved in many aspects of cancer progression. The DEAH-box polypeptide 36 (DHX36) has been identified as a dominant nucleic acid helicase which targets and disrupts DNA and RNA G4s in an ATP-dependent manner. However, the actual role of DHX36 in breast cancer remains unknown. In this study, we observed that the gene expression of DHX36 was positively associated with patient survival in breast cancer. The abundance of DHX36 is also linked with pathologic conditions and the stage of breast cancer. By using the xenograft mouse model, we demonstrated that the stable knockdown of DHX36 via lentivirus in breast cancer cells significantly promoted tumour growth. We also found that, after the DHX36 knockdown (KD), the invasion of triple-negative breast cancer cells was enhanced. In addition, we found a significant increase in the number of cells in the S-phase and a reduction of apoptosis with the response to cisplatin. DHX36 KD also desensitized the cytotoxic cellular response to paclitaxel and cisplatin. Transcriptomic profiling analysis by RNA sequencing indicated that DHX36 altered gene expression profile through the upstream activation of TNF, IFNγ, NFκb and TGFβ1. High throughput signalling analysis showed that one cluster of stress-associated kinase proteins including p53, ROCK1 and JNK were suppressed, while the mitotic checkpoint protein-serine kinases CDK1 and CDK2 were activated, as a consequence of the DHX36 knockdown. Our study reveals that DHX36 functions as a tumour suppressor and may be considered as a potential therapeutic target in breast cancer

Integrated transcriptome analysis identifies APPL1/RPS6KB2/GALK1 as immune-related metastasis factors in breast cancer

The aim of this study is to investigate the prognostic immune-related factors in breast cancer (BC) metastasis. The gene expression chip GSE159956 was downloaded from the gene expression omnibus database. Differentially expressed genes (DEGs) were selected using GEO2R online tools based on lymph node and metastasis status. The intersected survival-associated DEGs were screened from the Kaplan–Meier curve. Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) annotation analyses were performed to determine the survival-associated DEGs. Immune-related prognostic factors were screened based on immune infiltration. The screened prognostic factors were verified by the Cancer Genome Atlas (TCGA) database and single-sample gene set enrichment analysis (ssGSEA). As a result, twenty-eight upregulated and three downregulated genes were generated by the survival analysis. The enriched GO and KEGG pathways were mostly correlated with “regulation of cellular amino acid metabolic process,” “proteasome complex,” “endopeptidase activity,” and “proteasome.” Six of 19 (17 upregulated and 2 downregulated) immune-related prognostic factors were verified by the TCGA database. Four immune-related factors were obtained after ssGSEA, and three significant immune-related factors were selected after univariate and multivariate analyses. Based on the risk score receiver operating characteristic, the three immune-related prognosis factors could be potential biomarkers of BC metastasis. In conclusion, APPL1, RPS6KB2, and GALK1 may play a pivotal role as potential biomarkers for prediction of BC metastasis

Upregulated Expression of Indoleamine 2, 3-Dioxygenase in Primary Breast Cancer Correlates with Increase of Infiltrated Regulatory T Cells In Situ and Lymph Node Metastasis

IDO has been reported to induce immunotolerance and promote metastasis in solid malignancy, but the mechanisms involved were not fully understood. In this study, the expression of IDO in primary breast cancer was examined and the correlation between the expression levels of IDO and the densities of Foxp3+ Tregs in situ was studied. The IDO stably-expressing CHO cells(IDO/CHO) were generated to evaluate the induction of Foxp3+ Tregs after coculturing with CD3+ T cells in vitro. The IDO expression in cancer was higher than that in benign diseases both at RNA and protein levels. The IDO expression was significantly upregulated in tumors of more advanced stages and with more extensive lymph node metastasis, and displayed positive linear correlation with the density of Foxp3+ Tregs. We further demonstrated that CD4+CD25+CD127− Tregs could be amplified by coculturing CD3+ T cells with IDO/CHO cells in vitro which displayed increasing Foxp3 expression both at mRNA and protein levels. Our results implied that up-regulation of IDO in primary breast cancer may inhibit local immune surveillance and promote metastasis by favoring development and infiltration of Foxp3+ Tregs in the tumor microenvironment

Tim-3 promotes tube formation and decreases tight junction formation in vascular endothelial cells

As a negative immune checkpoint molecule, T cell immunoglobulin domain and mucin domain containing molecule-3 (Tim-3) has been found to serve a crucial role in immune escape and tumour progression. Previous studies have reported that Tim-3 is important to endothelial cells and it has also been demonstrated to be involved in numerous types of human disease, including melanoma, lymphoma, rickettsial infection and atherosclerosis; however, its exact mechanism of action remains largely unknown. In the present study, Tim-3 was overexpressed in vascular endothelial HMVECs and HUVECs and in vitro assays were used to determine that Tim-3 promoted cell proliferation, migration, invasion and tube formation through activating cyclin D1, Ras homolog gene family member A and vascular endothelial growth factor receptor 2 (VEGFR2). Additionally, Tim-3 decreased tight junction (TJ) formation and the transepithelial resistance of endothelial cells by decreasing the expression levels of TJ protein 2, Occludin and claudin 1. In conclusion, these findings suggested that Tim-3 may exert a positive role in angiogenesis and a negative role in TJ formation in vascular endothelial cells, which may provide novel strategies for the treatment of Tim-3 associated diseases

Datasheet5_Construction and analysis of a survival-associated competing endogenous RNA network in breast cancer.zip

BackgroundRecently, increasing studies have shown that non-coding RNAs are closely associated with the progression and metastasis of cancer by participating in competing endogenous RNA (ceRNA) networks. However, the role of survival-associated ceRNAs in breast cancer (BC) remains unknown.MethodsThe Gene Expression Omnibus database and The Cancer Genome Atlas BRCA_dataset were used to identify differentially expressed RNAs. Furthermore, circRNA-miRNA interactions were predicted based on CircInteractome, while miRNA-mRNA interactions were predicted based on TargetScan, miRDB, and miRTarBase. The survival-associated ceRNA networks were constructed based on the predicted circRNA-miRNA and miRNA-mRNA pairs. Finally, the mechanism of miRNA-mRNA pairs was determined. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of survival-related mRNAs were performed using the hypergeometric distribution formula in R software.The prognosis of hub genes was confirmed using gene set enrichment analysis.ResultsBased on the DE-circRNAs of the top 10 initial candidates, 162 DE-miRNAsand 34 DE-miRNAs associated with significant overall survival were obtained. The miRNA target genes were then identified using online tools and verified using the Cancer Genome Atlas (TCGA) database. Overall, 46 survival-associated DE-mRNAs were obtained. The results of GO and KEGG pathway enrichment analyses implied that up-regulated survival-related DE-mRNAs were mostly enriched in the “regulation of cell cycle” and “chromatin” pathways, while down-regulated survival-related DE-mRNAs were mostly enriched in “negative regulation of neurotrophin TRK receptor signaling” and “interleukin-6 receptor complex” pathways. Finally, the survival-associated circRNA-miRNA-mRNA ceRNA network was constructed using 34 miRNAs, 46 mRNAs, and 10 circRNAs. Based on the PPI network, two ceRNA axes were identified. These ceRNA axescould be considered biomarkers for BC.GSEA results revealed that the hub genes were correlated with “VANTVEER_BREAST_CANCER_POOR_PROGNOSIS”, and the hub genes were verified using BC patients' tissues.ConclusionsIn this study, we constructed a circRNA-mediated ceRNA network related to BC. This network provides new insight into discovering potential biomarkers for diagnosing and treating BC.</p