Molecular Mechanism Of The Synthetic Lethal Effect Of SWI/SNF Complex Subunit ARID1A And Aldehyde Dehydrogenase 2 In Cholangiocarcinoma

Cholangiocarcinoma is the second most common tumor in the liver derived from bile duct epithelial cells,accounting for about 10%-15% of all hepatobiliary malignancies.Due to the lack of effective non-surgical treatment and early clinical diagnostic markers,patients with cholangiocarcinoma have a poor prognosis,with an overall 5-year survival rate of less than 5%,and a median survival time of only 3years.With the gradual rise of the concept of precision medicine,the research of synthetic lethal therapy has developed rapidly and has achieved clinical success.The principle is: inhibiting the simultaneous inactivation of two non-lethal genes and ultimately leading to cancer cell death.Due to the development of next-generation sequencing technology,a large number of gene mutations have been identified in different types of cancers.ARID1 A as the gene encoding the SWI/SNF complex subunit BAF250 a has a mutation frequency of up to 20% in cholangiocarcinoma.In this study,we used bioinformatics methods to screen the potential synthetic lethal gene paired: acetaldehyde dehydrogenase 2(ALDH2)and ARID1 A.To verify the synthetic lethal effect of ARID1 A and ALDH2 in cholangiocarcinoma,we were constructed two ARID1 A knock-out cholangiocarcinoma cell lines(HCCC9810 and Hu CCT1)using CRISPR/Cas9 technology.In the cell clone formation experiment,we used the efficient,cheap,and easily available acetaldehyde dehydrogenase 2 inhibitor Disulfiram(Disulfiram,DSF)to verify the synthetic lethality phenotype at the level of ARID1 A wild-type and knock-out cholangiocarcinoma cells;to explore the molecular mechanism of the synthesis lethal,we used flow cytometry to detect the increase in reactive oxygen species(ROS)content in cholangiocarcinoma cells after ARID1 A gene knockout,combined treatment with acetaldehyde dehydrogenase 2 inhibitors,the content of ROS in cholangiocarcinoma cells further accumulates.Based on this phenomenon,it is hypothesized that ROS is the cause of death of cholangiocarcinoma cells after co-treatment with DSF and ARID1 A knockout.To verify the hypothesis,we used ELISA experiments and flow cytometry to prove that ROS directly caused DNA damage to ARID1 A knockout cholangiocarcinoma cells and induced apoptosis,on the other hand,Western blot results showed that ASK1 signaling pathway activated by ROS in ARID1 A knockout type cholangiocarcinoma cells,and the apoptosis executive proteins Caspase3 and PARP1 has activated through the mitochondrial pathway to induce apoptosis.Based on bioinformatics analysis,this study screened out the synthetic lethal effects gene pair ARID1A/ALDH2 in cholangiocarcinoma cells.We clarified from the perspective of oxidative stress and DNA damage at the cellular level by constructing a stable transfer knockout cell line for cholangiocarcinoma.The molecular mechanism of the ARID1A/ALDH2 gene in inducing the death of cholangiocarcinoma cells provides treatment strategies for cancer patients with ARID1 A inactivating mutations and provides theoretical basis and application practice for the precise treatment of cholangiocarcinoma.