| BackgroudRenal cell carcinoma(RCC)is one of the common tumors of urinary system.There will be an estimated 76,080 new cases and 13,780 deaths in the United States in 2021.Clear cell renal cell carcinoma(ccRCC)is the most common histological subtype,accounting for 75%-80%of cases.The early,localized diseases have a relatively high cure rate with 5-year survival at more than 90%,but once it progresses to distant metastatic disease,it drops dramatically to 12%for patients.30%of patients are metastatic at diagnosis and almost 30%of the remaining patients will develop metastases detected during the follow-up,indicating a relatively poor prognosis.Although the application of targeted therapies such as the vascular endothelial growth factor(VEGF)receptor tyrosine kinase inhibitor(TKI)and mammalian target of rapamycin(mTOR)inhibitors has improved progression-free survival(PFS),it also leads to drug resistance and tumor metastasis and recurrence.Subsequently,emerging immune checkpoint inhibitors alone or in combination anti-cytotoxic T-lymphocyte antigen-4(CTLA4)and anti-programmed death 1(PD-1)have expanded treatment options,but the long-term response rates are low.Therefore,the identification and validation of new biomarkers and the in-depth explore the molecular mechanisms of ccRCC proliferation and metastasis are extremely urgent and meaningful.Research confirms that ccRCC is fundamentally a metabolic-related disease.As is wellknown,cancer cells predominantly produce energy by lactic acid fermentation even under normoxia,rather than by glycolysis followed by the tricarboxylic acid(TCA)cycle in mitochondria,which is known as the classic Warburg effect.The metabolic reprogramming including decreased TCA cycle and upregulation of the VHL/HIF(Von Hippel-Lindau/hypoxia inducible factor)pathway increased the fatty acid(FA)synthesis gene expression and cholesterol for cancer cells and a large number of raw materials that aid in cancer cells progression.Clear cell tumors are defined histologically as a large amount of neutral lipids such as cholesterol esters and triglycerides accumulate in ccRCC tissue,which is the basic pathological feature of lipid deposition in cells.ccRCC clearly display a propensity for lipid deposition rather than lipid catabolism.Studies have shown that its proliferation and metastasis are closely related to FA synthesis,oxidative catabolism,cholesterol uptake and transport.The large quantities of lipid storage in ccRCC has two well-characterized functions including affecting energy homeostasis and the release of lipid species for cell membrane synthesis during proliferation.In addition,the accumulation of lipids also plays an important role in cell signal transduction and activity,pro-inflammatory,phenotypic conversion,and drug treatment resistance.Therefore,targeting lipid deposition will provide a new therapeutic direction for the next step to unveil the complex mechanisms of tumors.In this study,we used TCGA(The Cancer Genome Atlas)public database to construct a prognostic risk model of ccRCC patients among lipid storage related genes,and through subsequent verification and multi-parameter analysis,we screened and extracted that AUP1(lipid droplet regulating very low-density lipoprotein assembly factor)is significantly highly expressed in ccRCC and is closely related to the clinical prognosis.Combined with in vivo,in vitro experiments and RNA-seq,we revealed that AUP1 induced lipid accumulation in ccRCC by mediating FA and cholesterol pathways,and realized lipid reprogramming,thus promoting the progression of ccRCC.Further analysis of the biological function and the underlying mechanism is helpful to better understand the biological process of lipid metabolism in ccRCC cells.AUP1 can be used as a new effective potential target and prognostic marker for the treatment of ccRCC.Objective1.To analyze the prognosis related risk factors,screen and mine the potential prognosis related risk genes by constructing a clinical prognosis related model for ccRCC patients.2.To determine the expression levels of AUP1 in ccRCC and its biological functions in regulating tumor cell proliferation,migration and invasion.3.To explore the mechanism by which AUP1 mediates cell cycle and apoptosis to regulate cell proliferation and induce EMT to affect tumor invasion and metastasis.4.To explore the molecular mechanism by which AUP1 regulates lipid metabolism and induces lipid accumulation in a number of different ways,thereby providing a new target for the diagnosis,treatment and prognosis of ccRCC.MethodsPart Ⅰ Construction of a clinical prognostic model and the biological role of AUP1 in clear cell renal cell carcinoma1.Construction of prognosis related risk model:The mRNA sequence expression data of 539 ccRCC patient tissues and 72 normal kidney tissues were downloaded from TCGA database and 79 lipid storage related genes were obtained from GSEA(Gene Set Enrichment Analysis)website.Multi-parameter analysis and prognostic risk model construction were performed using R language and related software packages,the validity of the model was verified by survival curve and ROC(receiver operating characteristic)curve analysis,and the nomogram were generated by combining clinicopathological parameters,risk scores,single/multivariate Cox regression analysis,and co-expression analysis to predict the 5-,7-,and 10year survival of patients.2.Screening the target genes:The risk genes with high expression and poor prognosis in ccRCC tissues were selected according to the relevant indexes in the model,and AUP1 was finally extracted as the target gene for subsequent experimental validation and mechanistic study by combining the functional properties of the gene and PubMed literature search without relevant reports.3.Clinical tissue samples were collected and the expression of AUP1 at the mRNA and protein levels in ccRCC and normal tissues was assessed by qRT-PCR and immunohistochemical staining.4.In vitro cell lines screening and the construction of stable transfected strain:Western Blot was used to detect the expression of AUP1 in various renal cancer cell lines ACHN,A498,786-O,Caki-1 and normal renal tubular epithelial cells HK-2.ACHN and A498 were used to construct a stable transfected strain of lentiviral knockdown AUP1 cells,and the transfection efficiency was verified by qRT-PCR and Western Blot.5.Functional experiments in vitro:After the cells knocked down the expression level of AUP1,the changes of cell proliferation level were detected by CCK-8 and EdU cell proliferation assay,the ability of cell colony formation was detected by clone formation assay,the migration level of cells was detected by wound healing assay and the migration and invasion ability of cells was assessed by Transwell assay.Part Ⅱ The molecular mechanism of AUP1 regulating lipid metabolism and inducing lipid accumulation in clear cell renal cell carcinoma1.To analyze the underlying biological mechanisms by detecting the differences in mRNA level expression between AUP1 knockdown and control groups by RNA-seq technology.2.Intracellular lipid levels were measured by the Oil Red O assay,the Triglyceride Assay Kit and the Cholesterol Assay Kit respectively.3.The changes of cell cycle,apoptosis,EMT(epithelial mesenchymal transition)related proteins were detected by Western Blot.4.Combined with RNA-seq and lipid multi-pathway Venn diagram convergence analysis,the downstream target SOAT1 was extracted and validated by qRT-PCR and Western Blot.Clinical tissue samples were collected,and the expression of SOAT1 at the mRNA and protein levels in the tissue samples and in vitro cell lines were detected by qRT-PCR,immunohistochemistry,and Western Blot.5.The cell lines were transfected with si-RNA to knockdown SO AT1,and the knockdown efficiency were verified by qRT-PCR and Western Blot.The cell proliferation changes were detected by CCK-8 assay,the cell clonogenic ability were detected by clonogenic assay,and the cell migration and invasion were detected by Transwell assay.6.Rescue experiments:Based on the stable transfected cell lines with AUP1 knocked down by lentivirus as well as control cells,transfection of plasmids to overexpress SOAT1 was continued,the transfection efficiency were verified by qRT-PCR and Western Blot,after which the proliferation level in each corresponding cell was assessed by CCK-8 assay,and the lipid levels in the corresponding cells were measured using the Triglyceride Assay Kit and the Cholesterol Assay Kit.7.The qRT-PCR assay and primer screening were performed for the downstream lipid metabolism-related proteins of AUP1,followed by Western Blot to detect changes in the downstream key lipid metabolism-related proteins.In combination with TCGA database analysis,Western Blot was used to detect its expression differences in renal cancer cell lines ACHN,A498 and normal renal tubular epithelial cell line HK-2.8.The expression of AUP1 in ccRCC and adjacent normal tissues was assessed by tissue microarray and immunohistochemical staining.Part Ⅲ The effect of AUP1 inhibition on the in vivo xenograft model of clear cell renal cell carcinoma1.BALB/c nude mice xenograft tumor models were constructed and divided into two groups:the AUP1 knockdown group by lentivirus and the negative control virus group.2.The subcutaneous tumor volume and body weight changes of nude mice were monitored in real time,and the nude mice were sacrificed on the 28th day after tumor inoculation,and the transplanted tumors were weighed and photographed.3.Tissue sections of transplanted tumors were made,and HE(hematoxylin eosin)staining and immunohistochemical staining were performed to detect the related protein level changes.ResultsPart Ⅰ Construction of a clinical prognostic model and the biological role of AUP1 in clear cell renal cell carcinoma1.69 of the 79 lipid storage related genes were differentially expressed between ccRCC and normal kidney tissues,including 39 upregulated and 30 downregulated genes.Univariate Cox regression analysis revealed 39 prognosis related genes,including 14 risk genes.17 genes were selected for model construction by LASSO regression analysis and divided into high-risk and low-risk groups according to the median risk score,survival curve analysis confirmed that the high-risk group had a poor survival,and ROC curve analysis indicated that the model could accurately predict the survival of ccRCC patients.Further combined with clinicopathological parameters by multivariate Cox regression analysis revealed that risk score,age,tumor stage,and grade were independent risk factors for patient prognosis,and the nomogram was drawn based on the above variables which could accurately predict patient 5-,7-,and 10-year survival rates.Six genes were highly expressed(logFC>0)and had poor prognosis(hazard ratio,HR>1)in the model,according to the prognostic analysis related indices,literature search and gene functional attributes,AUP1 was extracted as the target gene.2.The mRNA and protein expression levels of AUP1 in ccRCC tissues were significantly higher than those in adjacent normal tissues and closely correlated with pathological grade,which was consistent with the results of TCGA database.The expression of AUP1 in the ccRCC cell lines ACHN and A498 was significantly higher than that of HK-2,a normal renal tubular epithelial cell line.3.Stable transfection strains with lentiviral were effective in reducing the mRNA and protein levels of AUP1 in ACHN and A498 cells compared to the control cells.4.The decreased expression of AUP1 inhibited the proliferation and colony formation of ccRCC cells and weakened the migration and invasion abilities of the cells.Part Ⅱ The molecular mechanism of AUP1 regulating lipid metabolism and inducing lipid accumulation in clear cell renal cell carcinoma1.A total of 1964 differentially expressed genes were found by RNA-seq,of which 558 were downregulated and 1406 were upregulated.GO(Gene Ontology)functional enrichment analysis revealed that the main biological processes involved in the differentially expressed signalling molecules downstream of AUP1 are:cellular response to fatty acid,response to fatty acid,DNA replication,regulation of transcription involved in G1/S transition of mitotic cell cycle,cholesterol efflux,regulation of epithelial cell differentiation/proliferation,lipid localization and lipid transport.KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway database and Reactome pathway database enrichment analysis revealed that the main pathways involved were cell cycle(especially the G1/S phase transition)and DNA replication.2.AUP1 mediates both fatty acid and cholesterol metabolism in ccRCC.Oil Red O assay,intracellular triglyceride and cholesterol assays intracellular triglyceride and cholesterol levels were reduced after lentiviral knockdown of AUP1.3.AUP1 promotes the proliferation of ccRCC cells by regulating the cell cycle and inhibiting apoptosis,and promotes the migration and invasion of ccRCC cells by regulating EMT.The expression of the cell cycle related proteins cyclin D1,cyclin-dependent kinase 8,and cell division cycle 6 was decreased by AUP1 knockdown as determined by Western Blot.The expression of the apoptotic protein Bax,the apoptosis-executing protein cleaved caspase3 were increased,while the anti-apoptotic protein Bcl2 was downregulated.E-cadherin expression was upregulated,while the expression of N-cadherin,vimentin and MMP9 were downregulated.4.Convergent analysis of lipid multi-pathway Venn diagram and q-PCR,Western Blot confirmed that the expression level of SO AT1 was significantly decreased accompanied by the decreased expression of AUP1.The mRNA and protein expression levels of SOAT1 in ccRCC tissues and ACHN and A498 cell lines were significantly increased compared with those in adjacent normal tissues and normal tubular epithelial cells,HK-2,and the protein expression increased in a stepwise manner as the pathological grade of the tumors progressed.The mRNA and protein levels of SOAT1 in ACHN,A498 cells were effectively reduced by transfection with si-RNA.Inhibition of SOAT1 expression reduces the proliferation,colony formation,migration and invasion capacity of ccRCC cells.5.AUP1 induces lipid accumulation by targeting SOAT1 to partially promote ccRCC progression,and the increased lipid content rescued cell death and promoted cell proliferation.The efficiency of transfection plasmids was detected by q-PCR and Western Blot,and the mRNA and protein levels of SO AT1 were significantly overexpressed in ACHN and A498 cell lines.CCK-8 cell proliferation assay and intracellular triglyceride and cholesterol content assays showed that overexpression of SO AT1 enhanced the proliferation of ccRCC cells and increased the triglyceride and cholesterol content in ACHN and A498 cells.Meanwhile,upregulation of SO AT1 partially reversed the inhibition of cell proliferation and the decrease of intracellular triglyceride and cholesterol levels due to the knockdown of AUP1.6.AUP1 induces lipid accumulution in ccRCC cells by promoting fatty acid de novo synthesis,inhibiting the β oxidation of fatty acid and lipid transport,and regulating lipolysis,thus enabling reprogramming of lipid metabolism,but not by affecting the intracellular cholesterol synthesis pathway.q-PCR was performed to determine the lipid metabolism related genes in RNA-seq,and PRKAA2,a key enzyme inhibiting fatty acid synthesis,and STARD5,a lipid transporter,were significantly upregulated after AUP1 knockdown.Western Blot confirmed that the protein expression levels of PRKAA2,STARD5 and CPT1A,the key enzyme for fatty acid β-oxidation,increased significantly along with the knockdown of AUP1,while the protein levels of MGLL,the key enzyme for lipolysis,decreased significantly,while HMGCR,the key enzyme for cholesterol synthesis,did not change significantly.7.Combined with TCGA database analysis,we found that MGLL expression was significantly higher and STARD5,PRKAA2,CPT1A expression was significantly lower in ccRCC cell lines ACHN,A498 cells compared with normal renal tubular epithelial cell line HK-2 by Western Blot.MGLL and STARD5 could be used as potential biomarkers for ccRCC.8.Tissue microarray technology further confirmed that AUP1 expression was significantly increased in ccRCC tissues compared to the adjacent normal tissues.Part Ⅲ The effect of AUP1 inhibition on the in vivo xenograft model of clear cell renal cell carcinoma1.The effect of inhibition of AUP1 on the xenograft tumor growth in vivo.Compared with the control,knockdown of AUP1 significantly impaired the growth rate of xenografted tumors in nude mice,and the volume and weight of the transplanted tumors were significantly reduced.2.Given the function of AUP1 in lipid storage and regulation of lipid metabolism,there was no significant change in body weight of nude mice after inhibiting the expression of AUP1.3.The effect of lipid metabolism regulation on transplanted tumors in nude mice.The expressions of AUP1 and its downstream SO AT1 were detected by HE and immunohistochemical staining.In comparison to the control group,the expression levels of both AUPl and SOAT1 were reduced along with the suppressed growth rate of transplanted tumours,indicating that the reduced regulation of lipid metabolism inhibited the proliferation of transplanted tumours in vivo.Conclusion1.The expression of AUP1 in ccRCC tissues was significantly higher than that in normal tissues,and was closely related to stage and grade.Reducing the expression of AUP1 could inhibit the proliferation,clone formation,migration and invasion of ccRCC cells.2.AUP1 mediates both fatty acid and cholesterol metabolism in ccRCC cells.And promote the proliferation of ccRCC cells by regulating the cell cycle and inhibiting apoptosis,and promote the migration and invasion of ccRCC cells by regulating EMT.3.AUP1 induces lipid accumulation by targeting SOAT1 to partially promote the progression of ccRCC.4.AUP1 promotes the occurrence and development of ccRCC by promoting the de novo synthesis of fatty acids,inhibiting fatty acid β-oxidation and lipid transport,and regulating lipolysis,thus realizing the reprogramming of lipid metabolism,but it does not depend on the endogenous cholesterol synthesis pathway. |
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