| Objective:In our previous study,it was found that adipose could induce the radioresistance of cholangiocarcinoma(CCA)cells by using the CCA cell/adipose co-culture system;and SULT2A1 was further screened from the CCA cells co-cultured with adipose cells by high-throughput proteomics combined with bioinformatics analysis,which is one member of the soluble sulfonyltransferase superfamily related to the detoxification and excretion mechanism of bile acids(BAs).In order to clarify the relationship between the BAs metabolism and the radiosensitivity of CCA cells,and to investigate the biological effects and potential regulatory mechanisms of BAs in regulating the radiosensitivity of cells.Methods:Firstly in the present study,the mice model was constructed by exposeing hepatobiliary region to a single dose of 30 Gy X-ray irradiation using X-RAD SmART small animal radiotherapy simulator,followed by transmission electron microscope and blood biochemical analysis confirmation.Then,targeted metabonomics was applied to explore the radiation-responsive BAs in the mice model.Subsequently,CCK-8 method was performed to identify the toxic effects of radiation-responsive BAs on CCA cell lines,and flow cytometry assay was adopted to detect the effects of BAs on cell cycle distribution and apoptosis.The clonogenic assay was then performed,and Chenodeoxycholic Acid(CDCA)was chosen for the following study,because it was the only radiation-responsive BAs that had a significant effect on the radiosensitivity of CCA cells.Afterwards,immunofluorescence staining of phosphorylated γ-H2AX focus and neutral single cell gel electrophoresis were used to determine the specific effects of CDCA on DNA double strand breaks(DSBs)in irradiated CCA cells.Western blot assay was used to detect the effects of CDCA on the protein expression of DNA damage repair related factors such as Rad50,Mre11 and Ku-80.Furthermore,pathway specific antagonists,immunofluorescence staining and flow cytometry were used to determine whether or not CDCA regulated the radiosensitivity of CCA cells in a nuclear receptor farnesol X receptor(FXR)dependent way.Finally,high-throughput RNA sequencing combined with bioinformatics analysis was used to explore the downstream key effector molecules and potential molecular mechanisms of CDCA/FXR signal pathway involved in the regulation of radiosensitivity of cholangiocarcinoma cells.Results:Two weeks after X-ray irradiation,relatively normal liver function as well as significant ultrastructural variations were observed in the hepatobiliary tissues in mice model.And the results from targeted metabonomics showed that primary BA CDCA and secondary bile acid ursodeoxycholic acid(UDCA)decreased significantly,while secondary bile acid taurine deoxycholic acid(TDCA)and glycine deoxycholic acid(GDCA)increased significantly,compared with the control group.CCK-8 experiment showed that 200 μM of CDCA could survive 80%of cholangiocarcinoma cells,while UDCA,TDCA,and GDCA had no obvious toxic effects on CCA cells.Thus 200 μM of CDCA was adopted for the following functional experiments.Compared with the radiation treatment alone,CDCA could significantly alleviate the radiation-induced cell cycle G2/M phase arrest and the radiation-induced cell apoptosis in CCA cells,while other radiation-responsive bile acids failed to manifest similar effects.Therefore,the follow-up experiments will mainly explore the biological effect of CDCA on the radiosensitivity of CCA cells.The results of colony formation assay showed that CDCA could significantly increase the survival fraction of cholangiocarcinoma cells after irradiation.By fitting the curve to multi-target-single-hit model,the sensitization enhancement ratio(SER)was calculated as 0.83<1,which indicated decreased radiosensitivity of CCA cells.CDCA can significantly reduce the formation of phosphorylated y-H2AX focus in nucleus,and alleviate the DSBs,as well enhance the expression of DNA damage and repair related proteins in CCA cells after irradiation.CDCA can activate FXR,presents as the translocation of FXR from cytoplasm to nucleus in RBE cells.The FXR specific antagonist Guggulsterone can to some extent reverse the effect of CDCA on cell cycle distribution and apoptosis of cholangiocarcinoma cells after irradiation.Compared with the 8Gy X-ray irradiation group,496 differentially expressed genes were screened by RNA sequencing in the CDCA combined irradiation group,of which 150 genes were down-regulated and 346 genes were up-regulated.Bioinformatics analysis showed that the above differential genes were mainly involved in the biological processes such as regulating cholesterol biosynthesis,cholesterol biosynthesis,solid alcohol biosynthesis and so on.cell localization analysis showed that the differential genes were mainly located in the structural parts of cells,such as semi-desmosome,extracellular space,plasma membrane and so on.The molecular function analysis of differential genes shows that most of the differential genes are mainly involved in cellular and molecular functions,such as tyrosine protein kinase binding,collagen binding,receptor binding and so on.In addition,the results of KEGG signal pathway analysis of differential genes showed that PI3K-AKT signal pathway,proteoglycan and tumor related signal pathway,relaxin signal pathway,estrogen signal pathway,TGF-β signal pathway,steroid biosynthesis,fatty acid biosynthesis and other signal pathways may also be involved in the process of CDCA regulating radiosensitivity of cholangiocarcinoma cells.Finally,the(GSEA)results of gene enrichment analysis also showed that PI3K-AKT signal pathway,fatty acid chain elongation synthesis signal pathway,JAK-STAT signal pathway,cell cycle related signal pathway and other pathways may also be involved.Conclusions:The primary bile acid CDCA might regulate the radiosensitivity of CCA cells to X-Ray,probably by activating the nuclear receptor FXR and downstream signalings.The CDCA/FXR bile acid metabolic pathway might be a novel therapeutic target for CCA. |
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