The Study Of The Molecular Mechanism Underlying The Effect Of ABHD5 And NPC1L1 On The Proliferation And Chemoresistance Of Colorectal Cancer

BackgroundsColon cancer is one of the highest incidence gastrointestinal tumors worldwide in human,and the morbidity and mortality are high for many years.At present,in addition to surgery,chemotherapy is the main means of colon cancer treatment,but the widespread presence of chemotherapy resistance has limited patient benefit.Therefore,in-depth study of the malignant progression of colon cancer and the specific mechanism of chemoresistance is very important to find new therapeutic or chemotherapy sensitization targets,and ultimately improve the prognosis of colon cancer patients.Cellular metabolic reprogramming is one of the prominent features of malignant tumors.Abnormal lipid metabolism is significantly related to the increase of tumor mortality.In order to understand the influence of dyslipidemia on tumors,the main components of serum lipids--triglycerides and cholesterol need to be research firstly.ABHD5（Abhydrolase Domain Containing 5）is coenzyme of ATGL（Adipose Triglyceride Lipase）which plays an important role in the process of triglyceride hydrolysis.ABHD5 has previously been identified as an important tumor suppressor gene,but its role in tumor chemotherapy response is unclear.nowadays,lipid lowering agents are clinically performed by reducing cholesterol,which can be divided into the following three types according to different mechanisms:inhibiting cholesterol synthesis-statins,inhibiting cholesterol absorption-Ezetimibe,and cholesterol esterification inhibitor-Avasimibe.Cholesterol plays an important role in maintaining normal physiological functions,but also participates in the malignant phenotype of tumors.But at the same time,there are studies have found that cancer is associated with lower cholesterol levels in some cases,so it’s important to learn what role of different lipid-lowering drugs playing in colon cancer.Objectives1.To explore the role and mechanism of ABHD5 in the sensitivity of FU in colorectal cancer.2.Explore the effects of different lipid-lowering drugs on colorectal cancer proliferation.Methods1.Analyze the relationship between ABHD5 and the response of fluorouracil（FU）-based therapy by in vivo and in vitro assays.2.Analyze the correlation of ABHD5 with FU absorption and uracil metabolism by liquid chromatography.3.Acquiring the colon cancer data from the public database and analyzing the enrichment pathway of ABHD5^high/low by GSEA.4.The correlation between ABHD5 knockdown and Ribonuclease T2（RNASET2）knockdown on pyrimidine-related metabolites were analyzed by liquid chromatography-mass spectrometry.The relationship between ABHD5 and RNASET2 was explored by rescue experiment.5.Western blot of the extracted lysosomes and cell immunofluorescence assay were used to detect whether the ABHD5 located in lysosomes.6.CO-IP and yeast two-hybrid experiment to research the binding form between ABHD5 and RNASET2.7.HuProtTM human protein chip and CO-IP assay to detect the binding form between ABHD5 and protein disulfide isomerase A5（PDIA5）（inactivated RNASET2）.8.The protein docking method predicts the binding pattern between ABHD5,RNASET2 and PDIA5,and then the relationship between ABHD5,RNASET2 and PDIA5is verified by competitive binding experiments.9.In vitro and in vivo experiments to explore the effects of three kind of lipid-lowering drug such as Avasimibe,Ezetimibe and Fluvastatin on the proliferation and invasion of colon cancer.10.The knocked out NPC1L1 HCT116 cells were constructed.The effects of NPC1L1on the proliferation of colon cancer were studied by CCK-8 assay and colony formation assay.Results1.ABHD5 defience increased the sensitivity of colon cancer to FU therapy.Absorption of FU were increased and uracil production were decreased in ABHD5 KD cells compared to control.2.ABHD5^low group was significantly enriched in lysosomal gene set.RNASET2knockdown and ABHD5 knockdown could reduce the production of pyrimidine-related metabolites.Knocking down RNASET2 in ABHD5 overexpression SW480 cells could reverse FU uptake and uracil production.3.ABHD5 binds indirectly to RNASET2 in lysosomes and directly to PDIA5.Further experiments revealed that ABHD5 competes with RNASET2 for binding to theβ-sheet domain of PDIA5.4.Fluvastatin can inhibit the proliferation and invasion of colon cancer,and there is no significant effect on the proliferation and invasion of colon cancer by Ezetimibe and Avasimibe.5.CCK-8 assay and colony formation experiment showed that specific knockout of NPC1L1 could promoting colon cancer proliferation and colony formation.Conclusions1.Deficiency of ABHD5 increase the sensitivity of colon cancer to FU therapy.2.ABHD5 sustains RNASET2 activity.Activated RNASET2 promotes autophagy degradation of RNA to produce a large number of pyrimidine products,and the accumulation of uracil leads to a decrease in exogenous FU absorption,which leads to decreased sensitivity of FU treatment.3.RNASET2 directedly binding to and be inactivated by PDIA5,while ABHD5competes with RNASET2 for binding to theβ-sheet region of PDIA5,protects RNASET2from being inactivated by PDIA5.4.Different mechanisms of lipid-lowering drugs have different effects on colon cancer proliferation and invasion ability5.Specific knockout of NPC1L1 promotes colon cancer proliferation,suggesting that NPC1L1 participates in the tumorigenesis of colon cancer through a non-cholesterol-dependent classical pathway.