At the first, we perform an integrated high-throughput bioinformatics analysis to demonstrate significantly biomarkers involved in breast cancer. We carried out a microarray analysis to find the differentially expressed genes that could affect breast cancer development. After finding the DEGs, we choose ADAMTS5 for further analyses and investigations. Also, we performed following bioinformatics analyses to find a systematic view of the regulatory mechanism of up and down regulated genes in this dataset on the BC development, specially ADANTS5: mRNA – mRNA interaction, mRNA – lncRNA interaction, mRNA – microRNA interaction, miRNA – lncRNA interaction, GSEA, and GO. Based on mentioned analyses, Also, we find that the VTI1B mRNA have a significant RNA interaction with the ADAMTS5, as a down-regulated tumor suppressor gene in the BC. Also, we find that lncRNA BAIAP2-AS1 has a significant interaction with ADAMTS5, as an up-regulated tumor suppressor lncRNA. ADAMTS5 also is a potential target of hsa-miR-181a-5p, as a competitive endogenous RNA (ceRNA) of lncRNA CRNDE. We find a multi-component ceRNA network that regulated by has-miR-135b-3p. From this network, we selected the CRNDE - hsa-miR-135b-3p – ADAMTS5 axis and predicted that lncRNA CRNDE and ADAMTS5 could indirectly affect expression of each other by changes in expression level and binding affinity of hsa-miR-135b-3p in breast cancer patients.
Based on this hypothesis, we evaluated the expression level of ADAMTS5 in human BC samples in order to gain some understanding of the function of this network and the changes in gene expression in this axis. Our real-time PCR experiment revealed that the expression level of ADAMTS5 is decreasing in breast cancer patients. Also, these two RNA could be the diagnostic and prognostic biomarkers, based on ROC and clinicopathological analyses. Also, we find that the lower expression level of lncRNA BAIAP2-AS1 has a significant correlation with the higher death rate of the patients.
Generally, the possible role of Has-miR-135b-3p in some different human diseases have been investigated. Based on previous studies, this micro-RNA could have some effect on Breast cancer (50), Epithelial Ovarian cancer (51), and Adenocarcinoma (52). However, the main characteristics of this miRNA in different cancer types – including Breast cancer - is still unclear. Based on miRWalk and lncBase analysis, we demonstrated that this miRNA could regulate the ADAMTS5 and lncRAN CRNDE. CRNDE is the gene symbol for colorectal neoplasia differentially expressed (non-protein-coding), a long non-coding RNA (lncRNA) gene that expresses numerous splice variants and has a tissue-specific expression pattern. According to previous studies, this lncRNA could have some essential role in cancer, neurobiology, and development (53). This non-coding RNA is involved in enteral metabolism by regulating different genes, responding to insulin/IGF signaling. CRNDE nuclear transcripts are specifically downregulated by insulin, IGF1, and IGF2, by both the PI3K/Akt/mTOR and Raf/MAPK signaling pathways (54). This lncRNA also could promote glioma cell growth and invasion through mTOR signaling pathway (55). Interestingly, the effect of CRNDE on the growth of myeloma cell could perform by suppressing miR-451 (56). About the functions of this lncRNA in several disease, various results have been reported. CRNDE by suppressing miR-348 could promote hepatic carcinoma cell migration, invasion, and proliferation (57). Suppressing miR-145 by this lncRNA could promote proliferation of Gastric cancer cells (58). This RNA by suppressing PUMA expression, enhance the cervical cancer progression (59). Specially in breast cancer, previous studies have been investigated about some different networks. For example, previous studies revealed that CRNDE by inhibiting miR-136 in BC cell lines could activate Wnt/β-catenin signaling and overexpression of this RNA in Breast cancer is reported (60). Our study discussed about a novel and different ceRNA network and specially, a single CRNDE – hsa-miR-135b-3p – ADAMTS5 axis in a multi-component ceRNA network. Based on our investigation, both ADAMTS5 and CRNDE could have the tumor suppressor effect on the human breast cancer.
Based on genecards.com, VTI1B is a protein-coding gene that codes for vesicle transport through interaction with T-SNAREs 1B. Hermansky-Pudlak syndrome and familial hemophagocytic lymphohistiocytosis 4 are two conditions linked to VTI1B. Response to increased platelet cytosolic Ca2+, and wtCFTR as well as late endosome and lysosome, are some of its linked processes (norm and CF). In this study, we performed the DE analysis of the VTI1B in the BC samples for the first time. There was no previous research about the correlation of VTI1B’s expression level with the BC development. About lncRNA BAIAP2-AS1, previous studies revealed that BAIAP2-AS1 activated by E2F1 and HepG2 and PLC5 cell growth and metastasis were suppressed by BAIAP2-AS1 silencing. In general, BAIAP2-AS1 altered the miR-361-3p/SOX4 axis to encourage the growth of HCC (61). Xianli Gong et al. in a same gene expression profiling demonstrated that BAIAP2-AS1 is a differentially expressed RNA in the HBV-related HCC (62). Furthermore, Xiaogang Mao et al.in 2018 revealed that BAIAP2-AS1 is a potential cervical squamous cell carcinoma biomarker and can be considered as a novel predictor of the patients’ survival (63). However, there was no previous study about the effect of changes in the expression level of BAIAP2-AS1 in the BC. In this study, we find that BAIAP2-AS1 has a significant high-expression in the BC patients. Also, based on our bioinformatics investigation, there was a significant positive correlation between the expression of BAIAP2-AS1 and the survival rate of the patients. So, this lncRNA could be a potential predictor of the BC survival. This lncRNA has a significant negative co-expression with ADAMTS5. So, this lncRNA could have a significant suppressor effect on the ADAMTS5’s expression level.
To finding more accurate and valid results, it is suggested that the same experiments be perform on the samples from different populations and examine the possibility of being biomarker in the different disease. This study demonstrates a possible ceRNA network that could have effect on Breast cancer. Additionally, as we select and analyzed the has-miR-135b-3p completely bioinformatically, we suggest that the expression level of this micro-RNA be analyzed in next investigations and the biomarker capability of this miRNA be evaluated. High or low expression of this miRNA could have some essential information about the oncogenic or tumor suppressor effect of this miRNA on Breast cancer, especially in Isfahan. Further studies could make these results clearer and have more accurate information about this network and the correlation of it in different cancer types, including BC. Furthermore, we highly recommended that the expression level of BAIAP2-AS1, VTI1B, and lncRNA CRNDE be evaluated by qRT-PCR experiment on the human clinical tumor samples, or in the different BC cell lines. Also, we suggest that the RNA interaction of BAIAP2-AS1 with ADAMTS5 be evaluated by luciferase assay experiments for validation of this study.