miR-149-3p-mediated TIMP3 restoration suppresses tumor aggressiveness in sorafenib-resistant liver cancer cell.

Hepatocellular carcinoma (HCC) is a highly lethal malignancy in which acquired resistance to sorafenib remains a major therapeutic challenge. To investigate mechanisms of resistance, we performed transcriptomic profiling of sorafenib-resistant HCC cells, which revealed enrichment of processes related to tumor progression, including mitotic regulation, chromatin remodeling, and apoptotic signaling. Notably, these cells displayed marked downregulation of tissue inhibitor of metalloproteinases-3 (TIMP3), a tumor suppressor known to regulate invasion and epithelial-mesenchymal transition (EMT). Functional studies showed that TIMP3 overexpression (OV) suppressed cell viability, stemness-associated proteins, and a key cell cycle regulator, while upregulating apoptosis-related proteins. Conversely, TIMP3 knockdown (KD) enhanced proliferation, stemness, and EMT. EMT markers were reduced in TIMP3-OV cells but increased with TIMP3-KD, consistent with spheroid sprouting assays, highlighting TIMP3 as a brake on aggressiveness. To explore upstream regulators, we integrated in silico predictions with validation, identifying miR-149-3p as a novel repressor of TIMP3. miR-149-3p overexpression reduced TIMP3 levels and promoted EMT and invasion, whereas inhibition of miR-149-3p restored TIMP3 expression and suppressed migration. Clinical analyses using TCGA datasets and HCC tissue microarrays confirmed significantly lower TIMP3 expression in tumors compared with normal liver tissue, and showed inverse correlations between TIMP3 and proliferative or stemness markers, including Ki67 and CD44. Collectively, these findings establish a mechanistic axis in which miR-149-3p-mediated suppression of TIMP3 promotes sorafenib resistance, EMT, and stemness in HCC. This work identifies TIMP3 as a pivotal determinant of tumor aggressiveness and suggests restoring TIMP3 or targeting downstream pathways as strategies to overcome resistance.