Silencing of SRSF1 to induce cholangiocarcinoma cell death

Abstract

Cholangiocarcinoma or CCA is a malignance cancer arising from abnormal growth of bile duct epithelial. CCA present the high incidence and mortality rate in Southeast Asian countries, especially in Thailand. CCA development does not have any specific symptoms. Therefore, CCA difficult to detect or diagnosis in early state, then developed into advance stages with highly metastasis which resulting in ineffective treatment and leading to high mortality rate. Several strong evidences presented the association of CCA development and progression with aberrant alternative splicing. These processes regulated by Serine/Arginine-rich splicing factors or SRSFs. Dysregulation of SRSFs can generated the aberrant transcript variants and cancer related protein isoforms of many important genes in CCA. Some of the spliced genes mentioned in CCA related to anti-apoptosis and promoted cancer cells evading from death. Previous studies reported high expression of SRSF1 in various cancers and the result from our group also found that SRSF1 was upregulated in CCA patient tissues and in CCA cell lines. This study aimed to investigate the role of SRSF1 on CCA cell death. Particularly, to clarify the specific type of death. SRSF1 mRNA and protein were verified that predominantly upregulated in KKU-055 and KKU-213A (CCA cells) when compared with MMNK-1 (cholangiocyte). Next, to study the effect of SRSF1-silencing by 2 specific siRNAs (siSRSF1-1 and siSRSF1-2), SRSF1 expression was depleted in both CCA cell lines. Especially, the higher efficiency was observed in siSRFS1-2 transfection. For the effect of SRSF1-silencing on CCA cell death, SRSF1-silencing increased the number of death cell and the specific morphologies of death cell including apoptosis, autophagy and necrosis were observed under transmission electron microscope (TEM). For apoptosis, SRSF1-silencing was slightly increase apoptotic cell number and caspase 3/7 enzyme activity which confirmed by upregulation of BAX and downregulation of Bcl-2. For autophagy-dependent cell death, SRSF1-silencing was downregulated p62 protein and upregulated ATG5 and Beclin-1 mRNA, and increased the LC3B-II and LC3B-I protein expression ration. From these results suggest that high expression of SRSF1 promotes CCA cell evading from death which verified by SRSF1 silencing by siRNAs can induce CCA cell to apoptosis and autophagy-dependent cell death. Therefore, these results might be used as supportive information for applying the SRSF1 targeting as an alternative treatment strategy for CCA.

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