| BackgroundColorectal cancer(CRC)is a comon gastrointestinal cancer.The incidence and mortality rates of CRC are among the forefront of the cancer spectrum.Statistics from cancer society of United States showed that colorectal cancer incidence in men is after prostate cancer and lung cancer;while in women the incidence is after breast cancer and lung cancer.The mortality of CRC cancer rank the third in both male and female cancers.In China,the incidence of colorectal cancer and mortality rank the fifth among all the cancers.Moreover,with changing lifestyles,colorectal cancer incidence and death rate increased year by year.Early colorectal cancer patients can be treated by surgical resection and chemotherapy,and the prognosis is good.However,the advanced colorectal cancer especially metastasis has occurred is still lack of effective treatment,and the prognosis is poor.Therefore,further studies on the molecular mechanisms of colorectal cancer development will help to find new targets for diagnosis of colorectal cancer and provide experimental evidence for clinical treatment.During the development of tumorigenesis,tumor cell gene expression profile changes,including increased expression of oncogenes and decreased tumor suppressor genes.Epigenetic abnormalities is one of the main causes of cancer aberrant gene expression.Histone acetylation is the most common and important epigenetic modification,wich is involved in chromatin remodeling and gene expression regulation.The formation and removal of histone acetylation is regulated by histone acetyltransferase(HAT)and histone deacetylase(HDAC).It is reported that a variety of tumor development is often accompanied by abnormal histone acetylation and HDAC abnormal expression.HDAC inhibitors can effectively inhibit tumor development,which provides potential targets for cancer therapy.These reports indicate that HDAC-mediated histone acetylation deregulation is one of the key factors in tumor development.SIRT6 belongs to Sirtuin(SIRT)family,which are homologues of yeast silent formation regulator 2(Sir2).This family belongs to Class III histone deacetylase,whose functions are dependent on NAD+.Seven different Sirtuin proteins,named SIRT1-7 have been reported in human cells.SIRT family members regulate the transcription of target genes mainly through the removal of histone acetylation,and thus participate in a variety of biological processes such as neuroprotection,cell apoptosis,cellular senescence,sugar lipid metabolism,inflammation and oxidative stress regulation etc.Recent studies showed that SIRT6 plays a tumor suppressing role in a variety of tumors.Clinical studies showed that SIRT6 protein is down-regulated in liver cancer,head and neck squamous cell carcinoma,breast cancer etc.Studies in cultured cells and in animals showed that SIRT6 retulates a number of cancer-related pathways:SIRT6 maintains genome stability,retulates glucose metabolis in cancer cells,promotes cancer cell apoptosis and inhibits cancer cell proliferation,thereby inhibits tumor occurrence and development.However,SIRT6 expression in colorectal cancer,and the functions of SIRT6 in colorectal cancer stem cell behaviors remain elusive.SIRT6 regulates gene transcription mainly through histone H3K9 deacetylation to,thus regulates tumor development.SIRT6 inhibit the expression of anti-apoptotic protein survivine by deacetylates histone acetylations at its promoter,thereby promotes liver cancer progression.By repressing Hifla activity,SIRT6 regulates cancer cell glucose homeostasis.By inhibiting the expression of glycolytic pathway,SIRT6 inhibits cancer cell glycolysis pathway.SIRT6 also inhibits the expression of ribosomal proteins,thereby inhibits protein translation and cell proliferation.However,SIRT6-regulated target gene in colorectal cancer stem cell remain unclear.In this thesis,we systematically studied the role of SIRT6 during colorectal cancer development.First,we detected the protein level of SIRT6 in 62 colorectal tissues and adjacent tissues using immunohistochemistry.We analyzed the correlations between SIRT6 expression and patient age,disease stage,tumor differentiation.Next,we determined the effects of SIRT6 on cell proliferation,migration and invasion in colorectal cancer stem cells.Finally,by using RNA-seq and functional analysis,we identified CDC25A,TGFB2,SMAD2 and SMAD3 as potential SIRT6 target genes.Therefore,we further studied the regulation of SIRT6 on the expression of these genes.This thesis might provide targets for the diagnosis and treatment for colorectal cancer.Part Ⅰ:The expression of SIRT6 in colorectal cancer tissueObjective:To detect the expression of SIRT6 in colorectal cancer tissues and adjacent normal tissues,and to analyze the correlation between SIRT6 with colorectal cancer development.Methods:To establish the association of SIRT6 and colorectal cancer development,we first assessed the SIRT6 expression levels in 62 paired samples of colorectal cancers and their adjacent samples.The relationship between the expression of SIRT6 andcolorectal cancer development and progression was then analyzed.Results:1.The results showed that SIRT6 expression was significantly down-regulated in colorectal cancer tissue compared to adjacent non-tumor tissue(P<0.01).2.There is no correlation bet-ween SIRT6 expression and age,gender,or tumor size(P>0.05).3.The percentage of SIRT6 strong staining in stage Ⅰ,Ⅱ,Ⅲ,Ⅳ samples is 57.1%,27.3%,11.2%,and 0.0%respectively(P=0.030).4.The percentage of SIRT6 strong staining in grade 1,grade 2,and grade 3 samples are 50.0%,20.0%,and 7.2%(P=0.009).Conclusion:1.SIRT6 expression was significantly down-regulated in colorectal cancer tissue.2.There is no correlation between SIRT6 expression and age,gender,or tumor size.3.SIRT6 is down-regulated in late stage colorectal tissue.4.SIRT6 is down-retulated in high pathological grade colorectal cancer tissue.Part Ⅱ:The effect of SIRT6 on the proliferation and invasion of colorectal cancer stem cellsObjective:To charicterize the expressin of SIRT6 in colorectal cancer stem cells.To study the effect of SIRT6 on the proliferation,and invasion of SW480 cancer stem cells.Methods:The cancer stem cells(CSCs)population was separated from the SW480 cell line and colorectal cancer tissue using the marker CD 133.The expression of SIRT6 was detected by quaitative polymerase chain reaction and immunoblotting.Control lentivirus or lentivirus expressing SIRT6 was infected into SW480 cancer stem cells.Then,cell proliferation was detected by CCK8.Cell cycle was detected by flow cytometry.Clone formation was also detected.Matrigel-based transwell study was used to detect the effect of SIRT6 on the invasion of SW480 cancer stem cells.Results:1.SW480 CSCs expressed significantly decreased levels of SIRT6 compared with their corresponding non-CSCs population.The mRNA level of SIRT6 in CSCs isolated from colorectal cancer tissue was significantly decreased compared with their corresponding non-CSCs.2.Compared with the control group,the proliferation rate was significantly increased in SW480 CSCs(CD133+)overexpressing SIRT6.3.The SW480 CSCs expressing SIRT6 had increased numbers of cells in the G0/G1 phase(31±5%in control group vs 51±6%in SIRT6 expressing group)and decreased numbers of cells in the S phase(49±6%in control group vs 28±5%in SIRT6 expressing group,P<0.01).4.Clone formation assays showed that SW480 CSCs expressing SIRT6 had decreased colony numbers ompared with control cells(64±8 in control vs 36±8 in SIRT6 expressing group,P<0.01)5.The Matrigel invasion assays showed that the SW480 CSCs expressing SIRT6 had decreased invasion ability compared with control cells(0.70±0.04 fold,P<0.01).Conclusions:1.The expression of SIRT6 is inhibited in colorectal cancer stem cells.2.SIRT6 inhibits the proliferation of SW480 CSCs.3.SIRT6 induces G0/G1 phase retardation in SW480 CSCs.4.SIRT6 inhibits the colone formation of SW480 CSCs.5.SIRT6 inhibits the invasion of SW480 CSCs.Part Ⅲ:The identification of SIRT6 target genesObjective:To identify SIRT6 target genes in SW480 CSCs,thereby to clarify the mechanism of SIRT6 role in regulating SW480 CSCs behaviors.Methods:SW480 CSCs expressing SIRT6 were subjected to RNA-seq.Among the differentially expressed genes,CDC25A,TGFB2,SMAD2,and SMAD3 were closely associated with the cell cycle and cell migration.Therefore,they were picked for further analysis.To determine whether CDC25A,TGFB2,SMAD2,and SMAD3 are SIRT6 target genes,the expression level of these genes was detected by quantitative PCR and immunoblotting after SIRT6 overexpression.Further,the binding of SIRT6 with the promoters of CDC25A,TGFB2,SMAD2 and SMAD3 wsa determined by chromatin immunoprecipitation.Finally,SIRT6 was expressed and acetylation level of histone H3 lysine 9(H3K9)were detected by chromatin immunoprecipitation.Results:1.RNA-seq has identified 806 upregulated genes and 621 downregulated gene in SIRT6-expressing SW480 CSCs.Function annotation showed that the differentially expressed genes are associated with pathways of metabolism pathway,cell cycle,colorectal cacner and p53 pathway.We finally picked CDC25a,TGFB2,SMAD2 and SMAD3 genes for further study.CDC25a is closely related with cell cycle regulation,while TGFB2,SMAD2 and SMAD3 are key molecules in TGF-β signaling transduction.2.Real-time qPCR data showed that the mRNA levels of CDC25a,TGFB2,SMAD2 and SMAD3 significantly decreased in SIRT6 expressing SW480 CSCs(0.32±0.05 fold,0.58±0.08 fold,0.40±0.07 fold,and 0.54±0.06 fold).The immunoblotting data showed that the protein levels of CDC25a,TGFB2,SMAD2 and SMAD3 significantly decreased in SIRT6 expressing cells.3.The promoter regions of CDC25a,TGFB2,SMAD2 and SMAD3 were much higher in SIRT6 enriched group than IgG group in chromatin immunoprecipitation assays(7.28±0.49 fold,5.83±0.91 fold,6.59±0.84 fold,and 5.60±0.73 fold).4.H3K9 acetylation levels at the promoter of CDC25a,TGFB2,SMAD2 and SMAD3 genes decreased significantly in SIRT6 expressing cells(0.62±0.12 fold,0.73±0.08 fold,0.68±0.07 fold,and 0.72±0.09 fold).Conclusion:1.SIRT6 inhibits the expression of CDC25a,TGFB2,SMAD2 and SMAD3 in SW480 CSCs.2.SIRT6 binds to the promoter of CDC25a,TGFB2,SMAD2 and SMAD3.3.SIRT6 deacetylates H3K9 at the promoter regions of CDC25a,TGFB2,SMAD2 and SMAD3. |
