Silencing serine/arginine-rich splicing factor 1 to induce apoptosis and autophagy-dependent cell death in cholangiocarcinoma through the correction of oncogenic splicing errors.

Abstract

Cholangiocarcinoma (CCA) is a bile duct cancer often linked to liver fluke infections. In Thailand, it has a high incidence and mortality rate due to imprecise symptoms, late diagnosis, and limited treatments. Our previous research identified gene-splicing errors as a factor in CCA progression. This study focused on the role of serine/arginine-rich splicing factors (SRSFs) in CCA. SRSF1-SRSF12 expressions were analyzed using reverse transcription polymerase chain reaction (RT-PCR), confirming upregulation with RT quantitative PCR, western blotting, and dataset analysis. One SRSF was selected for silencing in CCA cell lines (KKU-055 and KKU-213A) using small interfering RNAs. Cell proliferation and survival were assessed through various assays, while apoptosis and autophagy markers were analyzed using western blotting, immunofluorescence, RT-PCR, and transmission electron microscopy (TEM). Splicing errors in death-related genes were also examined. The results showed that SRSF1 was significantly overexpressed in CCA, especially among gastrointestinal cancers. Silencing SRSF1, particularly in KKU-213A cells, reduced cell proliferation and increased cell death. Apoptosis was marked by increased caspase-3 and BAX, decreased Bcl-2, and cytochrome C release. Autophagy was indicated by a higher LC3B-II/ LC3B-I ratio, lower p62, and increased Beclin-1 and ATG5. TEM further confirmed apoptotic and autophagic cell changes. Splicing errors in key death-related genes, including Δ133p53, AGR2vH, MKNK2-b, PKM2, MCL-1L, and BIN1+12a, were also detected. These findings suggest that SRSF1 plays a key role in CCA by regulating apoptosis and autophagy. Its silencing promotes cancer cell death, partly by correcting oncogenic splicing errors, making it a potential target for CCA treatment.