| Objective:In vitro（cells）experiments:To observe the effect of high glucose（D-glucose:25 m M）on the proliferation and migration of gastric cancer（GC）cells and its effect on Pin1/BRD4/NAP1L1/P21 signal pathway,and to clarify the potential mechanism of Pin1/BRD4 gene on the proliferation and migration of gastric cancer cells under the condition of high glucose.In vivo（animals）experiments:To investigate the effects of Pin1/BRD4 on the proliferation and metastasis of GC tissues in mice with streptozotocin-induced Diabetes mellitus（DM）.Methods:In vitro（cells）experiments:The cells were divided into the following groups:Ctrl group（Control group,D-Glucose:5.6 m M）,HG group（High glucose group,D-glucose:25 m M）,Mannitol group（hypertonic group,D-glucsoe:5.6m M+Mannitol:19.4 m M）,HG-Sh Pin1 group（GC cells were stably transfected with the Sh Pin1 vector）,HG-Sh NC group（GC cells were stably transfected with the Sh-NC vector）and HG-JQ1 group(GC cells were treated with JQ1:10-6M).Ed U and CCK-8 assays were measured GC cells proliferation;Transwell and Wound-healing experiments measured GC cells migration;Cell cycle and apoptosis were detected by flow cytometry;Western blotting（WB）and Real-time quantitative PCR（q RT-PCR）were used to detect the expression of Pin1,BRD4,NAP1L1,P21 and specific proliferation-related molecule PCNA in GC cells.GC cells were transfected with specific Pin1-Sh RNA（shorthairpin RNA,Sh RNA）lentivirus and treated with BRD4inhibitor JQ1(10-6M),to observe the effect of Pin1/BRD4 pathway on the proliferation and migration of GC cells.In vivo（animals）experiments:The diabetic mouse model was established by intraperitoneal injection of 100 mg/kg streptozotocin（STZ）in 4-week-old nude mice,and the corresponding MKN45 cell lines in each group were injected to subcutaneous or tail vein to observe the effect of Pin1/BRD4 on the growth and metastasis of GC cells in vivo.Animals were combined in groups for experiments:Ctrl group（Normoglycemic group）,DM group（diabetes group）,DM-Sh Pin1 group（mice were injected with MKN45-Sh Pin1）,DM-Sh-NC group（mice were injected with MKN45-Sh-NC）,DM-JQ1（mice were treated with JQ1:50mg/kg）and Ctrl-Sh-NC group;Tumor length and width were measured using a Vernier caliper every three days.Every week,tumor growth and metastasis were monitored by in vivo imaging system;Fasting plasma glucose（FPG）was measured using the glucose oxidize method;HE staining was used to observed morphological and histological changes;the expression of Pin1,BRD4,NAP1L1,P21 and the expression of specific proliferation related molecules PCNA in tissue level was observed by immunohistochemical method.Results:In vitro（cells）experiments:（1）HG microenvironment can significantly promote the proliferation and migration of gastric cancer cells.Compared with Ctrl group,HG group significantly increased the ability of proliferation and migration of GC cells.Silencing Pin1 gene by transfection of lentivirus and inhibiting BRD4 gene by JQ1(10-6M)intervention can significantly inhibit the proliferation and migration of GC cells induced by high glucose.Compared with HG group,HG-Shpin1 group and HG-JQ1 group,the metabolic proliferation activity and migration level of GC cells decreased significantly.（2）HG exposure can promote GC cells to pass the arrest point of cell cycle G1/S phase,accelerate the process of cell cycle and inhibit apoptosis.Compared with Ctrl group,the number of cells in G0/G1 phase in HG group decreased significantly,the number of cells in S phase increased significantly,and the apoptosis rate decreased significantly,antagonizing Pin1 or BRD4 gene could significantly inhibit cell cycle progression and promote the level of apoptosis.（3）Compared with the Ctrl group,HG could significantly increase the expression of Pin1,BRD4 and NAP1L1 protein and inhibit the expression of P21 protein GC cells;antagonizing Pin1/BRD4 gene could significantly inhibit the expression of NAP1L1,while the expression of P21 was significantly increased;compared with the HG group,the expression of BRD4 was inhibited by JQ1,and there was no significant difference in the expression of Pin1 protein.In vivo（animal）experiments:（1）Compared with Ctrl group,the blood glucose level of all groups increased significantly after injection of STZ（100mg/kg）.There was no significant difference in blood glucose level among DM group,DM-Sh Pin1group,DM-Sh-NC group and DM-JQ1 treatment group.（2）The results of tumor formation in nude mice showed that compared with normal blood glucose group（Ctrl group and Ctrl+Sh-NC group）,the tumor mass and growth of diabetic mice group（DM group and DM+Sh-NC group）was the largest and fastest,while that of DM+JQ1group and DM+Sh-Pin1 group was significantly smaller and slower than that of diabetic mice group（DM group and DM+Sh-NC group）.（3）The results of mouse tail vein lung metastasis test showed that hyperglycemia could significantly promote gastric cancer metastasis.Compared with Ctrl-Sh-NC group,DM-Sh-NC group had obvious lung metastasis 4 weeks after injection of tumor cells.Compared with DM+Sh-NC group,lung metastasis in DM+JQ1 group and DM+Sh-Pin1 group decreased significantly.（4）The results of immunohistochemical assay showed that compared with normal blood glucose group（Ctrl group and Ctrl+Sh-NC group）,the expression of Pin1,BRD4,NAP1L1 and PCNA in hyperglycemia group（DM group,DM+Sh-NC group）was significantly increased,while the expression of P21 was significantly decreased,by inhibiting the expression of Pin1 or BRD4,NAP1L1 and PCNA,the expression of P21 was significantly increased,but there was no significant change in the expression of Pin1 in the case of inhibition of BRD4.Conclusions:Through this study,we found that Pin1/BRD4 plays an important role in the process of high glucose promoting tumor growth.Pinl and BRD4 may be the key targets for the prevention and treatment of the growth and metastasis of GC,which have potential theoretical and clinical significance to improve the prognosis of diabetic patients with GC. |
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