Mir 18a 5p agomir

To confirm the expression of miR−18a−5p and HER2 in BC cells and non-cancerous breast cells, human mammary epithelial cell line (MCF−10A) and human breast cancer cell lines (MDA-MB−231, MDA-MB−453, and MCF−7) were acquired from the BeNa Culture Collection (BNCC, China). MCF−10A cells were cultured in MEGM (BNCC), MCF−7 cells in DMEM (BNCC), while MDA-MB−453 and MDA-MB−231 cells were cultured in L−15 medium (BNCC). 10% fetal bovine serum (FBS; BNCC) was added to the culture medium and the cells were maintained at 37°C and 5% CO₂.
The agomiR NC, mimic negative control (NC), miR−18a−5p mimic, and miR−18a−5p agomiR were obtained from GenePharma (China). HER2 overexpression vectors and empty vectors were constructed using pcDNA3.1, obtained from GenePharma (China). Lip2000Transfection Reagent (Biosharp, China) was used to transfect the recombinant plasmids listed above into both MDA-MB−231 and MCF−7 cells. Cell transfection was performed for 48 h and assessed by qPCR analysis.

The study was approved by the Animal Ethics Committee of Wannan Medical College. Four-week-old female thymus-free BALB/c nude mice were randomly divided into the following four groups with three mice in each group to examine tumorigenicity: (i) agomir control group, (ii) GAS5 overexpression + agomir control group, (iii) miR-18a-5p agomir group, and (iv) GAS5 overexpression + miR-18a-5p agomir group. First, T24 cells (stably overexpressing GAS5 or empty vector) were transfected with miR-18a-5p agomir (GenePharma, China) or control agomir (GenePharma) for 48 h. After treatment, the cells were collected and resuspended in PBS, and a single-cell suspension of 2 × 10⁶ cells was inoculated subcutaneously into the right axilla of mice. In addition, to ensure the expression of miR-18a-5p in tumors, we also performed experiments in which agomir-18a-5p NC/agomir-18a-5p was injected directly into implanted tumors at a dose of 10 nmol per mouse (in 20 μL of PBS) twice. The width (W) and length (L) of the tumors were measured every 7 days. Tumor volume was expressed as L × W²/2. The mice were killed after 4 weeks. All tumors were debrided, weighed, and fixed.