Objective To investigate the molecular mechanism by which long non-coding RNA (lncRNA) titin antisense RNA 1 (TTN-AS1) regulates the microRNA (miR)-134-5p/epidermal growth factor receptor (EGFR) axis in breast cancer cell proliferation, apoptosis and glycolysis.
Methods Western blot was used to detect EGFR protein expression. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure the expression levels of lncRNA TTN-AS1, miR-134-5p and EGFR mRNA in breast epithelial cells MCF-10A and breast cancer cell lines MCF-7, MDA-MB-231 and SKBR-3. MDA-MB-231 cells were selected for subsequent experiments, and were divided into control, si-lncRNA TTN-AS1, si-lncRNA TTN-AS1+miR-134-5p-inhibitor and si-lncRNA TTN-AS1+EGFR-mimic groups. Cell proliferation rates were determined using the CCK-8 assay. Apoptosis rates were assessed by flow cytometry. Lactic acid, glucose and adenosine triphosphate (ATP) kits were used to detect glucose intake, lactic acid and ATP production in each group, respectively. Dual-luciferase reporter assays were performed to identify the binding sites between lncRNA TTN-AS1 and miR-134-5p, as well as between miR-134-5p and EGFR.
Results Compared with MCF-10A cells, the expression levels of lncRNA TTN-AS1 and EGFR mRNA were significantly higher, while miR-134-5p expression was significantly lower in breast cancer cell lines (P < 0.05). Among these, MDA-MB-231 cells exhibited the highest expression levels of lncRNA TTN-AS1 and EGFR mRNA, and the lowest expression level of miR-134-5p (P < 0.05). Compared with the control group, the expression level of miR-134-5p was significantly increased, while the expression levels of lncRNA TTN-AS1, EGFR mRNA, and EGFR protein were significantly decreased in the si-lncRNA TTN-AS1 group (P < 0.05). Compared with the si-lncRNA TTN-AS1 group, the expression levels of EGFR mRNA and EGFR protein were significantly increased, and miR-134-5p expression was significantly decreased in the si-lncRNA TTN-AS1+miR-134-5p-inhibitor group and si-lncRNA TTN-AS1+EGFR-mimic group (P < 0.05). Compared with the control group, cell proliferation rates were significantly reduced in the si-lncRNA TTN-AS1 group (P < 0.05). Compared with the si-lncRNA TTN-AS1 group, cell proliferation rates were significantly increased in the si-lncRNA TTN-AS1+miR-134-5p-inhibitor group and si-lncRNA TTN-AS1+EGFR-mimic group (P < 0.05). Compared with the control group, apoptosis rates were significantly increased in the si-lncRNA TTN-AS1 group (P < 0.05). Compared with the si-lncRNA TTN-AS1 group, apoptosis rates were significantly reduced in the si-lncRNA TTN-AS1+miR-134-5p-inhibitor group and si-lncRNA TTN-AS1+EGFR-mimic group (P < 0.05). Compared with the control group, glucose uptake, lactate production and ATP generation were significantly decreased in the si-lncRNA TTN-AS1 group (P < 0.05). Compared with the si-lncRNA TTN-AS1 group, glucose uptake, lactate production and ATP generation were significantly increased in the si-lncRNA TTN-AS1+miR-134-5p-inhibitor group and si-lncRNA TTN-AS1+EGFR-mimic group (P < 0.05). The dual-luciferase reporter assay confirmed that lncRNA TTN-AS1 had a targeted binding site with miR-134-5p, and miR-134-5p has a targeted binding site with EGFR.
Conclusion The lncRNA TTN-AS1 may negatively regulate the miR-134-5p/EGFR axis, thereby affecting the proliferation, apoptosis and glycolysis of breast cancer.
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