| Background:Wilms tumor is one of the most common malignant abdomen tumor in children.It is about 95%in pediatric kidney tumors,and the incidence rate is about 1/10000.The malignancy of the tumor is high,the growth rate is fast,and the metastasis is early.Although the current 5-year survival rate of children with Wilms' tumor can be significantly improved by surgical resection,combined with postoperative chemotherapy and radiotherapy,the current treatment of Wilms' tumor still faces many problems,such as the overcoming of multi-drug resistance,the reduction of chemotherapy side effects and the improvement of treatment efficiency.At present,the understanding of the pathogenesis and metastasis mechanism of Wilms' tumor is not sufficient,and there is no corresponding effective targeted therapy.Therefore,it is extremely urgent to find specific predictors and prognostic targets for the prognosis of Wilms' tumor.In recent years,the role of lipid metabolism in cancer has received increasing attention,and regulation of lipid metabolism has important clinical value in other tumors.Due to the high metabolic properties of tumor cells and their nutritional deficiencies in chemotherapy,fatty acid metabolism can often provide the necessary energy for tumor cells.Fatty acids are the basic synthetic components of phospholipids,important structural molecules of cell membranes.Therefore,the rapid proliferation of tumor cells requires a large amount of fatty acids.Fatty acids are also involved in the synthesis of many important cancer-promoting lipid signaling molecules including phosphoinositides,lysophosphatidic acid,and prostaglandins.Previous studies have shown that key genes ATP-citrate lyase,acetyl-CoA carboxylase,fatty acid synthase and acyl groups in fatty acid metabolism pathways have been observed in various tumors such as lung cancer,prostate cancer,gastric cancer,and bladder cancer.The expression and activity of coenzyme A desaturase are improved and closely related to poor prognosis.Tumor cell growth can be inhibited by down-regulating the expression of these metabolic enzymes or inhibiting metabolic enzyme activity by using specific inhibitors.Fatty acid synthase(FASN)is a key enzyme in the fatty acid synthesis pathway and energy metabolism of cell substrates.It plays a key role in the synthesis of long-chain fatty acids by acetyl-CoA,malonyl-CoA and NADPH.FASN is a complete enzyme system consisting of two identical 272 kDa multifunctional polypeptides.In most non-adipose tissues,the expression and activity of FASN are largely inhibited by adequate dietary fat intake,but are abnormally elevated in many human cancers such as ovarian cancer,prostate cancer,colon cancer,and bladder cancer.And it is associated with poor prognosis of the tumor.In the treatment of tumors,the treatment of targeting FASN has also attracted more and more attention.In colon cancer,by interfering with the expression of FASN,it can regulate the related cellular pathways such as AKT and JNK,and inhibit the proliferation,migration and invasion of tumor cells.In bladder tumors,inhibition of PKM2 indirectly leads to a decrease in the expression of FASN,and through the AKT/mTOR signaling pathway,it serves the purpose of treating tumors.Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Alpha(HADHA)is the alpha subunit of mitochondrial trifunctional protein(TFP),which catalyzes the last three steps of mitochondrial beta oxidation of long chain fatty acids.Mitochondrial TFP consists of four alpha(HADHA)and four beta(HADHB)subunits:HADHA is responsible for catalyzing long-chain 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities,while HADHB catalyzes 3-ketoacyl Coenzyme A thiolase activity.In addition to fatty acid ?-oxidation,HADHA is also involved in other lipid metabolism processes such as lipid biosynthesis,ketone formation and ketone hydrolysis.The study found that HADHA is involved in the pathogenesis,prognosis and chemoradiation resistance of human cancer.The expression of HADHA is down-regulated in breast cancer,especially in metastatic and recurrent breast cancer.Compared with the non-tumor control group,the expression of HADHA reduced in hepatocellular carcinoma,and HADHA may be a potential predictor of lung cancer chemotherapy response to platinum-based chemotherapy.Recent studies have also found that HADHA is involved in miRNA processing,autophagy,and apoptosis.Whether lipid metabolism is involved in the development of Wilms' tumor has not been studied.Therefore,exploring the role and regulation mechanism of key enzymes of lipid metabolism in Wilms' tumor will help to further elucidate the pathogenesis of Wilms' tumor and provide new ideas for its targeted therapy.Materials and MethodsThe tumor tissues and adjacent normal tissues of 20 children with Wilms' tumor who were treated and operated in Shandong Provincial Hospital were collected(stage I:10 cases;stage II:7 cases;stage III:3 cases);Cases(stage I:3 cases;stage II:3 cases;stage III:3 cases)were used to perform Label-free detection.And differentially expressed proteins in tumor tissues and normal tissues adjacent to the tumor were found;STRING database was used to analyze the interaction of differential proteins;mass spectrometry Data were comprehensively analyzed by IPA;GO/KEGG enrichment analysis was used to find the function of differential proteins;DAVID database was used for protein functional classification and screening out lipid metabolism-related proteins;KEGG was used to analyze the important signaling pathways involved;The classification identified proteins for further study.The 20 cases above were chosen.Oncomine database and qRT-PCR were used to verify the expression of FASN and HADHA from the genetic level.Western blotting,immunohistochemistry and immunofluorescence were used to verify the differential expression of FASN and HADHA proteins in tumors and adjacent normal tissues.65 cases of paraffin-embedded specimens of children with Wilms' tumor who were treated and operated in Shandong Provincial Hospital from January to January 2012 were chosen(stage I:32 cases;stage II:21 cases;stage III:12 cases),The relationship between histopathological typing/clinical pathological and FASN/HAD HA expression was analyzed.Kaplan-Meier survival curve was used to analyze the relationship between the expression of FASN/HADHA and the prognosis of children.SK-NEP-1 cell line was selected to compare the expression of FASN and HADHA in SK-NEP-1 cells and 293T cells;lentivirus interfered with the expression of FASN and HAHDA in SK-NEP-1 cells,and cell transfection efficiency was detected by qRT-PCR.CCK-8 and Western blotting were used to detect the proliferation of cells in each group after transfection;Western blotting was used to detect FASN,p-AKT,AKT,P62,Beclin-1,LC3,cleaved-caspase-3,pro-caspase-3,cleaved-parp-1,vemintin,E-cadherin and N-cadherin;Flow cytometry and AO-EB were used to detect the apoptosis and autophagic death,and inhibition of autophagy using 3-MA was used to verify whether there exists autophagic death or not.Transwell cell migration/invasion assays were used to detect changes in cell migration/invasiveness.The change in sensitivity of cells to vincristine chemotherapy after HADHA interference was verified.Results1.Analysis of lipid metabolism using proteomics in Wilms' tumorThe results of Label-free mass spectrometry showed that there were 437 differentially expressed proteins in the Wilms' tumor tissue compared with the corresponding normal tissues adjacent to the tumor.Among them,219 proteins were highly expressed and 218 proteins were lowly expressed(P<0.05).437 proteins were included in the STRING database,of which 418 proteins were related to each other.GO/KEGG enrichment analysis found that differentially expressed proteins exist in various cell biological behaviors,such as mitochondrial loss,metabolic pathway,cell death and acetylation modification.DAVID protein function analysis showed that there are 19 proteins related to lipid metabolism.Up-regulated proteins are FASN,ISYNA1,ALOX15;Down-regulated proteins are HADHB,ACAA1,ACAA2,CPT1A,ECHS1,ACSF2,EHHADH,PC,ACADM,ACADSB,ECH1,GM2A,DECR1,HADHA,NDUFAB1,H3F3A.IPA analysis can be found that deregulated protein in Wilms' tumor is closely related to cell death,cell cycle and lipid metabolism.Lipid metabolism plays an important role in tumors;and signal pathway analysis has found that dysregulated proteins play an important role in fatty acid metabolism pathways and are involved in the classical pathways,tumor apoptosis and autophagy of multiple tumors.From the regulatory sub-effect network,it can be seen that FASN is involved in a process including cell migration,cell proliferation,and tumor necrosis at an important position in the total regulatory network,and is associated with various signaling pathways.KEGG enrichment gives the top 20 pathways with minimal p values,including classical pathways for fatty acid metabolism and fatty acid degradation.We found that HADHA is located at an important position in the KEGG signaling pathway and is involved in this process.In summary,we screened the key proteins FASN and HADHA for lipid metabolism in Wilms,tumor2.The role and mechanism of FASN in Wilms' tumor(1)FASN is highly expressed in Wilms' tumor and is associated with clinical stage/tissue pathology and poor prognosisThe results of Label-free mass spectrometry showed that FASN was highly expressed in Wilms' tumor tissue compared with the corresponding normal tissues adjacent to the tumor.Oncomine database found that FASN was highly expressed in tumors(P=0.005).qRT-PCR further confirmed that the expression of FASN was significantly higher than that of paraneoplastic tissues(P<0.05).Western blotting,immunohistochemistry and immunofluorescence showed the expression of FASN protein in tumor tissues was significantly higher than that in paraneoplastic tissues.Analysis of clinicopathological parameters showed that there was no significant correlation between the high expression of FASN and the age of children(P=0.083),gender(P=0.620),lymph node metastasis(P=0.086),and histopathological typing(P=0.032).but it was closely related to the clinical stage of the child,and there was a statistical difference between the stage III and stage I(P=0.001),stage II and stage I(P=0.007).Prognostic analysis of paraffin-embedded specimens in 65 children with Wilms' tumor found that high FASN expression was closely associated with poor prognosis in children(P=0.016).(2)Down-regulation of FASN can inhibit the proliferation,migration and invasion of SK-NEP-1 by inhibiting AKT signaling pathway,and promote apoptosis and autophagic cell death.The results of Western blotting showed that the protein expression level of FASN in SK-NEP-1 was significantly higher than that in 293T cells(P<0.05).The proliferation of cells in the FASN interference group showed that the proliferative capacity of SK-NEP-1 cells was significantly inhibited with using CCK-8(P<0.05),and the expression of Ki67 was also significantly inhibited.Western blotting was used to detect the expression of apoptotic proteins and autophagy-related proteins:the expression of cleaved-caspase-3/pro-caspase-3 and cleaved-parp-1 was significantly increased in the FASN interference group;autophagy-related proteins LC3 and Beclin-1 Increased,P62 decreased;flow cytometry showed that early apoptotic cells and late apoptotic cells increased significantly after transfection,the difference was statistically significant(P<0.05);acridine orange/ethidium bromide staining results also suggest the number of dead cells after transfection was significantly increased(P<0.05).After the use of the autophagy inhibitor 3-MA,the level of autophagy was reduced and cell death was reduced,indicating that cell death caused by knockdown of FASN expression in SK-NE-1 cells was autophagic cell death.In the FASN-interfering group,E-cadherin expression was associated with increased epithelial-mesenchymal transdifferentiation,vimentin and N-cadherin expression were decreased(P<0.05);Transwell cell migration assay results showed that the FASN interference group passed through the chamber was significantly reduced,indicating that knockdown of FASN expression in SK-NE-1 cells inhibited tumor cell migration.The results of Western blotting showed that the expression of p-AKT in FASN interference group was significantly decreased(P<0.05),indicating that FASN may regulate the biological behavior of Wilms' tumor cells through AKT signaling pathway.3.The role of HADHA in Wilms' tumor and its role in tumor chemotherapy(1)HADHA is lowly expressed in Wilms' tumor,but children with high expression have higher malignancy and poor prognosis.Label-free mass spectrometry results showed that HADHA was lowly expressed in Wilms' tumor tissue compared with the corresponding normal tissues.qRT-PCR further confirmed that the expression level of HADHA mRNA in tumor tissues was significantly lower than that in paraneoplastic tissues(P<0.05).The results of Western blotting,immunohistochemistry and immunofluorescence showed that the expression of HADHA protein in tumor tissues was significantly lower than that in tumor tissues.Analysis of clinicopathological parameters showed that the expression of HADHA was not associated with age(P=0.137),gender(P=0.209),lymph node metastasis(P=0.479),and there was no correlation between HADHA expression and tumor stage.Stage III and stage I P = 0.406;stage II and stage I P = 0.300),HADHA is closely related to histopathological typing(P = 0.030).Prognostic analysis of 65 children with Wilms' tumor showed that high expression of HADHA was closely associated with poor prognosis in children(P=0.046)(2)Down-regulation of HADHA can inhibit the proliferation of SK-NEP-1 and promote its apoptosis by inhibiting AKT signaling pathway.The level of autophagy is inhibited,and the chemosensitivity of vincristine can be increased by inhibiting protective autophagy.The protein expression level of HADHA in SK-NEP-1 was significantly higher than that in 293T cells(P<0.05).After down-regulating the expression of HADHA,the proliferation of CCK-8 cells showed that the proliferation of SK-NEP-1 cells was significantly inhibited(P<0.05);the expression of Ki67 was also inhibited,suggesting a decrease in cell proliferation.Western blotting showed that the expression of cleaved-caspase-3/pro-caspase-3 and cleaved-parp-1 was significantly increased in the HADHA-interfering group;early apoptosis cells and late apoptotic cells found in the HADHA interference group by flow cytometry were significantly increased,the difference was statistically significant(P<0.05).In the HADHA-interfering group,the autophagy-related proteins LC3 and Beclin-1 were decreased,and P62 was increased.When SK-NEP-1 cells were treated with vincristine,the level of autophagy was enhanced,and the protective autophagy may be considered.When the HADHA interference group was treated with the same concentration of vincristine,the relative reduction of autophagy levels in the HADHA-interfering group was found,indicating that the cells after HADHA interference increased the sensitivity to vincristine.Flow cytometry showed that the death of tumor cells was significantly increased after HADHA interference.Therefore,it is considered that down-regulating the expression of HADHA can play a role in sensitizing vincristine chemotherapy by inhibiting protective autophagy.Western blotting results showed that the expression of p-AKT was significantly decreased in the HADHA interference group(P<0.05).It is indicated that down-regulation of HADHA can regulate the function of Wilms' tumor cells by inhibiting AKT signaling pathway.Conclusion1.Mass spectrometry-based non-labeled quantitative proteomics suggests the presence of 437 differentially expressed proteins in Wilms' tumors and adjacent tissues.These differentially expressed proteins or dysregulated proteins are involved in the generation of Wilms' tumor and have the potential to become diagnosis,prognostic markers,and targets of stratified therapy for Wilms'tumor;2.FASN is up-regulated in Wilms' tumor tissues and is closely related to clinical stage and prognosis.Down-regulation of FASN can inhibit Akt signaling pathway,with the inhibition of proliferation,migration and invasion of SK-NEP-1 cells.At the same time,the up-regulating of autophagy and apoptosis in Wilms' tumor occored which promoted the tumor cell death.The FASN gene played a role in the tumor suppression.3.HADHA is down-regulated in Wilms,tumor tissues,but its high expression is closely related to the clinical stage and prognosis of children.Down-regulation of HADHA can inhibit Akt signaling pathway,with the inhibition of proliferation and cell death of SK-NEP-1 cells.Meanwhile,down-regulating HADHA can enhance chemoresistance to vincristine by inhibiting protective autophagy. |
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