Genomic analyses identify biological processes of miR-125 mediated breast cancer cells


 miRNAs contain suppressive and oncogenic properties to regulate the progression of cancers. The expression of miR-125b expression is reported to be consistently low in breast cancers. However, the function and mechanism of miR-125b are not fully understood in breast cancers. In our study, our objective is to identify the DEGs and biological processes in the miR-125b mediated breast cancer cells by analyzing the RNA-seq data. The GSE123358 dataset was produced by the Illumina HiSeq 2000 (Homo sapiens). The KEGG and GO analyses showed the mitochondria and endoplasmic reticulum oxidative phosphorylation pathways are the main processes in miR-125b mediated breast cancer. Moreover, we identified ten interactive molecules including UQCRQ, CALR, HNRNPU, ATP5G1, NDUFB11, UQCRH, HSP90B1, TGOLN2, SAP18, and XPOT. Thus, our study may benefit the treatment of breast cancer by using miR-125b.


Introduction
Breast cancer is a heterogeneous disease, which is the most common cancer in women 1 . Recently, treatments have been involved in more biologically-directed therapies to decrease the adverse effects 2 .
Early breast cancer is considered curable due to the reason that cancer only spreads to the axillary lymph nodes 3 . Improvements in multiple therapies contribute to enhancing chances for cure in most patients 4 .
Conversely, metastatic diseases are not curable by using the available therapeutic methods 5 . For metastatic cancer, the main goals of therapy are to prolong survival and improve quality of life 6 . Small RNAs are de ned by their length and association with AGO family proteins, which are divided into three classes including miRNA, siRNA, and piRNA 7 . miRNA contains about 22 nucleotides and are created by RNase III proteins, Drosha, and Dicer 8 . miRNA functions as a guide by matching its target mRNAs, whereas AGO proteins function as translational repressors and mRNA decay 9 . The miRNA regulation involves multiple steps such as transcription and Drosha/Dicer procession 10 . Most protein-coding genes include at least one conserved miRNA-binding site, which may be directly regulated by miRNAs 11 . miR-125 is an important mediator in cancers, which binds to the RNA-induced silencing complex (RISC) to degrade the target mRNAs or inhibit the translation through binding the 3'UTRs 12 . miR-125 contains a synergistic effect with a similar miRNA cluster 13 . Moreover, miR-125 has the ability of lncRNA to repress the expression 14 .
In this study, we analyzed the effects of miR-125b on breast cancer cells by using the RNA-seq data. We gured out several DEGs and functional biological processes. We also introduced the gene enrichment and the protein-protein interaction (PPI) network to nd the interacting molecules. The signi cant signaling found in our study will improve the treatment of breast cancer.

Data resources
Gene dataset GSE123358 was obtained from the GEO database. The data was produced by the Illumina HiSeq 2000 (Homo sapiens) (Special Histopathology Laboratory, National University of Singapore, Lvl 3, NUH Main Building, 5 Lower Kent Ridge Road, Kent Ridge, Singapore). The analyzed dataset includes 12 high miR-125b breast cancer cells (H) and 9 low miR-125b breast cancer cells (L).

Data acquisition and processing
The data were organized and conducted by R package as previously described [15][16][17] . We used a classical t-test to identify DEGs with P< 0.05 and fold change ≥1.5 as being statistically signi cant.
The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) KEGG and GO analyses were conducted by R package (clusterPro ler) and Reactome. P<0.05 was considered statistically signi cant.

Protein-protein interaction (PPI) networks
The Molecular Complex Detection (MCODE) was used to construct the PPI networks. The signi cant modules were produced from constructed PPI networks. The pathway analyses were performed by using Reactome (https://reactome.org/), and P<0.05 was considered signi cant.

Results
Identi cation of DEGs by the impact of miR-125b on breast cancer cells To determine the mechanism of miRNA-125b on the transcriptome of human breast cancer cells, we analyzed the sorted cells with maximum and minimum miR-125b expression. A total of 48 genes were identi ed with the threshold of P< 0.05. The top ten of up-and-down-regulated genes by the impact of miR-125b on cancer cells are identi ed by the heatmap and volcano plot ( Figure 1). The top DEGs were indicated in Table 1.

PPI network construction
To further determine the relationship of the DEGs, we constructed the PPI network by String network and Cytoscape tool as described [17][18][19] . The combined score was set > 0.2 as a cutoff to construct the PPI network by using the 48 nodes and 32 edges. Table 2 showed the top ten molecules with the highest degree scores. The top two cluster modules were indicated in Figure 3. We further analyzed the DEGs and the PPI network by Reactome (Figure 4 and Supplemental Table S1). We also determined the top ten signi cant biological processes through Reactome map including "ATF6 (ATF6-alpha) activates chaperone genes", "ATF6 (ATF6-alpha) activates chaperones", "Respiratory electron transport, ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins", "Formation of ATP by chemiosmotic coupling", "Response of EIF2AK1 (HRI) to heme de ciency", "Cristae formation", "The citric acid (TCA) cycle and respiratory electron transport", "Unfolded Protein Response (UPR)", and "Cellular responses to stress".  22 . Interestingly, mitochondria metabolism involves various biological processes [23][24][25][26] , which is an effective target for cancer.

Discussion
Reprogramming metabolic pathways could ameliorate the metabolic inhibitors to suppress cancers, such as triple-negative breast cancer 27 . The endoplasmic reticulum (ER) attributes to the maintenance of cellular functions including organelle and lipogenesis, which plays essential roles in the progression of cancer 28-30 . Milad Ashra zadeh et al also found the autophagy and ER stress are promising methods to treat breast cancer 31 .
Base on the ndings of KEGG and GO, we also identi ed ten signi cant molecules that involve in the miR-125b mediated breast cancer. UQCRQ was identi ed as the signi cant protein in triple-negative breast cancer 32 . Eduardo Cruz-Ramos et al found the CALR levels were increased in breast cancer cells lacking estrogen receptor alpha (ERα), in comparison with those that express Erα 33 . G-protein-coupled receptors (GPCRs) and Regulators of G protein signaling (RGS) are key regulators that modulate a variety of human diseases including arthritis, bone, and heart diseases [34][35][36][37][38][39][40][41][42][43] . Interestingly, calreticulin has been found to interact with several GPCR proteins such as AT1R and V2R 44  In conclusion, this study showed the impact of miR-125b on breast cancer. The mitochondria and endoplasmic reticulum oxidative phosphorylation pathways are mainly processes in miR-125b mediated breast cancer. Therefore, this study may be bene cial in the treatment of breast cancer by using miR-125b.

Funding
This work was not supported by any funding.

Declarations of interest
There is no con ict of interest to declare.   The PPI network analysis of DEGs from the low and high expression of miR-125b in breast cancer cells Cluster 1 (A) and cluster 2 (B) were the top two clusters and were constructed by MCODE in Cytoscape.