Relationship Between DBP And Lipid Deposition In Clear Cell Renal Cell Carcinoma

Clear cell Renal Cell Carcinoma（ccRCC）is a malignant epithelial cell carcinoma.The accumulation of lipid in cancer cells is one of its characteristic cell phenotypes.It is of great clinical and social significance to find out the related factors of lipid accumulation in ccRCC for the prevention of ccRCC and the adoption of targeted measures to delay the development of ccRCC,so as to improve the quality of life and living standards of ccRCC patients.Previous studies have shown that the decrease or absence of peroxisome fatty acidβ-oxidation can also lead to cell steatosis.D-bifunctional protein（DBP）is an enzyme protein in theβ-oxidation pathway of peroxisome fatty acids.Bioinformatics analysis showed that the expression level of DBP mRNA in ccRCC cells was lower than that in normal tissues.So does the expression of DBP in ccRCC cells down regulate?If it is down-regulated,is lipid accumulation in cells of ccRCC related to the down-regulation of DBP?It is worth studyingObjective:To explore the relationship between DBP and lipid deposition in clear cell Renal Cell Carcinoma（ccRCC）.Methods:Immunohistochemistry（IHC）,in situ hybridization（ISH）and oil red O staining were used to detect the expression of DBP at protein and mRNA levels and lipid deposition in clear ccRCC tissues and matched adjacent tissues.After overexpression of DBP and knockdown of DBP,Western blot,quantitative RT-PCR and oil red O staining were used to observe the effect of DBP on 786-o cell line Effect of intracellular lipid quality.1. Specimen collection and processingFresh Cancer（Ca）and adjacent（Adj）tissues were collected from the second hospital of Hebei Medical University.Paraffin embedded section is used for IHC and ISH.After scanning the whole film,calculate the H-score value,and carry out relative quantitative expression of DBP.OCT embedded section is used for oil red O staining to observe the lipid deposition in the tissue.2.Cell processingCultivation of ccRCC 786-O cell line derived from renal tubules,with the tumor suppressor gene VHL^（-/-）deleted.After overexpressing and knocking down the DBP（48 h）of 786-O cells in the logarithmic growth phase,collect the cells for Western blot and quantitative RT-PCR analysis to detect whether the DBP has been successfully overexpressed or knocked down;and 6 days after the cells overgrow,Perform oil red O staining to detect the lipid level of cellsResults:1. Pathological characteristics of Ca and Adj tissue in patients with ccRCC:HE staining showed that in Ca,the normal renal tissue structure disappeared and showed nest like structure,the cell membrane was clear but the cytoplasm was transparent and light stained;in Adj,the normal renal tubule structure was found,the lumen of renal tubule was complete,the cytoplasm was full,and it was eosin.2. There was a large amount of lipid deposition in Ca of ccRCC patientsThe results of oil red O staining showed that there were more red granules in Ca than in Adj and the red color was deep.3. Down regulation of DBP protein expression in ccrcc patientsThe results of IHC showed that DBP protein was mainly expressed in normal renal tubular epithelial cells,and in ccRCC cancer tissues,DBP expression was down-regulated.The relatively quantitative H-Score value showed that the protein content of DBP in cancer tissue of ccRCC patients was significantly reduced（P=0.022<0.05）.4. Decreased expression of DBP mRNA in cancer tissues of patients with ccRCCThe results of ISH and relative quantitative H-Score showed that the expression of DBP mRNA was down-regulated in Ca of ccRCC（P=0.005<0.05）5. Changing the expression of DBP can affect the lipid content of786-O cells5.1 Overexpression of DBP can reduce the lipid content of 786-O cells.After PcDNA3.1-DBP plasmid is transfected into 786-O cells,the number of red particles stained with oil red is small and the color is light.5.2 After DBP Si RNA was transferred into 786-O cells,theexpression of DBP decreased and the number of red particles stained with oil red increased.Conclusion:1.The expression of DBP in ccRCC decreased with lipid deposition2.The decrease or increase expression of DBP will lead to the increase or decrease of lipids in 786-O cellsTherefore,the lipid deposition in cancer tissue of ccRCC patients may be related to the decrease of DBP expression...