Tehran University of Medical Sciences Immunology and Genetics Journal 2645-4831 8 0 2025 02 24 111 123 Mona Sadeghalvad Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Hamid-Reza Mohammadi-Motlagh Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran Kamran Mansouri Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran Ali Mostafaie Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran Farshid Noorbakhsh Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Sadaf Alipour Breast Disease Research Center, Cancer Institute, Tehran University of Medical Sciences, Tehran, Iran; Department of Surgery, Arash Women’s Hospital, Tehran University of Medical Sciences, Tehran, Iran Foroozan Ghalkhani Imam Reza Hospital, AJA University of Medical Sciences, Tehran, Iran Ramesh Omranipour Breast Disease Research Center, Cancer Institute, Tehran University of Medical Sciences, Tehran, Iran Seid-Shahram Miraghaee Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran Sarah Kiani Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran Nima Rezaei Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; Research Center for Immunodeficiencies, Children’s Medical Center Hospital, Tehran University of Medical Sciences, Tehran, Iran; Network of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN), Tehran, Iran 2025 02 24 2025 02 24 Background: Cancer-associated fibroblasts (CAFs) play an important role in the initiation and progression of tumor cells. These cells can trigger signaling pathways involved in tumor progression. HOTAIR, an increased long non-coding RNA (lncRNA) in breast cancer, has a vital role in the tumorigenesis and development of breast cancer cells. Methods: In this study, a fibroblast cell culture medium was used to investigate its possible role in inducing the HOTAIR expression and PI3K/Akt/mTOR pathway in breast cancer cells. CAFs and normal fibroblasts (NFs) were isolated from tumors of 6 patients with breast cancer and subjects with healthy breasts, respectively. The MCF-7 cells were cultured in a medium obtained from CAFs (CAF-CM) or NFs (NF-CM), and then the expression of HOTAIR and PI3K/Akt/mTOR in MCF-7 cells was assessed using Real-Time PCR. HOTAIR was silenced in MCF-7 cells using siRNAs and then cultured in CAF-CM or NF-CM. Subsequently, the phosphorylation status of PI3K/Akt/mTOR proteins was analyzed by western blotting. Results: Fibroblast culture medium enhanced the expression of HOTAIR and activation of the PI3K/Akt/mTOR pathway in breast cancer cells. By HOTAIR silencing, reduced activity of the PI3K/Akt/mTOR pathway, as well as the lower effect of fibroblast culture medium in the induction of PI3K/Akt/mTOR pathway, was seen. Conclusion: HOTAIR can play a role as a mediator in inducing the PI3K/Akt/mTOR pathway in breast cancer cells by the effect of cancer-associated fibroblast cells. https://igj.tums.ac.ir/index.php/igj/article/view/196 https://igj.tums.ac.ir/index.php/igj/article/download/196/173