| Objective:HSP90 α,a subtype of HSP90,is located in the cytoplasm and consists of 730 amino acids.HSP90AA1 is a functional gene of HSP90 α.The main purpose of this study is to explore the role of HSP90AA1 in the phenotype of proliferation and migration of gastric cancer cells in vitro and in vivo.As a targeted inhibitor of Hsp90,17-DMAG plays an increasingly important role in tumor therapy.17-DMAG has obvious inhibitory effect on various phenotypes of malignant tumor.In this experiment,17-DMAG and docetaxel were combined to treat gastric cancer cells to observe the effect of the combination on the phenotype of gastric cancer cells,to clarify its synergistic effect with docetaxel in the treatment of gastric cancer.Methods: Part Ⅰ:(1)The expression level of HSP90 α in cancer and adjacent tissues was determined by protein extraction;(2)q PCR and Western Blot were performed on AGS,SGC-7901,MGC-803,HGC-27 and GES-1 cell lines to find out the cell lines with the highest and lowest expression level of HSP90α;si RNA was transfected to knock down the cell lines with the highest expression level of HSP90AA1,and plasmid was transfected to the cell lines with the lowest expression level of HSP90AA1 to establish HSP90AA1 overexpression cell lines;(3)By using PCR and Western Blot experimental techniques,HSP90AA1 in gastric cancer cell line was verified at the transcription level and functional protein expression level,and the efficiency of transfection should be determined;(4)CCK-8 was used to detect the proliferation rate of gastric cancer cells,and the colony forming ability of gastric cancer cells was verified by cell colony forming assay;(5)The effects of HSP90AA1 on the migration and invasion of gastric cancer cells were evaluated by scratch test and Transwell test;(6)Western Blot was used to detect the expression of the proteins as E-cadherin,N-cadherin,vimentin and snail.These proteins were closely related to epithelial mesenchymal transition(EMT),which could be used to determine the role of HSP90AA1 in this kind of biological behavior.Part II: Female BALB / C nude mice aged6-8 weeks were selected and randomly divided into control group,empty plasmid group and HSP90AA1-sh RNA group,with 5 nude mice in each group.These mice were injected with HGC-27 gastric cancer cells,HGC-27 empty plasmid,and HGC-27 HSP90AA1-sh RNA intraperitoneally,to establish the model of intraperitoneal tumor implantation.The mice were divided into control group,NC group,and HSP90AA1-sh RNA group.The tumor volume was measured at 3;6;9;12;15;18;21;24,27,30 days after intraperitoneal injection,and the growth curve was drawn.After 30 days,all the mice were killed by cervical spondylectomy,and the tumor tissues were dissected and taken out,while the tumor mass and volume should be measured at the same time.The expression of HSP90AA1;Ki-67;PCNA was detected by WB and PCR.Part Ⅲ:(1)CCK-8 was used to detect the proliferation of various gastric cancer cell lines when 17-DMAG was applied in different concentrations and different time;(2) Flow cytometry was used to detect the effect of different concentrations of 17-DMAG on the apoptosis of gastric cancer cell lines at the same time;(3) As 17-DMAG was combined with docetaxel,CCK-8 was used to detect the proliferation of gastric cancer cells and normal gastric epithelial cells,and IC50 and CI were calculated;(4)As 17-DMAG was combined with docetaxel,flow cytometry was used to detect the changes of gastric cancer cell cycle and single drug group,and the expression of cycle related proteins was verified;(5)As 17-DMAG was combined with docetaxel,the apoptosis of gastric cancer cells was detected by flow cytometry,and the expression of apoptosis related proteins was verified;(6) As 17-DMAG was combined with docetaxel,the migration of gastric cancer cells was detected by scratch test,and the expression of migration related proteins was also verified.Results: Part Ⅰ:(1)HSP90αwas highly expressed in gastric cancer tissues,and the highest expression level of HSP90α was found in HGC-27 cell line,which was used to construct the target gene knockout cell line.The expression level of HSP90αwas the lowest in SGC-7901 cell line,which was used to construct the target gene overexpression cell line.PCR and WB showed that the transfection was successful;(2)According to the results of CCK-8: The proliferation of SGC-7901 was significantly increased 48 hours after HSP90AA1 overexpression transfection;The proliferation of HGC-27 decreased significantly 72 hours after HSP90AA1 knockdown transfection;The results of cell clone formation assay showed that the number of cell colonies increased significantly in the HSP90AA1 overexpression group and decreased significantly in the HSP90AA1 knockdown group;(3)Scratch test and Transwell test: As HSP90 a AA1 was overexpressed,the ability of cell migration and invasion was enhanced.Part Ⅱ:(1) A mouse model of intraperitoneal implanted tumor was established successfully,the success rate was 100%(15 / 15);(2)The tumor volume growth curves of control group and NC group coincided basically;(3)In HSP90AA1-sh RNA group,the volume of implanted tumor was significantly smaller than that of the other two groups;(4) In the control group and NC group,the protein expression levels of Ki-67 and PCNA were basically same;(5)WB results showed that the expression levels of Ki-67 and PCNA in HSP90AA1-sh RNA group were significantly lower than those in control group and NC group.Part Ⅲ:(1) 17-DMAG inhibited the proliferation of HGC-27 in a time-dose-dependent manner,which was more effective than other gastric cancer cell lines;(2)17-DMAG could induce apoptosis of gastric cancer cell lines in a concentration dependent manner after 24 hours;(3) Compared with normal gastric epithelial cells,17-DMAG inhibited the proliferation of HGC-27 more significantly and had a synergistic effect with docetaxel.The IC50 of 17-DMAG combined with docetaxel was 0.468μmol,and the index of CI was 0.67;(4)The effect of 17-DMAG on cell cycle arrest of HGC-27 was not obvious,and the cell cycle arrest of 17-DMAG combined with docetaxel was not as good as that of docetaxel alone;(5)17-DMAG can promote the apoptosis of HGC-27,and the combination of 17-DMAG and docetaxel has a synergistic effect,and the expression level of apoptosis related proteins is increased;(6)17-DMAG can inhibit the migration of HGC-27,and it has synergistic effect with docetaxel,and the expression level of migration related proteins is decreased.Conclusion:(1)Compared with cancer adjacent tissues,the expression of HSP90AA1 was higher in cancer tissues of those patients of gastric cancer,and had clinical relevance with gastric cancer patients;(2)HSP90AA1 has tumor gene activity in gastric cancer,which can promote the proliferation,migration and EMT in vitro experiment;(3)The results in nude mice showed that HSP90AA1 had a close relationship with tumor proliferation;(4)17-DMAG has shown the characteristics of anti-tumor phenotype in vitro experiments of gastric cancer cells;(5) 17-DMAG combined with docetaxel has synergistic effect on proliferation,migration and other phenotypes of gastric cancer;(6) The effect of 17-DMAG on the cell cycle of gastric cancer is not obvious.It is considered that 17-DMAG can inhibit cell proliferation by promoting apoptosis or other ways. |
