| Clear cell renal cell carcinoma（ccRCC）is the most common pathological subtype of renal cancer,accounting for about 70%-80%of renal cancer,and its metastasis rate can reach 60%,seriously damaging human health.Epithelial-mesenchymal transition（EMT）is the main feature of tumor cell metastasis and is involved in the migration and invasion of tumor cells.Although Transforming growth factor β（TGF-β）-mediated EMT has been widely studied,the relationship between abnormal lipid metabolism during ccRCC progression and EMT-mediated tumor cell metastasis remains unclear.Abnormal lipid metabolism of tumor cells is mainly manifested as excessive absorption of exogenous Fatty acids（Fatty acid,FA）,synthesis of endogenous FA and the decrease of Fatty acid oxidation（Fatty acid oxidation,FAO）level of tumor cells,which eventually leads to excessive lipid accumulation,thus promoting the proliferation,survival and metastasis of tumor cells.The FAO process is mainly divided into mitochondrial pathway and peroxisome pathway,which mediate FA of different chain lengths into the oxidation process respectively.Previous studies mainly focused on the relationship between mitochondrial FAO and tumor progression,but the relationship between peroxisome FAO and ccRCC metastasis potential remains unclear.In this study,The Cancer Genome Atlas（TCGA）database was used to confirm that low expression of acyl-Coa oxidase 1（ACOX1）in The FAO pathway of peroxisomes was associated with reduced survival of ccRCC Further results showed that low expression of ACOX1 in ccRCC induced lipid accumulation,and further upregulation of mammalian target of rapamycin Rapamycin（mTOR）signaling pathway promotes the proliferation and EMT of ccRCC cells,and ultimately promotes the growth and metastasis of ccRCC cells.Objective:To investigate the promoting effect of low expression of ACOX1 on proliferation and metastasis of ccRCC cells,and to explore the related molecular mechanism.Methods:1.To clarify the relationship between ACOX1 and proliferation and metastasis of ccRCC（1）The relationship between ACOX1 expression level and overall survival and progression-free survival of ccRCC patients was detected by TCGA database.（2）ACOX1 was knocked down in the ccRCC skin metastasis cell line CakI-1 by lentivirus transfection method,and the stable cell line of shACOX1-CAKI-1 was constructed.The purinomycin resistance gene carried by lentivirus was used to screen CAKI-1 cells with low expression of ACOX1,and the control group was empty lentivirus.（3）ACOX1 knockdown was used to detect the migration,invasion and proliferation of cells.①Western Blot was used to detect the expression of EMT-labeled molecules in shACOX1-CAKI-1 cells.②Changes in the migration ability of CAKI-1 cells of shACOX1 were detected by Transwell chamber assay.③Changes in the invasion ability of CAKI-1 cells of shACOX1 were detected by Transwell chamber assay.④The proliferation of CAKI-1 cells after ACOX1 silencing was detected by EDU and clonogenesis assay.2.Verify that ACOX1 promotes the proliferation and metastasis of CAKI-1 cells by inducing lipid accumulation（1）The oil Red O assay kit determined the lipid accumulation level of shACOX1CAKI-1 cells.（2）The ABCD1 gene responsible for transporting FA into peroxisome was knocked down in CAKI-1 cells to verify that inhibiting peroxisome FAO promoted the proliferation and metastasis of CAKI-1 cells.①CAKI-1 cells were transfected with lentivirus,and CAKI-1 cell lines with stable knockdown of ABCD1 gene were screened.The control group was empty lentivirus.②The lipid accumulation level of shABCD1-CAKI-1 cells was detected by oil red O staining kit.③Western Blot was used to detect the expression levels of ABCD1 and EMT labeled molecules.④Changes in the migration ability of CAKI-1 cells of shACOX1 were detected by Transwell chamber assay.⑤Changes in the invasion ability of CAKI-1 cells of shACOX1 were detected by Transwell chamber assay.⑥The proliferation level of CAKI-1 cells after ABCD1 knockdown was detected by EDU kit and clonogenesis assay.3.Explore the signaling pathway that mediates ACOX1 silencing to promote the proliferation and metastasis of CAKI-1 cells（1）Normal renal tubule epithelial cell line HK-2 was selected.After ACOX1 knockdown,RNA-sequence detection was performed on the cells and the control group,and the differential metabolic pathways were detected by enrichment analysis.（2）Verify the promoting effect of shACOX1 mediated by mTOR signal on the proliferation and metastasis of CAKI-1 cells.①Western Blot was used to detect the expression of mTOR and the changes of phosphorylation in CAKI-1 cell lines after knockdown of ACOX1.②Western Blot was used to detect the expression of mTOR and the changes of phosphorylation in CAKI-1 cell lines after ABCD1 knockdown.③shACOX1-CAKI-1 cells were treated with rapamycin,an mTOR signaling inhibitor.④Western Blot was used to detect the expression of EMT-labeled molecules in shACOX1-CAKI-1 cells treated with rapamycin.⑤The migration of shACOX1-CAKI-1 cells after treatment with rapamycin was determined by Transwell chamber assay.⑥Transwell chamber assay was used to detect the invasion ability of shACOX1CAKI-1 cells treated with rapamycin.⑦EDU kit and clonogenesis assay were used to detect shACOX1-CAKI-1 proliferation after rapamycin treatment.Results:1.To clarify the relationship between ACOX1 and proliferation and metastasis of ccRCC（1）ccRCC patients with significantly lower ACOX1 expression in TCGA database had shorter overall survival and progression-free survival.（2）The stable cell line of SHACOX1-CAKI-1 was successfully constructed.（3）After ACOX1 knockdown,the expression level of EMT-related molecules in CAKI-1 cells increased,and the ability of migration,invasion and proliferation was enhanced.2.Verify that ACOX1 promotes the proliferation and metastasis of CAKI-1 cells by inducing lipid accumulation（1）Excessive lipid accumulation was observed in shACOX1-CAKI-1 cells compared with the control group.（2）shABCD1-CAKI-1 cells showed excessive lipid accumulation,up-regulated expression of EMT-labeled molecules,enhanced invasion and metastasis ability,and increased proliferation ability.3.Explore the signaling pathway that mediates ACOX1 silencing to promote the proliferation and metastasis of CAKI-1 cells（1）Enrichment analysis results showed that mTOR signal was enriched in shACOX1-CAKI-1 cells.（2）shACOX1 enhanced the levels of mTOR and P-MTOR in CAKI-1 cells,and rapamycin treatment reversed the promoting effect of ACOX1 silencing on the proliferation and metastasis of EMT in CAKI-1 cells.Conclusions:1.Reduced ACOX1 expression in renal clear cell carcinoma promotes tumor proliferation and metastasis.2.Increased lipid accumulation mediates the promoting effect of shACOX1 on the proliferation and metastasis of CAKI-1 cells.3.shACOX1-induced upregulation of mTOR signal promotes the proliferation and metastasis of CAKI-1 cells. |
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