| Objective:To explore the correlation of vasculogenic mimicry(VM)in malignant brain glioma with Golgi phosphoprotein 3(GOLPH3),investigate the effects of GOLPH3 on in vitro biological activities of malignant brain glioma cells and identify the mechanism of action of GOLPH3 in malignant brain glioma VM.Methods:(1)Clinical data and tumor tissues were collected from 40 patients who were admitted to the Affiliated Hospital of Guizhou Medical University for brain glioma(WHO grade I-IV)as the pathological diagnosis.CD34-PAS double staining was performed on the tumor tissues for clinicopathologic observation of VM;IHC staining was conducted to observe GOLPH3;possible correlations among GOLPH3,VM,and relevant clinicopathologic factors were studied based on statistical analysis.(2)GOLPH3 was encapsulated into a lentivirus designed for transfection of the malignant glioma cell line U-87 MG,and the U-87 MG cells were assigned to five groups according to transfection efficiency,including a blank control(CON),a negative control knockdown(NC-KD)group,a knockdown(KD)group,a negative control overexpression(NC-OE)group,and an overexpression(OE)group.(3)In vitro experiments: U-87 MG cells were divided into different groups in advance to observe the effects of GOLPH3 on the proliferation,apoptosis,invasion,and migration of glioma cells.(4)High-throughput RNA sequencing(RNA-seq)was used for library construction and quality control,followed by differential gene analysis and enrichment analysis of signaling pathways and molecular functions associated with GOLPH3.(5)Based on transcriptomic profiling,western blot(WB)analysis was conducted to measure the expression levels of GOLPH3,ephrin type-A receptor 2(Eph A2),vascular endothelial(VE)-cadherin,vascular endothelial growth factor receptor 2(VEGFR2),?-catenin,vascular endothelial growth factor(VEGF),matrix metalloproteinase-2(MMP-2),and E-cadherin in relation to the target molecular functions and signaling pathways.(6)Nude mouse models were established by subcutaneous transplantation of U-87 MG tumors to observe tumor growth,plot a tumor growth curve and further examine the correlations between GOLPH3 and VM.(7)Transmission electron microscopy(TEM)was utilized to observe VM structures in the tumor-bearing nude mouse model.Results:(1)CD34-negative and PAS-positive VM and GOLPH3 expression were detected in the patients with WHO grade I-IV brain gliomas(grade I-IV: n =6,14,9,and 11,respectively).(2)Higher-grade brain gliomas were associated with increased VM density and GOLPH3 staining intensity.WHO grade I-IV brain gliomas showed significant differences in VM density(?2 =7.939,P <0.01)and GOLPH3 expression(?2 =14.138,P <0.01).GOLPH3 was shown to have a linear,positive correlation with VM(P <0.01,r =0.788).VM and GOLPH3 were not significantly correlated with such risk factors as sex,age,and primary tumor site.There is a positive correlation between VM and the pathological grading of brain gliomas(P <0.01,r =0.854).GOLPH3 also exhibited a positive correlation with the pathological grading of brain gliomas(P <0.01,r =0.852).(3)Proliferation: Compared with the NC-KD group,the KD group showed reduced cell proliferation of U-87 MG cells(P <0.0001);no significant difference was observed in U-87 MG cell proliferation between the NC-OE and OE groups(P =0.0146,>0.01).Apoptosis: There was no significant difference in U-87 MG cell apoptosis between the NC-KD and KD groups(P=0.0187,>0.01);no massive apoptosis was detected,with the apoptotic cells in each group making up less than 5% of the tumor cell population;the number of apoptotic cells in the OE group was not significantly different from that in the NC-OE group(P=0.0153,>0.01);no massive apoptosis occurred as the apoptotic cells in each group only accounted for less than 5% of the tumor cell population.Invasion: Compared with the NC-KD group,the KD group exhibited a decline in cell invasion(P <0.05)(Fig.6);in comparison with the NC-OE group,no significant changes of cell invasion were observed in the OE group(P >0.05).Migration: Compared with the NC-KD group,the KD group saw a decline in cell migration at 8 h post-scratch(P <0.05);no significant differences were noted between the NC-OE and OE groups in cell migration at 8 h post-scratch(P >0.05).(4)Transcriptomic profiling: a.Data quality control: After implementation of data quality control,over 6 GB of clean bases were obtained in each group with Q20 bases >97% and Q30 bases >93%.b.Mapping to reference assemblies: Uniquely mapped reads and total mapped reads in each group were above 81% and 86%,respectively.c.Inter-sample correlation: The R2 value in each group was above 0.8.d.Comparison of differential genes: In the NC-KD and KD groups,there were 8,318 genes with different expression,including 4,227 genes with upregulated expression and 4,091 genes with downregulated expression;in the NC-OE and OE groups,there were 1,936 genes with different expression,including1,111 genes with upregulated expression and 825 genes with downregulated expression.By comparing the NC-KD/KD and NC-OE/OE groups,there were 1,188 genes with different expression.e.Enrichment analysis: Significant differences were detected in the top 30 biological processes,cell compositions,and molecular functions between the NC-KD and KD groups,as well as the NC-OE and OE groups.Besides,there were significant differences in the top 20 pathways between the NC-KD and KD groups and between the NC-OE and OE groups.Molecular functions and signaling pathways strongly associated with GOLPH3 mainly included cadherin binding,cell adhesion molecule binding,ECM-receptor interaction,and the PI3K-Akt signaling pathway.(5)Related protein expression: GOLPH3 protein expression was reduced after interference with(knockdown of)the GOLPH3 gene(P <0.01);GOLPH3 protein expression was increased when overexpression of the GOLPH3 gene occurred(P <0.001).Eph A2 expression was elevated following interference with(knockdown of)the GOLPH3 gene(P <0.001);Eph A2 expression was decreased after overexpression of the GOLPH3 gene(P <0.01).There was a reduction in MMP2 expression under the circumstance of interference with(knockdown of)the GOLPH3gene(P <0.001);MMP2 expression was improved in the presence of overexpression of the GOLPH3 gene(P <0.001).VE-cadherin expression was inhibited by the interference with(knockdown of)the GOLPH3 gene(P <0.001);when overexpression of the GOLPH3 gene occurred,there was an increase in VE-cadherin expression(P <0.01).?-catenin expression was increased after interference with(knockdown of)the GOLPH3 gene(P <0.01);overexpression of the GOLPH3 gene led to a reduction in ?-catenin expression(P <0.01).No significant differences were detected in VEGF expression under the circumstances of interference with(knockdown of)or overexpression of the GOLPH3 gene.VEGFR2 expression was decreased by interference with(knockdown of)the GOLPH3 gene(P <0.001);however,no significant changes in VEGFR2 expression were induced by overexpression of the GOLPH3 gene.E-cadherin expression was not significantly affected by interference with(knockdown of)or overexpression of the GOLPH3 gene.(6)Subcutaneous tumor-bearing animal models: Tumor growth was not detected in the CON group but in the NC-KD,KD,NC-OE,and OE groups.Compared with the NC-KD group,the KD group exhibited slower tumor growth and had a smaller tumor volume(P <0.01);compared with the NC-OE group,tumor growth was faster and the tumor volume was greater in the OE group(P <0.01).VM was observed in the subcutaneous tumor-bearing nude mice in each group.(7)Oncopathology of animal models: a.VM: VM structures were present in each group's subcutaneous tumor tissues;compared with the NC-KD group,VM structures in the KD group were reduced,and the difference was statistically significant(P <0.05);VM structures were increased in the OE group,which was significantly different from the NC-OE group(P <0.05).b.GOLPH3: GOLPH3 expression was present in all subcutaneous tumor-bearing nude mice.In the KD group,GOLPH3 positivity was reduced,indicating a statistically significant difference from the NC-KD group(P <0.05);compared with the NC-OE group,the OE group had increased GOLPH3 positivity,and the difference was statistically significant(P <0.05).(8)VM structures in tumor-bearing animal models under TEM: VM was not detected in the CON or KD group but in the NC-KD,NC-OE,and OE groups.Conclusions:(1)GOLPH3 expression and VM structures are present in WHO grade I-IV brain gliomas.In brain gliomas,there is a linear,positive correlation between GOLPH3 and VM;besides,GOLPH3 and VM also have linear,positive correlations with the degree of malignancy.(2)GOLPH3 plays a role in the biological activities(e.g.,proliferation,invasion,migration)of malignant brain glioma cells,which maintains such biological activities without promoting proliferation,invasion,or migration of tumor cells;GOLPH3 is probably not involved in the apoptosis of malignant brain gliomas.(3)GOLPH3 modulates VM in malignant brain gliomas by regulating the epithelial-to-mesenchymal transition(EMT)process(ECM remodeling)via the VE-cadherin/Eph A2/MMP-2/?-catenin pathway. |
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