In vivo validation of the functional role of MicroRNA-4638-3p in breast cancer bone metastasis

Abstract

Purpose Skeletal metastases are increasingly reported in metastatic triple-negative breast cancer (BC) patients. We previously reported that TGF-β1 sustains activating transcription factor 3(ATF3) expression and is required for cell proliferation, invasion, and bone metastasis genes. Increasing studies suggest the critical regulatory function of microRNAs (miRNAs) in governing BC pathogenesis. TGF-β1 downregulated the expression of miR-4638-3p, which targets ATF3 in human BC cells (MDA-MB-231). In the present study, we aimed to identify the functional role of miR-4638-3p in BC bone metastasis by the caudal artery injection of the MDA-MB-231 cells overexpressing mir-4638 in the mice. Methods MDA-MB-231 cells overexpressing miR-4638 were prepared by stable transfections. Reverse transcriptase quantitative PCR was carried out to determine the expression of endogenous miR-4638-3p and bone resorption marker genes. X-ray, micro-CT, and Hematoxylin & Eosin studies were used to determine osteolytic lesions, trabecular structure, bone mineral density, and micrometastasis of cells. Results The mice injected with MDA-MB-231 cells overexpressing miR-4638-3p decreased the expression of bone resorption marker genes, compared to MDA-MB-231 cells injection. Reduced osteolytic lesions and restored bone density by MDA-MB-231 cells overexpressing miR-4638-3p were observed. Similarly, the mice injected with MDA-MB-231 cells overexpressing miR-4638-3p showed a better microarchitecture of the trabecular network. A few abnormal cells seen in the femur of MDA-MB-231 cells-injected mice were not found in MDA-MB-231 cells overexpressing miR-4638. Conclusion The identified functional role of ATF3 targeting miR-4638-3p in BC bone metastasis in vivo suggests its candidature as BC therapeutics in the future.