| Objective:In recent years,it has been found that Gab2-associated binding protein 2 is highly expressed in many malignant tumors,and its expression is closely related to the occurrence and development of tumors;At the same time,studies have shown that Gab2 also has a high expression trend in glioma tissues,but the influence of its transcription level and expression level on the development of glioma is still inconclusive.Detection of Gab2 mRNA in glioma tissues,quantitative analysis of the transcription level of Gab2,and correlation analysis with clinical pathology and glioma imaging data will help to further explain the regulation mechanism of the expression level of Gab2 on glioma.Therefore,this study will detect Gab2 mRNA in glioma and paraneoplastic tissues,and analyze its relationship with clinical pathology and imaging data;To further establish a scientific and standardized glioma model of orthotopic transplantation in Sprague-Dawley rat;By reducing the expression of Gab2,To study the effect of reducing the expression of Gab2 on the proliferation and apoptosis of C6 glioma cells in vitro and SD-rat brain,and the effect on the expression of cytokines IL-6,TNF-α,VEGF and tumor related factors MMP-2,MMP-9 and Bax,Bcl-2,p53,Cleaved-caspase-3;To elucidate the preliminary relationship between the expression level of Gab2 and the occurrence and development of glioma,and to explore the effect of Gab2 expression on proliferation and apoptosis of glioma cells and its possible molecular mechanism,which lays a foundation for the effective treatment of glioma.Methods:1.A total of 40 patients with clinical gliomas were enrolled,and their clinical pathology and imaging data were summarized.The relative expression of Gab2 mRNA in 40 gliomas and paraneoplastic tissues was detected by qRT-PCR.The different expression of Gab2mRNA in gliomas and paraneoplastic tissues was analyzed.The relationship between Gab2 expression and clinicopathological features and peritumoral edema were analyzed;2.Using C6 glioma cells to study the functional regulation mechanism of Gab2 on glioma cells;C6 glioma cells were cultured in vitro and divided into three groups for Gab2-siRNA cell transfection to down-regulate the expression of Gab2 gene in cells:control group;empty vector group;Gab2-siRNA group.The expression of Gab2 in three groups was detected by western-blot 48h after transfection to test the transfection efficiency.The apoptosis of the three groups was detected by flow cytometry 72h after transfection.3.The glioma model was established by using C6 glioma cells and SD-rats to further study the regulation mechanism of Gab2 on the proliferation and apoptosis of glioma cells in vivo;SD-rats were divided into three groups:control group,Gab2-siRNA group,47 rats in each group,and 5 rats in blank group;The same number of C6 glioma cells and C6 glioma cells transfected with Gab2-siRNA were stereotactically inoculated into the right caudate nucleus of rats in the control group and the Gab2-siRNA group to construct animal models;Five rats in the blank group were replaced with physiological saline.Two weeks after modeling,rats in the control group(n=15)and Gab2-siRNA group(n=15)were randomly selected and sacrificed after perfusion.The tumor was taken from the skull,the weight and volume of the tumors were measured,and the tumor formation rate was calculated;After 3 weeks of modeling,rats in the control group(n=30)and Gab2-siRNA group(n=30)were sacrificed after perfusion and taking blood from the eyelids;5 rats in the blank group were also sacrificed after perfusion;The tumor was taken from the skull,the weight and volume of the tumors were measured,and the tumor formation rate was calculated;The statistical analysis was performed and compared to the modeling after 2 weeks.After 3 weeks of modeling,the pathological features of the tumor were determined by HE staining,and the source of the tumor was determined by GFAP staining.The expression levels of Bax and Bcl-2 proteins were detected by immunohistochemical staining;The expression levels of MMP-2 and MMP-9 were detected by qRT-PCR and western-blot;The expression levels of Bax,Cleaved-caspase-3 and p53 proteins in tumors were detected by Western-blot;ELISA was used to detect the expression levels of cytokines IL-6,TNF-α and VEGF in the serum of tumor-bearing rats in control group and Gab2-siRNA group and the ultrastructure of the tumor cells in two groups was observed by electron microscopy.Results:1.The expression of Gab2 in glioma tissues was significantly higher than that in paraneoplastic tissues(P<0.01).With the increase of pathological grade of glioma and the increase of peritumoral edema,the relative expression level of Gab2-mRNA was significantly different(P<0.01).However,there was no statistical significance in expression between different genders and ages(P>0.05).2.After transfection of C6 glioma cells with Gab2-siRNA,the expression level of Gab2 protein in C6 glioma cells of Gab2-siRNA group was significantly lower than that of control group and empty vector group(P<0.01).The expression of Gab2 protein in empty vector group was not significantly different from that in the control group(P>0.05).The apoptosis rate of the control group was not significantly different from that of the empty vector group(P>0.05);The apoptosis rate of Gab2-siRNA group was significantly higher than that of the control group and the empty vector group,with significant difference(P<0.01).3.The results of sectioning the tumors,detecting the volume and weight of the tumors,and calculating the tumor formation rate showed that the volume and weight of the tumors after 3 weeks of modeling were significantly higher than those after 2 weeks of modeling(P<0.01);At the same time,the volume and weight of the tumors in the Gab2-siRNA group were significantly lower than those of the control group(P<0.01);The tumor formation rate of the Gab2-siRNA group was slightly lower than that of the control group,but there was no statistical difference(P>0.05),there was no tumor formation in the blank group.4.HE staining showed that compared with the control group,the Gab2-siRNA group had less cell allotype,decreased nucleoplasm ratio,less pathological mitotic figures and new blood vessels.The GFAP staining of the two groups of tumors was positive,indicating that the tumor source was a source of glial cells and could be used for follow-up studies.5.Immunohistochemistry of Bax and Bcl-2 protein showed that Bax、Bcl-2 was expressed in both tumor tissues;compared with control group,the expression of Bcl-2 protein in Gab2-siRNA group down-regulated(P<0.05),the expression of Bax protein in Gab2-siRNA group significantly increased(P<0.01).6.The relative expression levels of MMP-2 and MMP-9 mRNA and the expression level of MMP-2 and MMP-9 protein in Gab2-siRNA group were significantly lower than those in control group(P<0.01).7.The expression levels of p53,Cleaved-caspase3 and Bax in Gab2-siRNA group were significantly higher than those in control group(P<0.01).8.The expression of cytokines IL-6,TNF-α and VEGF in Gab2-siRNA group was significantly lower than that in control group(P<0.05).9.Electron microscopy showed that the signs of apoptosis in the Gab2-siRNA group were significantly more common than those in the control group.Conclusions:1.The expression of Gab2 gene in glioma tissues was significantly higher than that in paraneoplastic tissues(P<0.01),and its expression was significantly affected by the pathological grade of glioma and the degree of peritumoral edema;It is suggested that Gab2 may only be highly expressed in tumor tissues,and it is closely related to the occurrence and development of glioma,and it is expected to become a new molecular target.2.Gab2-siRNA transfected C6 glioma cells with higher efficiency,significantly decreased the expression of Gab2 protein in C6 glioma cells(P<0.01);Decreasing the expression of Gab2 can significantly promote the apoptosis of C6 glioma cells in vitro(P<0.01).The C6 glioma model of the caudate nucleus in SD rat is a good model with short tumor formation time,high tumor formation rate and good model stability.3.Decreasing the expression of Gab2 in C6 glioma cells can significantly inhibit its proliferation and growth and promote its apoptosis in the brain of SD-rat,reduce the weight and volume of the tumor,which may be due to the down-regulation of the expression of MMP-2,MMP-9 and cytokines IL-6,TNF-α,VEGF,increased the ratio of Bax/Bcl-2 and activated the apoptotic signaling pathway p53/Bax/Cleaved-caspase-3. |
PDF Full Text Request