Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 0 0 2025 12 22 1 10 EN Sepideh Mokabberi Department of Molecular Cell Biology and Genetics, Bu.C., Islamic Azad University, Bushehr, Iran Nahid Babaei Department of Molecular Cell Biology and Genetics, Bu.C., Islamic Azad University, Bushehr, Iran Hadi Esmaeili Gouvarchin Ghaleh Applied Virology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical sciences, Tehran, Iran Gholamreza Farnoosh Applied Biotechnology Research Centre, New Health Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran 2025 04 04 2025 09 11 Lung cancer remains a major cause of cancer-related mortality worldwide, with current treatments such as surgery, chemotherapy, and immunotherapy facing limitations, including severe side effects and high costs. Hyperthermia (H) has emerged as a promising strategy to enhance tumor sensitivity to treatments and reduce toxicity. This study investigates the effects of H in enhancing the anti-tumor efficacy of pemetrexed (PEM) and altering the expression profile of hsa-MiR-548c-3p (tumor suppressor) in the A549 cell line. A549 cells were cultured in DMEM medium and divided into four groups: Control, and treatment with H, PEM, and a combination of H and PEM. Subsequently, cell viability, apoptosis percentage, release rate of LDH, production of ROS, and the expression level of hsa-MiR-548c-3p, CASP 8 and 9, and TYMS genes were measured. Results revealed that the combination of H and PEM treatment had greater antitumor effects compared to the other groups. The combination of H and PEM significantly reduced cell viability, increased the percentage of apoptosis, LDH release, and ROS production, and upregulated hsa-MiR-548c-3p, CASP 8, and 9, while downregulating TYMS. The findings suggest that H enhances the chemotherapeutic efficacy of PEM by upregulating hsa-MiR-548c-3p expression and promoting apoptosis in lung cancer cells, making it a promising complementary approach for overcoming drug resistance. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/4406 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/4406/2276