Dysregulation of miRNAs plays key roles in the initiation, differentiation and migration of several types of cancer, including BC (Bertoli et al. 2015). Our study aims to improve BC prognosis by validating DE-miRNAs in BC tissue compared with normal tissue and elucidate the mechanisms regulating particular target genes and pathways. In the current study, miR-130b was identified to be significantly over-expressed in human BC tissue than adjacent normal tissue. After verifying the expression, we investigated the overall survival of miR-130b in BC by using the Kaplan–Meier Plot, which indicated that miR-130b was positively correlated with a worse prognosis. These results elucidated that miR-130b might function as an oncogene when overexpressed in BC. Ana The LinkedOmics database results indicated a strong positive correlation between CDCA8 and miR-130b. We found THAP11 as a transcription factor regulating CDCA8. Accordingly, we hypothesized that THAP11 played key roles in the upstream regulatory mechanism of miR-130b, and regulated the expression level of miR-130b.
THAP11, also known as thanatos associated protein 11, belongs to a unique family of TFs, which recognized particular DNA sequences through atypical zinc finger motif and regulated cell cycle and cell growth (Cukier et al. 2016). THAP11 has been linked and differentially expressed in several cancers, such as gastric cancer, esophageal cancer, and colon cancer (Jin Y 2019; Parker et al. 2012; Zhang et al. 2020). Until now, the function of THAP11 in BC remains elusive.
Next, we attempted to explore the downstream regulatory mechanism of miR-130b in breast cancer via predicting and analyzing genes correlated with miR-130b. A series of functional analysis was used to identify the biological functions and related signaling pathways of miR-130b in BC. Based on the data of GSEA, miR-130b was positively upregulated genes in the TH signaling pathway, such as PFKP, STAT1, SRC, and NOTCH2. Our results suggested that miR-130b was over-expressed in BC, which upregulated oncogenes, including PFKP, STAT1, SRC and NOTCH2, and played an essential role in BC progression.
In our study, PFKP was analyzed as the most positively upregulated gene target in the TH signaling pathway with miR-130b (Pearson- correlation = 0.3212, p < 0.01) in BC. PFKP, also known as phosphofructokinase platelet, is the platelet isoform, which encodes a phosphofructokinase A family protein. PFKP might be a significant mediator for cell metabolism, participating in cancer metastasis and initiation (Lang et al. 2019). Recently, PFKP is recognized for high epidemic property in several kinds of aggressive tumors such as glioblastoma and BC (Lee et al. 2017; Moon et al. 2011). In addition, PFKP is closely related to cell survival of muscle-invasive bladder cancer (Sun et al. 2016). Wang J et al. (Wang J 2016) reported that high mRNA expression levels of PFKP were correlated with amassment of apoptotic proteins and cell proliferation in renal carcinoma, indicating the relationship between PFKP and the growth of renal carcinoma. Moreover, lots of evidence suggested that PFKP act as an oncogene in lung cancer and hepatocellular carcinoma (Park et al. 2013; Wang et al. 2015). Our study disclosed a novel mechanistic interaction between miR-130b and PFKP, supposed that the BC progression may be reduced by down-regulating miR-130b, which reduces the expression level of PFKP and constrains the cell initiation and metastasis.
Apart from PFKP, the other three oncogenes associated with miR-130b, including STAT1, SRC and NOTCH2 were worthy of conducting further investigations. Previous studies reported that most of them play a significant role in cell proliferation and differentiation by up-regulating in various human tumors and are closely associated with their overall survival. The expression of STAT1 is related to cell growth, regulation, immune evasion and metastasis (Ryan et al. 2020). Zellmer et al. reported that STAT1 is a latent therapeutic target for BC by promoting tumor progression (Zellmer et al. 2017). As a central messenger, SRC correlated with the regulation of cell growth, differentiation, survival, angiogenesis, and invasion in several important signaling pathways (Espada and Martin-Perez 2017; Yeatman 2004). Highly expressed and activated SRC found in several solid tumors, including breast cancer tissue was involved in promoting cell invasiveness and metastatic potential and decreased the overall survival of patients (Djeungoue-Petga et al. 2019). NOTCH2 regulates tumor growth, invasion and metastasis (Kim et al. 2012; O'Neill et al. 2007). It was found that BC cells with Notch2 knockdown had a slowed down migration and poor cell survival rate, indicating Notch2 may play an oncogenic role (Chao et al. 2014).
To get a deeper insight into the downstream target genes of miR-130b, we analyzed the databases from DIANA-mT, miRDB, miRWalk, Targetscan and GEPIA. 54 common genes were selected, and the PPI network suggested that AR, KIT and ESR1 were hub genes, indicating the pivotal molecular function of miR-130b. The androgen receptor (AR) played a key role in mediating androgens biological effect, that act as a prognostic indicator in tumor growth, metastasis, relapse, hormonal therapy and chemotherapy (Feng et al. 2017). Interestingly, many studies have suggested that the expression level of AR is related to higher prognostic in BC (Qu et al. 2013; Vera-Badillo FE 2014). Proto-oncogene c-Kit (KIT), encodes a receptor tyrosine kinase and participates in regulating cell migration, proliferation and survival (Janostiak et al. 2018). The function of c-KIT in the malignant transformation of tumors might be two faceted, either oncogene or tumor suppressor gene. Recent studies indicated that the malignant transformation of breast epithelium was promoted by downregulating c-KIT expression (Chui 1996; Talaiezadeh et al. 2012), and c-KIT could serve as a biomarker for atypical and malignancy proliferative ductal breast lesions (Janostiak et al. 2018). Estrogen receptor 1 (ESR1) encodes an estrogen receptor that mediates the transcription of many estrogen-inducible genes essential for several reproductive functions, such as cell growth, metabolism, sexual development, and pregnancy, it is also expressed in other non-reproductive tissues such as bone (Bockers et al. 2020; Carleton et al. 2020; Pakharenko et al. 2020). ESR1 mutations have proved to be a key factor in endocrine therapy resistance in BC with hormone receptor-positive. ESR1 mutant provided an enrichment of subclonal cell populations in circulating tumor cells and metastatic sites,which may be partly due to an enhanced “aggressive phenotype”(Dustin et al. 2019). It could be used as a predictive biomarker in hormone-receptor BC, especially in the metastatic setting (Carausu et al. 2019). Based on the finding of current studies, miR-130b might be significantly correlated with BC via the above molecular mechanisms.
In summary, we revealed that the expression level of miR-130b was upregulated in BC tissue compared to normal tissue, which was significantly correlated with a poor prognosis of BC, signify that miR-130b functions as a significant oncogene in BC. Moreover, our study demonstrated that the expression level of miR-130b was regulated by THAP11 and further facilitated BC's development by regulating crucial downstream genes of the TH signaling pathway, including PFKP, STAT1, SRC, and NOTCH2. On the other hand, we predicted the potential downstream target hub genes of miR-130b, AR, KIT, and ESR1. This study for the first time, reported the novel molecular mechanisms between miR-130b and BC to the best of our knowledge. Therefore, we identified that miR-130b might act as a prognostic biomarker in BC. However, further in-depth research on molecular mechanisms of miR-130b will be helpful to provide a novel therapeutic approach for the treatment of BC.