Role And Mechanism Of LINC01667 In Regulating Fatty Acid Metabolic Reprogramming In Hepatocellular Carcinoma

OBJECTIVE: To define the role and mechanism of LINC01667 in regulating fatty acid metabolic reprogramming in hepatocellular carcinoma Methods:(1)Based on TCGA data and local cohort,the expression abundance of LINC01667 was detected by q-PCR,the expression localisation of LINC01667 was clarified by in situ hybridisation,and its correlation with clinicopathological indexes of HCC patients was analysed to clarify the possibility of its use in the clinical diagnosis and prognostic assessment of HCC patients as a biomarker;based on siRNA interference,lentiviral transfection and other techniques,cell lines with overexpression and low expression of LINC01667 were constructed.and lentiviral transfection,we constructed LINC01667 overexpression and underexpression HCC cell lines,and applied CCK8,transwell and flow cytometry to detect cell proliferation activity,penetration activity and apoptotic cell percentage,in order to clarify the biological effect of LINC01667 in HCC cells;siRNA transfection of HCC cells combined with sorafenib and regorafenib intervention,in order to clarify the biological effects of LINC01667;siRNA transfection of HCC cells combined with sorafenib,regorafenib interventions to clarify the relationship between LINC01667 and chemoresistance to anti-HCC drugs;applying Gal Nac technology to construct Gal Nac-siRNA small molecule targeting drugs,and preliminarily exploring the possibility of using LINC01667 as a novel anti-HCC drug target in nude mice homozygous tumour model.(2)Apply ChRIP-seq,PCR array and dual luciferase reporter assay to screen and validate the binding DNA and binding regions of LINC01667;RNA pulldown-MS combined with dataset prediction analysis to screen and validate the transcription factors recruited by LINC01667;and use ChIP-PCR and RIP-PCR in tandem to validate LINC01667.technology to verify the structure of LINC01667-DNA-transcription factor complex;apply Western blotting and immunofluorescence technology to verify whether LINC01667 has an activating effect on NF-κB signalling pathway.(3)Based on the construction of LINC01667 overexpression HCC cell lines,the following techniques were applied sequentially:(i)Nile Red and BODIPY576/589 staining to detect the degree of accumulation of lipid droplets in the cell lines;(ii)transcriptomics and metabolomics to analyse the differential genes and metabolites in the cell lines;(iii)flow cytometry to detect the alteration of the uptake capacity of the cell lines for free long-chain fatty acids;and(iv)seahorse XFe24 technique.seahorse XFe24 technology to detect the altered level of oxidative phosphorylation of long-chain fatty acids,and then apply CCK8,transwell and nude mouse loaded tumour techniques to detect the effect of the LINC01667/FABP5 regulatory axis on the malignant phenotype of HCC.RESULTS:(1)LINC01667 is expressed in the nucleus and cytoplasm of cells,and its expression abundance in HCC tissues is significantly higher than that in paracancerous tissues.In addition,there are differences in the expression abundance of LINC01667 in HCC cell lines,but all of them are significantly higher than that in normal hepatocyte cell lines;the expression of LINC01667 has nothing to do with the clinicopathological data,such as age and BMI,but its expression in HCC tissues is high.The expression of LINC01667 is not related to age,BMI and other clinicopathological data,but it is highly expressed in HCC tissues,which has certain clinical diagnostic efficacy for HCC patients and can make a prognostic assessment of the survival time of the patients;LINC01667,as an oncogene,participates in the malignant progression of HCC,and it promotes the proliferation,migration,and invasion of the tumour cells and inhibits apoptosis;LINC01667 enhances the chemo-resistance of the HCC cells to sorafenib and regorafenib,and the small microbes that target Gal Nac-siRNA I,a small molecule targeting drug of LINC01667,showed a more sensitive anti-HCC effect.(2)LINC01667 activates the NF-κB pathway and promotes the progression of HCC by recruiting the transcription factor STAT1 to FABP5,binding to the FABP5 DNA promoter region,enhancing its transcriptional activity,and up-regulating the expression of FABP5.(3)LINC01667 enhances the uptake of long-chain fatty acids in HCC cells,promotes the accumulation of intracellular lipid droplets,and elevates the level of fatty acid oxidative phosphorylation and metabolism;the LINC01667/FABP5 regulatory axis promotes the progression of HCC by reprogramming the fatty acid metabolism of HCC.CONCLUSION: LINC01667 may act as an oncogene and participate in the malignant process of HCC by promoting the proliferation,migration and invasion of tumour cells and inhibiting apoptosis;(2)the molecular biological mechanism by which LINC01667 participates in the malignant process of HCC is that it enhances the transcription of FABP5 DNA,upregulates the expression of FABP5,and then reprograms the fatty acid metabolism of HCC,ultimately promoting the malignant progression of HCC,through the recruitment of the transcription factor SATA1 to the FABP5 DNA promoter region.expression,thereby reprogramming HCC fatty acid metabolism and ultimately promoting the malignant progression of HCC.