Mechanism Of SIRT3 In Lipid Metabolism Reprogramming In Kazakh Patients With Esophageal Squamous Cell Carcinoma

Objective:(1)To analyze the serum lipidomics characteristics of Kazakh patients with Esophageal squamous cell carcinoma(ESCC),and to link the changes of cancer lipid metabolism with the changes of gene expression,so as to provide new insights for the diagnosis and treatment of Kazakh ESCC patients in Xinjiang.(2)To analyze the expression difference of Sirtuin 3(SIRT3)in ESCC patients of Xinjiang Kazakh,and to elaborate the relationship between SIRT3 expression level and clinicopathological characteristics and prognosis of patients.(3)To explore the effect of SIRT3 on the malignant phenotype and Epithelial-mesenchymal transition(EMT)of esophageal squamous cell carcinoma cells.Lipidomics and proteomics were used to analyze the function,possible downstream target genes and pathways of SIRT3 in lipid metabolism reprogramming of esophageal squamous cell carcinoma cells.(4)Through in vivo and in vitro functional tests,to clarify the mechanism of SIRT3 by inducing the expression of AKT-dependent fatty acid metabolism-related genes and EMT.To pave the way for the study of individualized treatment of Kazakh patients with esophageal squamous cell carcinoma in Xinjiang.Methods:(1)(1)ultra-performance liquid chromatogram-tandem mass spectrometry,UPLC-MS/MS)was used to conduct targeted quantitative lipidomics analysis of serum samples from Kazakh ESCC patients and matched normal population in Xinjiang.(2)Illumina Hiseq2000 sequencer was used to analyze the transcriptomics of Kazakh ESCC tissues and adjacent normal tissues.(3)Integrating the results of lipidomics and transcriptomics,the genes related to differential lipid metabolites in Kazakh ESCC patients were screened,and the key genes of ESCC tissues and adjacent normal tissues were verified by Real Time Quantitative PCR(RT-q PCR).(4)The effect of Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK),a key gene related to serum differential metabolites,on lipid metabolism of ESCC cells was detected by UPLC-MS/MS.(2)Clinical information of 65 Kazakh patients were collected,and tissue samples were collected according to the seventh edition of TNM staging standard of esophageal cancer in 2009.Immunohistochemistry(IHC)and RT-q PCR were used to detect AMPK.SIRT3 expression in cancer and adjacent normal tissues.Pearson correlation analysis was used to explore the relationship between AMPK and SIRT3.Based on the correlation between AMPK and SIRT3 expression in Kazakh esophageal squamous cell carcinoma tissues and the high expression of SIRT3 in cancer tissues,the correlation between SIRT3 and clinicopathological parameters and prognosis of Kazakh esophageal squamous cell carcinoma patients were further analyzed.(3)(1)The effect of SIRT3 on the malignant phenotype of ESCC cells was detected by in vitro experiments;(2)UPLC-MS/MS was used to detect the effect of target gene SIRT3 on lipid metabolism of ESCC cells.Oil red "O" assay was used to detect the effect of SIRT3 on neutral lipid content in ESCC cells.(3)Quantitative acetylation proteomics was used to screen the proteins with statistically significant difference in expression level after SIRT3 knockdown in esophageal squamous cell carcinoma cells,and to make function and pathway annotation of proteins.(4)The expression levels of AKT,p-AKT,FASN,ACC1 and EMT-related proteins(E-cadherin,Vimentin,Snail)in esophageal squamous cell carcinoma cells after SIRT3 knockdown and overexpression were detected by Western blotting.(5)AKT inhibitor MK2206 was added to verify the effect of SIRT3 on proliferation,invasion,migration and neutral lipid content of esophageal squamous cell carcinoma cells.Western blotting was used to detect the effect of MK2206 on FASN,ACC1 and EMT-related proteins(E-cadherin,Vimentin,Snail).(6)The subcutaneous tumor formation model of TE-1 cells overexpressing SIRT3 was constructed in nude mice,and the changes of body weight and subcutaneous tumor volume of each nude mouse were observed.The expression of relevant indicators in subcutaneous tumors of different groups of nude mice was further detected by IHC to clarify the effect of SIRT3 gene on cell growth and metastasis.Results:The first part:(1)The absolute lipid quantification of the serum samples of the two groups was carried out by UPLC-MS/MS,and a total of 13 lipids were detected,among which triglyceride(TAG)was the most.Lysophosphatidyl choline(LPC),phosphatidyl ethanolamine(PE)and ceramide(Cer)were the lipids that showed significant differences between the two groups(P<0.05).The lipid chain length and chain unsaturation of TAG,LPC and PC were significantly down-regulated(P<0.05).According to VIP ≥1,P ≤ 0.05,a total of 249 differential lipid metabolites were screened out.Cer(18: 0/26: 1)was the most significantly up-regulated lipid,and TAG(58: 9)＿FA22: 5 was the most significantly down-regulated lipid.Transcriptome analysis of Kazakh ESCC patients and adjacent normal tissues by Illumina Hiseq 2000 platform showed that the differential genes involved in the pathways related to lipid metabolism,such as unsaturated fatty acid biosynthesis and carnitine biosynthesis,were enriched in tumor tissues(P < 0.05).RT-q PCR results showed that genes closely related to these pathways were differentially expressed between Kazakh ESCC and adjacent tissues(P<0.05).Integrated analysis of lipidomics and transcriptomics showed that unsaturated fatty acid biosynthesis,fatty acid metabolism,lipid degradation,cholesterol metabolism and AMPK signaling pathways were enriched in tumor tissues(P< 0.05).Considering the key role of AMPK in lipid metabolism,targeted lipid metabolomics analysis of AMPK-knockdown esophageal squamous cell carcinoma cells was performed by UPLC-MS/MS,indicating that AMPK may be related to lipid metabolism in Kazakh ESCC patients.The first summary of Part II:(1)RT-q PCR results showed that the m RNA expressions of AMPK and SIRT3 were up-regulated in Kazakh ESCC patients compared with adjacent normal tissues(P < 0.05);IHC results showed that AMPK(X 2=10.086,P=0.001)and SIRT3 were highly expressed in Kazakh ESCC tissues(X2=29.823,P<0.001);(2)Pearson analysis was used to analyze the correlation between AMPK and SIRT3,indicating that the expression level of SIRT3 in cancer tissues was also increased in patients with high expression of AMPK protein(P=0.001).(3)The expression of SIRT3 in ESCC tissues was correlated with lymph node metastasis(P=0.025)and differentiation degree(P=0.033)(P < 0.05).However,there was no significant correlation with age(P=0.137),gender(P=0.567),T stage(P=0.318)and TNM stage(P=0.184)(P>0.05).(4)The expression level of SIRT3 was significantly correlated with the prognosis(P=0.0367).COX multivariate analysis suggested that SIRT3 was involved in the development of Kazakh ESCC patients.The second summary of Part II: The results of cell function assay showed that knockdown of SIRT3 inhibited the proliferation,migration and invasion of tumor cells,promoted apoptosis and arrested cell cycle at G2/M phase(all P < 0.05).After overexpression of SIRT3 in TE-1 cells,overexpression of SIRT3 promoted the proliferation,migration and invasion of tumor cells,inhibited apoptosis and arrested cell cycle at G0/G1 phase(all P<0.05).The absolute quantitative analysis of lipid in SIRT3 knockdown group and control group by UPLC-MS/MS suggested that SIRT3 knockdown affected the reprogramming of lipid metabolism in esophageal cancer cells,involving glycerolipid,glycerolipid and sphingomyelin.Oil red O assay showed that SIRT3 knockdown inhibited the content of neutral lipids in ESCC cells compared with the control group(P < 0.05).Overexpression of SIRT3 promoted the content of neutral lipids in esophageal squamous cell carcinoma cells(P < 0.05).The results of acetylation proteomics after SIRT3 knockdown in esophageal squamous cell carcinoma cells showed that there were 440 different acetylation sites and 220 corresponding proteins(P<0.05).The intersection of 220 protein and lipid biosynthesis related genes(map01040)was extracted,suggesting that FASN and ACC1 may be candidate genes for SIRT3 regulation.KEGG pathway analysis showed that SIRT3 was significantly correlated with the PI3K/AKT pathway(P<0.05),suggesting that SIRT3 may mediate the synthesis of fatty acids through the PI3K/Akt pathway,thus playing a role in promoting cancer.Part III: Western Blot results showed that compared with the control group,SIRT3 knockdown down-regulated the expression of FASN,ACC1,P-Akt,Vimentin and Snail,and up-regulated the expression of E-cadherin(P < 0.05).Overexpression of SIRT3 promoted the expression of FASN,ACC1,p-Akt,Vimentin and Snail,and down-regulated the expression of E-cadherin(P < 0.05).The addition of AKT inhibitor(MK2206)reversed the effects of SIRT3 knockdown and overexpression on the malignant phenotype of tumor cells(P<0.05).In addition,AKT inhibitor was added to KYSE150 cells with SIRT3 knockdown,and the expression of p-Akt,FASN and ACC1 was reversed to the corresponding control level(P<0.05).This result was obtained in KYSE150 cells with SIRT3 overexpression(P<0.05).Oil Red O experiment also verified the above results,MK2206 reversed the changes of neutral lipid content caused by SIRT3 knockdown and overexpression(P < 0.05).Western blot showed that MK2206 reversed the effect of SIRT3 on the expression levels of E-cadherin,Snail and Vimentin(P<0.05).The tumor formation experiment in nude mice showed that the tumor volume of nude mice injected with SIRT3 overexpressed cells was larger than that of SIRT3 empty vector control group.IHC showed that compared with the SIRT3 empty vector control group,the expressions of SIRT3,Vimentin and Snail in the tumor of nude mice injected with SIRT3 overexpression cells were up-regulated(P<0.05).Conclusion:(1)Lipid metabolism reprogramming exists in cancer tissues of Kazakh ESCC patients,and AMPK plays an important role in lipid metabolism reprogramming in ESCC.(2)SIRT3 and AMPK protein expression level is relevant in the kazak esophageal squamous carcinoma tissues,SIRT3 involved in the development of the kazak patients with esophageal squamous cell carcinomas.(3)SIRT3 by induce AKT depend on the expression of fatty acid metabolism related genes and EMT,promote the progress of ESCC.