miR-301a-5p negatively affects Treg and Th17 related gene expression in contrast to miR-21 and miR-23b-5p in earlystage breast tumor tissue

Lyuba Miteva¹, Stoyan Nikolov², Noyko Stanilov³, Petio Chilingirov⁴, Spaska Stanilova¹

¹Department of Molecular Biology, Immunology and Medical Genetics; Medical Faculty, Trakia University, Stara Zagora, Bulgaria.

²University Hospital, Department of Surgery, Stara Zagora, Bulgaria.

³Breast Oncoplastic Unit, University College London Hospital, London, United Kingdom.

⁴Medical Oncology Clinic, Complex Oncology Center, Stara Zagora, Bulgaria.

Summary

Purpose: This study aimed to investigate the local levels of microRNA (miR) miR-23b-5p, miR-301a-5p, and miR-21 in relation to T regulatory (Treg)/T helper 17 (Th17) related gene expression in paired tissue samples of breast cancer (BC) patients and their association with clinical characteristics of BC.

Methods: Fresh primary tumor samples and adjacent normal tissue specimens were collected for miRNA and total RNA isolation. cDNA synthesis was performed by reverse-transcription PCR, followed by quantitative PCR (qPCR) to determine the relative quantity of miR-21, miR-23b-5p, and miR-301a-5p, as well as eight Treg/Th17 related target genes. Relative quantification analysis was conducted using the comparative Ct (Cq) method.

Results: The study found that miR-21 expression was significantly upregulated by over 6-fold in tumor tissue compared to normal tissue (p=0.002). Additionally, the levels of miR-23b-5p and miR-301a-5p were significantly higher in larger tumors (T2) compared to smaller tumors (T1) (p=0.014 and p=0.009, respectively). MiR-301a-5p expression showed a strong inverse relationship with TGFBR2 (r=-0.6668; p=0.0035) and IL10 (r=-0.6356; p=0.0061). Conversely, miR-21 expression was positively correlated with TGFBR2 (r=0.592; p=0.01), TGFB1 (r=0.6308; p=0.0066), IL10, and FOXP3 with borderline significance. Furthermore, miR-21 expression was positively correlated with IL6 gene expression (r=0.5669; p=0.018), RORC (r=0.5024; p=0.039), and IL12B (r=0.8999; p=0.0374).

Conclusions: The study confirms the oncogenic role of miR-21 and suggests that its function may be counteracted by miR-301a-5p as part of negative feedback mechanisms contributing to Th17-like Treg cancer-associated inflammation in early-stage, luminal breast cancer.

Keywords: breast cancer, miR-21, microRNA, transforming growth factor beta receptor II.

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