| Objective:Colon Carcinoma is a kind of quite multiple malignant tumor found most commonly inside alimentary canal,which severely threatens people's health for ranking the first five.Its risk factors include family medical history,gender,smoking,alcoholic,lack of exercise,obesity and consumption of red meat and processed meat,among which obesity leads to the changes of some harmful hormone and immunometabolism.The series of changes increase the risk of suffering from colon carcinoma.Fat mass and obesity-associated protein(FTO)is allel related to obesity in the meantime with the gene length of 400 kb including nine exons and eight introns it is also N6-methyladeno demethylase which is located at chromosome 16 q.FTO has a domain that can combine ferrous ion and?-ketoglutaric acid,so it belongs to a member of Alk B homologue superfamily,for which it is also called ALKBH9.Many researches have shown that FTO plays roles in adjusting the metabolic rate,controlling the consumption and balance of energy,gathering the weight and body fat and differentiation of stem cells.In addition,it also involves cardiovascular disease,alzheimer's disease and the occurrence and development of tumor.According to some references,FTO is capable of involving the signal path m TOR that is about energy metaboliam to affect carcinoma proliferation.Pyruvate Kinase Isotype M2(PKM2)which can be expressed in cells with demanding metabolism such as embryonic cell,stem cell and tumor cell is a kind of hypotype of Pyruvate Kinase M(PKM).At the same time,it is the key enzyme involving metabolic path of tumor's aerobic glycolysis.There is no relevant report about whether FTO affects biological behavior of colon carcinoma and aerobic glycolysis through adjusting the expressions of PKM2.Therefore,the research aims to explore and discuss the above hypothesis further.Methods:Above all,based on the data,RNAseq of colon carcinoma,downloaded from The Cancer Genome Atlas(TCGT),all the information was transformed by Log2.Meanwhile,connecting the information from other biological information database such as UALCAN,try to analyse the difference of the expression of FTO in colon carcinoma.The paired samples of colon carcinoma,103 cases,and clinical pathological information were collected from Department of Pathology in Sheng Jing Hospital of China Medical University.And then the differences of the expression of FTO in colon carcinoma were studied by immunohistochemical technique and the clinical relevant was analyzed.Later,the cell lines with high expression of FTO and the cell lines with low expression of FTO were screened from colonic epithelial normal cells(Hco Epic)and five cell lines with colon carcinoma(LOVO,SW620,SW480,Caco-2,CL187).Those chosen cell lines were FTO knocked down and FTO over expressed by lentivirus transfection technology while the stable cell lines with high expression and low expression were built.In order to observe the influence of biological behavior in colon carcinoma cells caused by FTO,Cell Counting Kit8(CCK-8)was used to study cell proliferation;Scratch Test was applied to test cell migration;Transwell was adopted to test cell invasion;Flow cytometry(FCM)was employed to know apoptosis.Finally,the protein expression of PKM2 in cell lines with stable high expression and low expression was tested by Western.After comparing the differences between the FTO stable high expression and low expression,distribution of PKM2 in cytoplasm and nucleus was studied by Immunofluorescence staining technique.Glycolysis in FTO stable high expression and low expression was tested by Seahorse-Glycolysis pressure test.In the meanwhile,shikonin,inhibitor of PKM2,was given to intervene the cell lines with the FTO stable high expression.And the situation of glycolysis in intervened cell lines was tested by Seahorse test.After that,the cell viability was observed by CCK-8 for further observation of the changes of cell viability and glycolysis pressure in FTO stable high expression caused by the inhibitor of PKM2.Results:Part?:1.The analyses of bioinformatics showed that the expression of FTO among the patients with colon carcinoma presented highly and FTO had something to do with prognosis and clinical pathological classification.2.The results of immunohistochemistry indicated that FTO in the nucleus of colon carcinoma tissue presented high expression and the expression of FTO has relationships with the ages of patients(P<0.05),gender(P<0.05),T stage(P<0.05),tumor grades(P<0.01).while it had no clear relationship with the tumor size,N stage,M stage,vascular invasion,neural invasion,CEA in blood and CA199.According to Single factor analysis,the high expression of FTO has a close relationship with the ages of patients(P<0.05),gender(P<0.05),T stage(P<0.05),tumor grades(P<0.01).And based on Logisitics,tumor grade is the independent risk factor of FTO high expression among patients of Colon Carcinoma.Part ?:1.Cell lines with FTO high expression,LOVO,and Cell lines with FTO low expression,CL187,were chosen from colonic epithelial normal cells(Hco Epic)and five cell lines with colon carcinoma LOVO,SW620,SW480,Caco-2,CL187.Those chosen cell lines were FTO knocked down and FTO over expressed by lentivirus transfection technology and the stable cell lines with low expression(LOVO-GL407 NC,LOVO-Y11316),and high expression(CL187-GL107,CL187-H15278)were built.2.Cell phenotype test showed that the over expressed FTO cells of colon carcinoma had stronger ability in proliferation,migration and invasion than the ones in vector group.While the cells of apoptosis in the above two groups had no clear difference.The knocked down FTO cells of colon carcinoma had sharply weaker ability in proliferation,migration and invasion than the ones in negative control group.While the cells of apoptosis in the above two groups had no clear difference.The results indicated that FTO might strengthen the abilities of colon cancer cells in proliferation,migration and invasion aspects.Part ?:1.The protein level of PKM2 in the over expressed FTO cells of colon carcinoma increased higher than the level in vector group from Western Test.On the contrary,the protein level of PKM2 decreased.2.From the immunofluorescence test,comparing the over expressed FTO cells of colon carcinoma with the ones in vector group,fluorescence intensity of PKM2 in nucleus enhanced.While comparing knocked down FTO cells of colon carcinoma with the ones in negative control,fluorescence intensity of PKM2 in nucleus weakened.They presented positive correlation.3.From Seahorse glycolysis pressure test,the ability of glycolysis in over expressed FTO cells of colon carcinoma increased clearly.In contrast,comparing knocked down FTO cells of colon carcinoma with the ones in negative control,the ability of glycolysis decreased.4.Shikonin,inhibitor of PKM2,was used to intervene cell lines with steady high expression.The results of CCK-8 showed that after applying different densities(0?M,0.5?M,1?M,1.5?M,2?M,2.5?M,5?M,7.5?M,)of shikonin were given to cells of CL187-H15278 24 hours and 48 hours,the cell viability decreased and the dependence of density increased with the increasing density.After 24 hours intervention,the IC50 value was 3.65?M.After 48 hours intervention,the IC50 value was1.84?M.Meanwhile,the cell viability of CL187-H15278 decreased as the density of shikonin increased.At the same time,shikonin concentrations of 1.5?M and 2.5?M were selected for subsequent experiments.5.After the different densities(1.5?M,2.5?M)of shikonin were given to cells of CL187-H15278 24 hours,Seahorse-Glycolysis pressure test indicated that shikonin with different densities could inhibit glycolysis ability of FTO over expressed cells of colon at different levels.The density was proportionate to the inhibition level.The inhibition function of expressed group was better than the negative control group.Conclusion:1.In colon carcinoma tissue,FTO presented high expression,which was relevant to some clinical pathological parameters including prognosis,the age of patients with Colon Carcinoma,gender,T-staging and tumor grade.2.Over-expression of FTO could improve the ability of proliferation,migration and invasion in colon carcinoma cells,while it didn't affect the ability of apoptpsis.3.FTO affected the level of aerobic glycolysis by influencing the level of expression of PKM2,which proved linkage relationship exited between FTO and PKM2.4.Shikonin,inhibitor of PKM2,could inhibit the level of glycolysis of colon carcinoma cells and proliferation of colon carcinoma cells.And it could influence malignant biological behavior of colon carcinoma from the aspect of energy metabolism. |
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