| Background and ObjectiveAs we all known,gallbladder cancer is the highest incidence of biliary tract malignancy in the world.At present,the overall incidence is only 2.5 per 100,000,but the incidence is insidious,the degree of malignancy is high,and the prognosis is very poor.Most patients progress once diagnosed,so the 5-year survival rate is only 5%,with an average survival time of 6 months.Gallbladder therapy has not been determined,the traditional long course of radiation and chemotherapy treatment the effect not beautiful,radical resection is the only possible way to cure,gallbladder patients early diagnosis is difficult,however,only 15%?47%of patients found feasible surgical radical resection,therefore,the diagnosis and treatment principles for gallbladder is still is early detection,early diagnosis and early treatment.NGAL,or neutrophil-associated apolipoprotein,is a new member of the family of lipid transport proteins.In recent years,it has been found that the free sulfhydryl group of the propion-folding terminal of NGAL can bind to MMP9 in the form of dimercaptobond,promote the activation of pro-mmp9,inhibit the endogenous degradation of MMP9,further slow down the inactivation of matrix metalloproteinase inhibitor(timp-1)on MMP9,and thus prolong the activity of MMP9.At the same time,NGAL can combine with the iron carrier to form a complex,so that the extracellular iron can be transported to the inside of the cell,thereby increasing the content of intracellular iron,inhibiting the expression of Bim protein that promotes apoptosis,and upregulating the level of beta-catenin protein to enhance the Wnt signaling pathway and promote the occurrence of tumors.More and more studies have found that NGAL is abnormally expressed in malignant tumors of the digestive system such as pancreatic cancer,gastric cancer,liver cancer and colorectal cancer,and is involved in the occurrence and development of tumors.Moreover,the mechanism of action of NGAL gene is tissue specific.Therefore,the study of the effect of silencing NGAL gene on the proliferation and metastasis of GBC-SD cells can provide new methods and ideas for gene therapy of gallbladder cancer patients.In this study,the expression of NGAL gene in human gallbladder cancer tissues and cells was detected by immunohistochemical method,and the NGAL gene in gallbladder cancer gcc-sd cells was silenced by siRNA to observe the effect on the proliferation and metastasis of GBC-SD cells.MethodsBy referring to the medical record system of the department of pathology in our hospital,wax samples that were pathologically diagnosed as gallbladder cancer were selected,and the expression of NGAL gene in gallbladder cancer tissues was detected by immunohistochemistry,and positive control was conducted with breast cancer samples.After siRNA silencing in GBC-SD cells of human gallbladder cancer,the expression of NGAL mRNA before and after transfection was detected by q-pcr.The expression of NGAL gene in BC-SD cells of human gallbladder carcinoma was detected by immunoblotting.Cell count kit-8 method was used to observe the effect of NGAL gene silencing on the proliferation of BC-SD cells in human gallbladder cancer.Cell scratch test was carried out to verify the migration of GBC-SD cells after transfection with gallbladder carcinoma.Flow cytometry was used to detect the effect of silencing NGAL gene on apoptosis of human gallbladder carcinoma(GBC-SD)cells.Transwell detecting the invasion ability of tumor cells after NGAL gene was silenced.Results:one-way anova and homogeneity test of variance were performed.Lsd-t test was used in pair-to-pair-comparison,and the difference of data was statistically significant,with P<0.05 as the standard.Results(1)immunohistochemical staining of 21 pathological sections showed that NGAL gene was positively expressed in 16 samples,with a positive rate of 76.19%(16/21).There was no or low expression of NGAL in the adjacent tissues of gallbladder cancer.Cellular immunohistochemistry showed that NGAL was positively expressed in the GBC-SD cells of human gallbladder cancer.(2)transfection of ngal-sirna into human gallbladder cancer gbc-sd cells,PCR results showed that the expression level of NGAL mRNA after transfection of ngal-sirna significantly decreased,and the difference was statistically significant compared with that of the blank control group and the NC control group(P<0.01).(3)western blotting results showed that ngal-sirna transfected into human gallbladder cancer GBC-SD cells,and the transfection group significantly inhibited the expression of NGAL protein in human gallbladder cancer GBC-SD cells compared with the control group and the NC group,with statistically significant difference(P<0.01)..(4)CCK8 results showed that the proliferation activity of bcc-sd cells significantly decreased after ngal-sirna transfection,which was statistically significant compared with the control group and the NC group(P<0.05).(5)the cell scratch experiment showed that the lateral migration ability of ngal-sirna transfected cells decreased,which was statistically significant compared with the control group and the NC group(P<0.01).(6)flow cytometry showed that the apoptosis of GBC-SD cells in gallbladder cancer was promoted after NGAL gene silencing,which was statistically significant compared with the control group and the NC group(P<0.05).(7)Transwell Chambers method of human gallbladder GBC-SD cells that transfection group of cells was significantly less than the blank control group,and NC group,the difference statistically significant(P<0.01).ConclusionsThe NGAL gene is positively expressed in human gallbladder cancer BC-SD cells,and NGAL-siRNA can effectively silencing BC-SD cells,reducing the expression of NGAL gene mRNA and its protein in gallbladder cancer cells,limiting the growth,migration and invasion of BC-SD cells,and accelerating their apoptosis.NGAL gene silencing effectively limits the proliferation and metastasis of GBC-SD cells in human gallbladder cancer,so NGAL may be a potential target for gene therapy of gallbladder cancer. |
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