#METABOLOMICS WORKBENCH zhaoyuan0315_20241104_174136 DATATRACK_ID:5344 STUDY_ID:ST003568 ANALYSIS_ID:AN005861 PROJECT_ID:PR002202 VERSION 1 CREATED_ON November 10, 2024, 9:45 pm #PROJECT PR:PROJECT_TITLE High mobility group A1 (HMGA1) promotes the tumorigenesis of colorectal cancer PR:PROJECT_TITLE by increasing lipid synthesis PR:PROJECT_TYPE LC-MS PR:PROJECT_SUMMARY Metabolic reprogramming is a hallmark of cancer, enabling tumor cells to meet PR:PROJECT_SUMMARY the high energy and biosynthetic demands required for their proliferation. High PR:PROJECT_SUMMARY mobility group A1 (HMGA1) is a structural transcription factor and frequently PR:PROJECT_SUMMARY overexpressed in human colorectal cancer (CRC). Here, we show that HMGA1 PR:PROJECT_SUMMARY promotes CRC progression by driving lipid synthesis in a AOM/DSS-induced CRC PR:PROJECT_SUMMARY mouse model. Using conditional knockout (Hmga1△IEC) and knock-in PR:PROJECT_SUMMARY (Hmga1IEC-OE/+) mouse models, we demonstrate that HMGA1 enhances CRC cell PR:PROJECT_SUMMARY proliferation and accelerates tumor development by upregulating fatty acid PR:PROJECT_SUMMARY synthase (FASN). Mechanistically, HMGA1 increases the transcriptional activity PR:PROJECT_SUMMARY of sterol regulatory element-binding protein 1 (SREBP1) on the FASN promoter, PR:PROJECT_SUMMARY leading to increased lipid accumulation in intestinal epithelial cells. PR:PROJECT_SUMMARY Moreover, a high-fat diet exacerbates CRC progression in Hmga1△IEC mice, while PR:PROJECT_SUMMARY pharmacological inhibition of FASN by orlistat reduces tumor growth in PR:PROJECT_SUMMARY Hmga1IEC-OE/+ mice. Our findings suggest that targeting lipid metabolism could PR:PROJECT_SUMMARY offer a promising therapeutic strategy for CRC. PR:INSTITUTE Henan University PR:LAST_NAME Xu PR:FIRST_NAME Zhi-Xiang PR:ADDRESS Jinming Road, Kaifeng, Henan province, 475000, China PR:EMAIL zhixiangxu08@gmail.com PR:PHONE 86-13270538760 #STUDY ST:STUDY_TITLE Differentially regulated metabolites in intestinal epithelial cells from ST:STUDY_TITLE Hmga1flox/flox and Hmga1△IEC mice ST:STUDY_SUMMARY Dysregulations of cell metabolism, such as elevated aerobic glycolysis and ST:STUDY_SUMMARY increased fatty acid metabolism, play a critical role in the tumorigenesis of ST:STUDY_SUMMARY colorectal cancer (CRC). To determine whether HMGA1 promotes CRC through ST:STUDY_SUMMARY regulating cell metabolism, we performed an untargeted metabolomics using ST:STUDY_SUMMARY intestinal epithelial cells (IECs) from Hmga1flox/flox and Hmga1△IEC mice. In ST:STUDY_SUMMARY comparison with the Hmga1flox/flox mice, the Hmga1△IEC mice displayed 187 ST:STUDY_SUMMARY up-regulated metabolites and 120 down-regulated metabolites in IECs. Enrichment ST:STUDY_SUMMARY analysis of the metabolites showed that these differential metabolites mainly ST:STUDY_SUMMARY enriched in 20 pathways. Fatty acid synthesis was the most significantly ST:STUDY_SUMMARY deregulated signal pathway in HMGA1 deficient IECs. We separated the altered ST:STUDY_SUMMARY metabolites into up-regulated and down-regulated groups, and showed the top five ST:STUDY_SUMMARY classes of metabolites in up-regulated and down-regulated groups in heatmaps. ST:STUDY_SUMMARY The results showed that fatty acyl metabolites were mainly down-regulated in ST:STUDY_SUMMARY HMGA1-deficient IECs. ST:INSTITUTE Henan University ST:LAST_NAME Xu ST:FIRST_NAME Zhi-Xiang ST:ADDRESS Jinming Road, Kaifeng, Henan province, 475000, China ST:EMAIL zhixiangxu08@gmail.com ST:PHONE 86-13270538760 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS Hmga1flox/flox-1 T2318024a Sample source:intestinal epithelial cells | Genotype:Wild-type RAW_FILE_NAME(Raw file name)=T2318024a SUBJECT_SAMPLE_FACTORS Hmga1flox/flox-2 T2318025a Sample source:intestinal epithelial cells | Genotype:Wild-type RAW_FILE_NAME(Raw file name)=T2318025a SUBJECT_SAMPLE_FACTORS Hmga1flox/flox-3 T2318026a Sample source:intestinal epithelial cells | Genotype:Wild-type RAW_FILE_NAME(Raw file name)=T2318026a SUBJECT_SAMPLE_FACTORS Hmga1△IEC-1 T2318027a Sample source:intestinal epithelial cells | Genotype:HMGA1-knockout RAW_FILE_NAME(Raw file name)=T2318027a SUBJECT_SAMPLE_FACTORS Hmga1△IEC-2 T2318028a Sample source:intestinal epithelial cells | Genotype:HMGA1-knockout RAW_FILE_NAME(Raw file name)=T2318028a SUBJECT_SAMPLE_FACTORS Hmga1△IEC-3 T2318029a Sample source:intestinal epithelial cells | Genotype:HMGA1-knockout RAW_FILE_NAME(Raw file name)=T2318029a #COLLECTION CO:COLLECTION_SUMMARY The mice were euthanized, washed with pre-cooled PBS, and dissected CO:COLLECTION_SUMMARY longitudinally. Colon tissues of the mice were isolated and cut into 0.5 - 1.0 CO:COLLECTION_SUMMARY cm fragments and flushed 15 - 20 times with pre-cooled PBS followed by CO:COLLECTION_SUMMARY incubation in 20 mL HBSS containing 2% fetal bovine serum, 1 mM EDTA, and 1mM CO:COLLECTION_SUMMARY dithiothreitol (DTT) in a 50 mL centrifugal tube with constant shaking at 200 CO:COLLECTION_SUMMARY rpm for 20 min at 37 °C. The washed colon tissue pieces were transferred into CO:COLLECTION_SUMMARY a new tube containing 20 mL HBSS buffer and incubated for another 20 min. The CO:COLLECTION_SUMMARY colon tissues were cleaned again with 5 mL DMEM medium containing 10% FBS and CO:COLLECTION_SUMMARY then transfered to a tube with 5 mL DMEM containing 10% FBS, 50 U/mL collagenase CO:COLLECTION_SUMMARY type VIII, and 50 U/mL DNase I, and incubated at 37 °C for 50 min with constant CO:COLLECTION_SUMMARY shaking at 200 rpm. The digested tissue fragments were swirled for 30s and the CO:COLLECTION_SUMMARY undigested tissue was removed through a 40μm filter. The filtered cell CO:COLLECTION_SUMMARY suspension was centrifugated at 410 x g for 5 min at 4°C and then re-suspended CO:COLLECTION_SUMMARY in 10 mL DMEM containing 15% FBS, 100 U/mL penicillin and 100 μg/mL CO:COLLECTION_SUMMARY streptomycin (Thermo Fisher Scientific) for further culture. CO:SAMPLE_TYPE intestinal epithelial cells #TREATMENT TR:TREATMENT_SUMMARY The samples were not subjected to any further treatment. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Take out the sample from the -80 °C refrigerator and thaw it on ice. Add 1mL of SP:SAMPLEPREP_SUMMARY the extraction solvent (MTBE: MeOH =3:1, v/v) containing internal standard SP:SAMPLEPREP_SUMMARY mixture. After whirling the mixture for 15 min, 200 μL of water was added. SP:SAMPLEPREP_SUMMARY Vortex for 1 min and centrifuge at 12,000 rpm for 10 min. 200 μL of the upper SP:SAMPLEPREP_SUMMARY organic layer was collected and evaporated using a vacuum concentrator. The dry SP:SAMPLEPREP_SUMMARY extract was dissolved in 200 μL reconstituted solution (ACN: IPA=1:1, v/v) to SP:SAMPLEPREP_SUMMARY LC-MS/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Nexera LC-30A CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 0.1 % formic acid CH:SOLVENT_B 100% acetonitrile; 0.1 % formic acid CH:FLOW_GRADIENT (A:B) - 95:5, V/V at 0 min, 80:20 V/V at 2.0 min, 40:60 V/V at 5 min, 1:99 V/V CH:FLOW_GRADIENT at 6 min, 1:99 V/V at 7.5 min, 95: 5 V/V at 7.6 min, 95: 5 V/V at 10 min CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40 °C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6600 TripleTOF MS:INSTRUMENT_TYPE Triple TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The data acquisition was operated using the information-dependent acquisition MS:MS_COMMENTS (IDA) mode using Analyst TF 1.7.1 Software (Sciex, Concord, ON, Canada). The MS:MS_COMMENTS source parameters were set as follows: ion source gas 1 (GAS1),50 psi; ion MS:MS_COMMENTS source gas 2 (GAS2), 50 psi; curtain gas (CUR), 25 psi; temperature(TEM), 550 MS:MS_COMMENTS °C; declustering potential (DP), 60 V, or−60 V in positive or negative modes, MS:MS_COMMENTS respectively; and ion spray voltagefloating (ISVF), 5000 V or−4000 V in MS:MS_COMMENTS positive or negative modes, respectively. The TOF MS scan parameters were set as MS:MS_COMMENTS follows: mass range, 50–1000 Da; accumulation time, 200 ms; and dynamic MS:MS_COMMENTS background subtract, on. The product ion scan parameters were set as follows: MS:MS_COMMENTS mass range, 25–1000 Da; accumulation time, 40 ms; collision energy, 30 or−30 MS:MS_COMMENTS V in positive or negative modes, respectively; collision energy spread, 15; MS:MS_COMMENTS resolution, UNIT; charge state, 1 to 1; intensity, 100 cps; exclude isotopes MS:MS_COMMENTS within 4 Da; mass tolerance, 50 ppm; maximum number of candidate ions to monitor MS:MS_COMMENTS per cycle, 18. MS:MS_RESULTS_FILE ST003568_AN005861_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END