Study Of Function And Molecular Mechanisms Of SIRT1 On Colorectal Cancer Metastasis

It is now generally accepted that metastasis is the main cause of death in patients with colorectal cancer(CRC).In recent years,genetic alterations in CRC have been extensively studied.However,the relevant factors that contribute to metastasis are still not well determined,which is urgently required for the targeted therapy of metastatic CRC.As a NAD+-dependent deacetylase,SIRT1 regulates the histone deacetylase,as well as non-histone deacetylase.Previous studies have reported that SIRT1 is upregulated in colorectal cancer,and its overexpression is associated with more aggressive capability in patients with CRC,as well as in CRC cell lines.Although these data suggest SIRT1 may take part in CRC metastasis,the role and molecular mechanisms of SIRT1 on CRC metastasis are less understood.In this study,we will study the function and molecular mechanisms of SIRT1 on CRC metastasis,thus providing a novel mechanistic role of SIRT1 in CRC metastasis and a rationale for clinically exploring the use of SIRT1 inhibitors in the therapy of metastatic CRC.Methods are as follows:(1)After exogenous expression of SIRT1 in low-metastatic SW480 cell line,the expression efficiency of SIRT1 was checked via western blot analysis,and the migratory and invasive capacities in vitro were analyzed by transwell assay.(2)After knockdown of SIRT1 in high-metastatic SW620 and LoVo cell lines(designated as SW620-shSIRT1 and LoVo-shSIRT1),the knockdown efficiency of SIRT1 was checked via western blot analysis,and the migratory and invasive capacities of SW620 and LoVo were measured by transwell assay.Wound healing assay was performed to detect the migratory capacity of SW620.(3)SW620-shSIRT1,LoVo-shSIRT1 and their corresponding control cells were injected into the spleen of each mouse to observe their metastasis capacity in vivo.(4)After knockdown of SIRT1,qRT-PCR was used to detect changes in the mRNA expression of epithelial-mesenchymal transition(EMT)associated genes,and immunofluorescence staining and western blot analysis were performed to detect changes in the protein expression of EMT-associated genes.Western blot analysis was used to detect changes in the protein expression of EMT-associated genes in SW620 cells treated with tenovin-6.(5)After knockdown of SIRT1,the changes in the mRNA and promoter activity expression of Fra-1 were examined by qRT-PCR and luciferase reporter assays respectively,and the changes in the protein expression of Fra-1 were detected by western blot analysis and immunofluorescence staining.(6)Western blot analysis and transwell assay were performed to study the role of Fra-1 in SIRT1 regulating EMT.(7)SIRT1 and Fra-1 expressions in human cancers were detected by immunofluorescence analysis,and the association between their expression status and the clinical characteristics of CRC patients were then assessed.The results show:(1)Exogenous expression of SIRT1 enhances colorectal cancer cells migration and invasion in vitro.(2)Knockdown of SIRT1 impairs colorectal cancer cells migration and invasion in vitro.(3)Knockdown of SIRT1 suppresses colorectal cancer cells metastasis in vivo.(4)Knockdown of SIRT1 promotes mesenchymal-epithelial transition(MET),suggesting that SIRT1 regulates EMT.(5)Knockdown of SIRT1 inhibits Fra-1 expression at mRNA and protein level,suggesting that SIRT1 regulates Fra-1 expression.(6)Knockdown of Fra-1 suppresses the role of SIRT1 on EMT,suggesting that SIRT1 regulates EMT in a Fra-1-dependent manner.(7)SIRT1 and Fra-1 predict poor survival of human colorectal cancer.Taken together,SIRT1 promotes epithelial–mesenchymal transition and metastasis in colorectal cancer by regulating Fra-1 expression,and may serve as a potential therapeutic target for metastatic CRC.