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Objective:Explore the expression of SIRT1 protein in gastric cancer tissues and cells.Analyze the correlation of SIRT1 protein expression and multidrug resistance protein P-gp and Top-Ⅱ alpha.Methods:Immunohistochemical to detect SIRT1,P-gp and Top-Ⅱ alpha protein in 15 cases of gastric cancer tissue and 15 cases of normal gastric mucosa tissues.Western blot test the expression of SIRT1 protein in normal gastric mucosa epithelial cell line GES-1 and human gastric cancer cell line SGC7901,MKN45,MKN28,HGC27,AGS and MGC803.Western blot test the expression of SIRT1,P-gp and Top-Ⅱ alpha protein in normal gastric mucosa epithelial cell line GES-1 and human gastric cancer cell line SGC7901,MKN45.After siRNA-SIRT1/liposome transfection gastric cancer cells SGC7901,western blot test the changes of expression of SIRT1,P-gp and Top-Ⅱ alpha protein and MTT test the changes of SGC7901 cells proliferation in vitro and the sensitivity of chemotherapy drugs 5-Fu.Results:In 15 cases of gastric cancer tissue have the positive expression of 15 cases of SIRT1 protein,13 cases of P-gp protein and 3 cases of Top-Ⅱ alpha protein.In 15 cases of normal gastric mucosa tissues have the positive expression of 6 cases of SIRT1 protein,5 cases of P-gp protein and 0 cases of Top-Ⅱ alpha protein.The relative quantity of SIRT1 protein expression average were 0.385,0.827,0.009,0.232,0.275,0.159,0.275 in GES-1,SGC7901,MKN45,MKN28,HGC27,AGS and MGC803,respectively.The relative quantity of P-gp protein expression average were 0.339,0.526,1.03 in GES 1,SGC7901 and MKN45 and Top-Ⅱ alpha protein were 0.093,0.889,and 0.158,respectively,siRNA-SIRT1 transfected SGC7901 for 8 hours and after 72 hours to test the relative quantity of SIRT1 protein expression average were 0.965,0.937,0.958,0.567,0.253,0.083 in control,BC-V,negative,siRNA-1,2,3 SIRT1,P-gp were 1.893,1.905,1.932,0.465,0.006,0.465 and Top-Ⅱ alpha were 0.09,0.07,0.073,0.085,0.168,0.085.MTT results showed that after SIRT1 protein expression was inhibited the SGC7901 proliferation has no obvious change in vitro and cell sensitivity to the chemotherapy drugs 5-Fu was increased significantly.Conclusion:SIRT1 expression in gastric cancer tissues and the expression of SIRT1 in gastric cancer tissue and cell carcinoma factors role,SIRT1 protein overexpression can promote P-gp protein expression lower Top-Ⅱ alpha protein expression in gastric cancer cells to chemotherapy of multiple drug resistance increase.
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Background Diabetes is one of the risk factors that leads to corneal neuropathy.Silent signal regulatory factor 1 (Sirt1) plays an important role in glucose metabolism,lipid metabolism,regulation of insulin secretion and is closely related to the nervous system disease.The relationship between Sirt1 and diabetic corneal neuropathy is not fully understood.Objective This study was to detect the expression of Sirtl in cornea and trigeminal ganglion with type 1 diabetes model mice and explore the association of Sirt1 expression with diabetic corneal neuropathy.Methods Eight C57BL/6-Ins2Akita/J male mice and eight wild-type C57BL/6 male mice in the same litter were selected as type 1 diabetes model group and control group,respectively.The mice of two groups were sacrificed in overdose anesthesia method at 12-month old.Histological examination of cornea and trigeminal ganglion was performed using hematoxylin and eosin staining.Expression and localization of Sift1 protein in cornea and trigeminal ganglion were detected using immunohistochemistry.Western blot assay and fluorescine quantitative PCR were respectively used to quantitatively analyze the expression of Sirt1 protein and Sirt1 mRNA.Results Trigeminal ganglion cells were uneven in size and shape with the loosened cellular arrangement and disorder neurofibrosis alignment,and the corneal epithelial cells were less in the C57BL/6-Ins2Akita/J mice,but the trigeminal ganglion cells and corneal epithelial cells were normal in wild-type C57BL/6 mice.Immunochemisty exhibited that Sirtl protein was expressed mainly in corneal epithelium and the expression of Sirtl protein was stronger in the C57BL/6 mice than that in C57BL/6-Ins2Akita/J mice.Fluorescine quantitative PCR assay showed that the gray scale value of Sirt1 mRNA in cornea in C57BL/6-Ins2Akita/J mice was lower than that of the wild-type C57BL/6 mice(0.56±0.03 vs.0.98±0.13) with significant difference (t =5.853,P =0.010).Western blot showed that the expression of Sirt1 protein in cornea was lower in C57BL/6-Ins2Akita/J mice than that of the wild-type C57BL/6 mice(0.78±0.017 vs.1.300±0.012) with significant difference(t =33.140,P =0.001).However,no significant differences were seen in the gray scale value of Sirt1 mRNA(2.45±0.18 vs.2.51±0.22) (t=0.587,P=0.599) and protein level(1.100±0.015 vs.1.110±0.017) (t =0.430,P=0.709) in trigeminal ganglion tissues between C57BL/6-Ins2Akita/J mice and wide-type C57BL/6 mice.Conclusions The corneal nerve and structure is abnormal in 12-month-old C57BL/6-Ins2Akita/J mouse.Sirt1 is involved in the pathogenesis of diabetic keratoneuropathy,suggesting that it may be a potential target.