Effect Of Tim-3 And Cholesterol In Human Breast Cancer Cells Study On The Relationship Between Tim-3 Gene Polymorphisms And Essential Thrombocythaemia

The first part Expression and significance of Tim-3 in human breast cancer cellsObjectives:Breast cancer is a result of the combined action of many factors,including external environment and genetic backgrouds.The in-depth study of the molecular mechanism of breast cancer is beneficial to find an effective way to treat the disease.T cell immunoglobulin and mucin-domain containing molecule-3(Tim-3),as an immune negative regulator,plays an important role in the process of tumor cell immunity.The aim of this study was to investigate the expression of Tim-3 in human breast cancer cells and its effect on cell biological behavior(proliferation,migration,invasion and apoptosis).Methods:Western blotting was used to detect the expression of Tim-3 in breast cancer cells.The expression of Tim-3 protein was detected after transfecting siRNA and ADV-Tim3 by western blotting and agarose gel electrophoresis to validate the effectiveness.The effect of Tim-3 on the breast cancer cells was detected by CCK-8 assay,wound healing assay,transwell assay and apoptosis assay.Results:The expression of Tim-3 in breast cancer cells was detected.After siRNA transfection,the proliferation,migration and invasion abilities of breast cancer cells decreased significantly while the early apoptosis rate improved significantly(P<0.05).After ADV-Tim3 transfection,the proliferation,migration and invasion abilities increased significantly while the early apoptosis rate decreased significantly(P<0.05).Conclusions:Tim-3 can express on breast cancer cells.Tim-3 can promote the proliferation,migration,invasion and inhibit the early apoptosis abilities of breast cancer cells.Tim-3 provides new target for the prevention and treatment of breast cancer.The second part Effect and mechanism of cholesterol in human breast cancer cellsObjectives:Studies have shown 'that obesity and metabolic syndrome are important factors threatening the occurrence and development of breast cancer.Hypercholesterolemia can lead to the proliferation and metastasis of breast cancer cell.However,breast cancer cell response induced by cholesterol has not yet been confirmed.This study was to investigate the effect and mechanism of two concentrations of cholesterol(20umol/L and 40umol/L)on breast cancer cells in vitro.Methods:To detect the effect of cholesterol on the proliferation?migration?invasion and apoptosis abilities of breast cancer cells by CCK-8 assay?wound healing assay?transwell assay and apoptosis assay.After inhibiting and promoting the expression of Tim-3 through the siRNA and ADV-Tim3 transfection,we detect the effect of cholesterol on the proliferation and invasion of breast cancer cells.Results:Cholesterol 20umol/L and 40umol/L could inhibit the proliferation of the breast cancer cells,promote the migration and invasion abilities,and inhibit the early apoptosis ability(P<0.05).After blocking and promoting the expression of Tim-3 by siRNA and ADV-Tim3 transfection,we found cholesterol still could inhibit the proliferation ability and promote the invasion ability compared with the control whether blocking or up-regulating the expression of Tim-3(P<0.05).Conclusions:Cholesterol can inhibit the proliferation and early apoptosis ability of breast cancer cells,promote migration and invasion abilities.Cholesterol inhibits the breast cancer cells' proliferation and promotes its invasion through the non-Tim-3 dependent pathway.The third part Study on the relationship between Tim-3 gene polymorphisms and essential thrombocythaemiaObjectives:T cell immunoglobulin and mucin-domain containing molecule-3(Tim-3)is preferentially expressed on terminal differentiated Th1 cells,which can induce the apoptosis of Thl cells and inhibit the secretion of IFN-y when combines with its ligand galectin-9.Studies have shown the association between chronic inflammatory status and the occurrence of myeloproliterative neoplasms.Tim-3,as an important inflammatory regulator,may be associated with the development of essential thrombocythaemia(ET).The aim of this study was to investigate whether the three single nucleotide polymorphisms(SNPs)of the Tim-3 gene(-1516G>T,-574G>T and + 4259T>G)were associated with the susceptibility of patients with ET.Methods:We selected 175 patients with ET and 151 healthy controls.Genotyping of the TIM-3-1516G>T,-574G>T and + 4259T>G SNPs was performed by a polymerase chain reaction---restriction fragment length polymorphism assay.The relationships between the genotypes of each SNP and the risk factors of ET such as routine blood indexes,age and JAK2 V617F mutation were also explored.Results:The genotype and allele frequencies of the-15116G>T?-574G>T and+4259T>G SNPs were significantly different between ET patients and the controls(P<0.05).A haplotype analysis found that the GGT and TGT haplotypes had significantly different distributions between the ET group and the control group(P<0.05).However,significant differences between the genotypes of the three SNPs and routine blood indexes,age,JAK2V617F mutation were not detected.Conclusions:The-1516G>T,-574G>T and + 4259T>G SNPs in Tim-3 gene may be related to the susceptibility of the patients with ET.