KAL suppresses cholangiocarcinoma growth via downregulating miR-21 and modulating the PTEN/AKT signaling pathway

This study aims to explore the mechanism by which KAL inhibits the proliferation and migration of cholangiocarcinoma (CCA) cells through the downregulation of miR-21, thereby modulating the PTEN/AKT signaling pathway. Cholangiocarcinoma cell lines HUCCT1 and RBE were cultured and transfected with KAL overexpression plasmids or miR-21 mimics. Transfection efficiency was validated by Western blot. Cell viability was assessed using the CCK8 assay, apoptosis levels were analyzed via flow cytometry, and cell invasion capability was evaluated through Transwell assays. PIP3 levels were measured using ELISA. Western blot was used to detect the expression levels of PTEN, AKT/mTOR signaling pathway proteins, and apoptosis-related proteins, including BCL-2 and Cleaved-Caspase3. Dual-luciferase reporter assays were performed to confirm the interaction between PTEN and miR-21-5p. Based on in vitro experimental results, HUCCT1 cells were selected for in vivo tumorigenicity experiments to assess the effects of KAL overexpression lentivirus on CCA tumor growth. Tumor size, volume, and weight were measured, and immunohistochemistry was used to detect the positive expression levels of KI67, PTEN, BCL-2, and Cleaved-Caspase3 in tumor tissues. PCR and Western blot analyses confirmed the effective transfection of KAL overexpression into HUCCT1 and RBE cells. Overexpression of KAL significantly inhibited CCA cell proliferation and invasion, reduced PIP3 production, suppressed the AKT/mTOR signaling pathway, and promoted apoptosis. PCR results demonstrated that miR-21 mimic transfection into HUCCT1 and RBE cells was successful. Overexpression of miR-21 reversed the inhibitory effects of KAL on CCA cell proliferation and migration, as well as the pro-apoptotic effects of KAL. Additionally, miR-21 suppressed the KAL-induced upregulation of PTEN and Cleaved-Caspase3 and promoted BCL-2 expression. Dual-luciferase reporter assays confirmed a targeted interaction between PTEN and miR-21-5p. In vivo tumorigenicity experiments showed that KAL overexpression significantly inhibited tumorigenesis in CCA cells. Tumor tissues from the KAL overexpression group exhibited significantly increased expression levels of Cleaved-Caspase3 and PTEN and decreased positive expression levels of KI67 and BCL-2, indicating suppressed proliferation and enhanced apoptosis in CCA cells. Overexpression of KAL inhibits CCA cell proliferation and promotes apoptosis. Overexpression of miR-21 reverses the effects of KAL on CCA cells, suggesting that KAL suppresses CCA growth through miR-21-mediated modulation of the PTEN/AKT signaling pathway.