Lymphatic metastasis-associated circRNA‒miRNA‒mRNA network for exploring the pathogenesis and therapeutic target of triple negative breast cancer based on whole-transcriptome sequencing analysis: an experimental verification study.

Background: The metastatic mechanisms of axillary lymph nodes (ALNs) in triple-negative breast cancer (TNBC) remain unclear. We aimed to identify the potential circRNA regulatory network in ALN metastasis.

Methods: We performed whole transcriptome sequencing (WTS) to determine the expression profiles of RNAs and screen out differentially expressed messenger RNAs (DEMs), microRNAs (DEMis), and circRNAs (DECs) between ALN-positive and ALN-negative TNBC patients. Functional enrichment analysis and Kaplan-Meier survival analysis were utilized to unearth the potential regulatory mechanisms of the DEMs. A competing endogenous RNA (ceRNA) network was constructed using computational biology. The expression levels of DECs in cell lines were confirmed by real-time polymerase chain reaction (RT‒PCR).

Results: Following WTS and differential expression analysis, 739 DEMs, 110 DEMis, and 206 DECs were identified between ALN-positive and ALN-negative TNBC patients. Functional analysis indicated that the DEMs mainly functioned in carcinogenesis and tumor progression-related pathways. ceRNA networks containing eight circRNAs, six miRNAs, and eighteen mRNAs were developed. In the ceRNA network, two mRNAs (RAB3D and EDARADD) that were significantly associated with better overall survival and one mRNA (GSR) that predicted favorable recurrence-free survival in TNBC patients were chosen for further analysis. Then, a survival-related ceRNA network containing two DECs (hsa_circ_0061260 and hsa_circ_0060876), two DEMis (hsa-miR-5000-3p and hsa-miR-4792), and three mRNAs (GSR, RAB3D, and EDARADD) was identified. Then, two candidate DECs were validated by real-time PCR.

Conclusion: Our research constructed a ceRNA network that provides novel insights into the molecular mechanism of ALN metastasis and potential therapeutic targets in TNBC.