| Background: Burkitt lymphoma(BL)is a highly aggressive non-Hodgkin’s lymphoma for which there are no effective,uniform treatment options for relapsed/refractory BL.Bone marrow cytology in BL patients shows a large number of lipid vacuoles in the cytoplasm,suggesting that abnormal lipid accumulation is most likely occurring in cells.Excess lipids support cell proliferation and invasion in terms of cell membrane biosynthesis,signaling,and energy supply.Fatty acid synthase(FASN),a key enzyme of de novo fatty acids,is abnormally highly expressed in a variety of haematological tumours.RNA-binding proteins(RBPs)are proteins that bind RNA through one or more RNA-binding domains and regulate all processes involved in RNA transcription,making RBPs of great relevance to cellular function and disease development.Enolase-1(alpha enolase 1,ENO1),a member of the enolase family,is an RBP that is highly expressed in tumour cells and is closely associated with tumour progression.Our group found that ENO1 was highly expressed in both BL clinical samples and cell lines,and promoted the proliferation and invasion of BL cells.Since ENO1 is a metabolic enzyme,we speculate that its overexpression is a result of metabolic reprogramming of BL cells,and whether its overexpression is associated with excessive lipid accumulation in BL cells is the scientific question of interest in this study.Objective:1.To detect the effect of ENO1 on the lipid content of BL cells.2.To clarify that ENO1 protein increases its protein expression by stabilizing FASN mRNA.3.To clarify the effect of FASN on BL cell lipid droplets and its effect on cell proliferation and invasion.Methods:1.Application of Oil Red O staining to examine the content of lipid droplets in cells.2.Extraction of triglycerides from cells using isopropyl alcohol,followed by quantification of triglycerides by colorimetric assay.3.RNA immunoprecipitation sequencing(RIP-seq)was used to predict the genes of the lipid metabolism pathway that potentially bind to the ENO1 protein.4.to detect the relative enrichment of FASN mRNA in the ENO1-RNA complex using RNA immunoprecipitation(RIP).5.apply RNA quantitative reverse transcription PCR(RT-q PCR)and protein blotting(Western Blot,WB)to detect changes in FASN mRNA expression levels and protein expression levels.6.Application of RNA stability assay to detect the effect of ENO1 protein on FASN mRNA stability7.Application of lentiviral packaging infection to construct ENO1 and FASN stable knockdown cell lines.8.Application of CCK8 and clone formation assays to detect the effect of FASN knockdown on cell proliferation and Transwell assay to detect the effect of FASN knockdown on cell invasion.Results:1.Oil Red O staining and colorimetric results demonstrated that ENO1 and FASN knockdown both reduced lipid droplets and triglyceride levels in BL cells.2.The relative enrichment of FASN mRNA in the ENO1-RNA complex was demonstrated by RIP-qRT-PCR to be more than 4 times that of the Ig G antibody group.3.qRT-PCR results demonstrated that ENO1 knockdown reduced FASN mRNA expression levels,and WB results demonstrated that FASN protein expression levels were also down-regulated.4.the results of RNA stability assay demonstrate that ENO1 protein can stabilize FASN mRNA.5.CCK8 and clone formation assays demonstrated that shFASN inhibited the proliferation ability of cells;Transwell assays demonstrated that shFASN inhibited the invasion ability of cells.Conclusions: In BL cells,the ENO1 protein increases the level of lipid droplets and triglycerides by binding and stabilizing FASN mRNA,promoting cell proliferation and invasion. |
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