| Objective:Epidermal growth factor receptor has been used for a long time as a molecular target for solid tumors,however,its clinical efficacy in glioma is poor,and the molecular mechanisms underlying the development and progression of EGFR overexpressing gliomas are not very clear.In this study,receptor expression enhancing protein 2 that may interact with EGFR was obtained through database screening,and transcriptomics was used to search for differentially expressed genes in glioma cells that interfere with REEP2,to construct a small molecule interaction network that combined a novel class of noncoding RNAs from competing endogenous RNAs perspective to explore that circular RNA,circ POSTN adsorbs miR-361-5p to functionally worked the key gene TPX2 in the regulation of proliferation,apoptosis and glycolytic metabolism of glioma,and to provide more molecular targets for the development and clinicopathological diagnosis of glioma.Methods:(1)Enriching the genes related to poor prognosis of gliomas by using the online database of Human Protein Atlas and screening the key gene REEP2 which differs greatly from normal brain tissue.(2)The differential expression of REEP2 and EGFR protein in 63 cases of gliomas and 3 cases of normal brain tissues were detected by immunohistochemical method,and the differential expression of REEP2 in gliomas was analyzed by clinical correlation.The histological localization and co-expression effect of REEP2 and EGFR protein in the above-mentioned gliomas were detected by double fluorescence immunostaining.(3)The difference of REEP2 expression levels in U251,LN229,U87,U178,SHG-44 glioma cells and NHA normal human astrocyte was detected by Western Blotting,and the cell lines needed for subsequent experiments were screened.(4)Transfection of glioma cells U251 and LN229 with sh-NC and sh-REEP2 plasmids by 48 hours,the differentially expressed genes of glioma cells interfering with REEP2 were studied by transcriptome sequencing.Common differentially expressed genes of the two cells were obtained under the conditions of 2-fold change and p < 0.05.GO enrichment,interaction analysis of KEGG signal pathway and PPI protein interaction were carried out.The differentially expressed down-regulated microtubule aggregation factor was obtained by RT-q PCR.TPX2 as the follow-up research object.(5)Synthesize si-NC and si-TPX2 small interfering RNA,detect the effect of interfering TPX2 on the proliferation of glioma cells by MTT test,and detect the expression level of Bcl-2,Bax and the change of caspase-3 activity by Western Blotting.(6)Analyze the expression of miR-361-5p and circ POSTN predicted by TPX2 with binding sites by using the Star base 3.0 on-line bioinformatics and the fluorescent enzyme report gene experiment.Detect the expression of their RNA in 25 cases of gliomas,3 cases of normal brain tissue,LN229,U251 and NHA cells by RT-q PCR.Analyze the correlation between the two and the expression of TPX2.(7)Western Blotting experiment and glucose metabolism kits were used to detect the expression changes of HK2 and LDHA,the key genes of glycolysis and metabolism,the levels of glucose,lactic acid and ATP in cell culture supernatant,the production changes of lactate dehydrogenase A and ROS in cells.(8)LN229 glioma cells were infected with sh-NC and sh-circ POSTN lentiviral vectors for 48 hours,and subcutaneous tumorigenic model of nude mice were used to explore the effect of circ POSTN on malignant proliferation of gliomas,the effect of circ POSTN adsorbing miR-361-5p to interfering with TPX2 on malignant proliferation of gliomas was discussed through in vivo experiments.Results:(1)201 prognostic-related genes from 153 glioma patients with TCGA were reported by HPA.Six genes specifically expressed in brain tissue,including REEP2,CEND1,NACAD,STAC2,PTPRN and SCHIP1,were identified.The differential expression of REEP2 m RNA was higher than each other,and its overexpression associated with poor survival rate of glioma patients.(2)Immunohistochemical staining showed that the expression of EGFR and REEP2 protein increased significantly with the increase of WHO pathological grade of glioma.REEP2 protein was mainly diffuse in glioma cytoplasm.Clinical correlation analysis showed that REEP2 expression level was closely related to age and pathological grade of glioma patients.Double immunofluorescence staining showed that REEP2 protein was slightly less fluorescent than EGFR,and REEP2 protein was mainly diffuse in glioma cytoplasm.Co-expression fluorescence intensity gradually increased with the pathological grade of glioma,suggesting that the co-expression of REEP2 and EGFR may be an indicator of the pathological grade of glioma.(3)Western blotting results showed that REEP2 protein of U251 and LN229 glioma cells were higher than that of control of NHA cells,and the difference was significant(p < 0.05).Therefore,these two cells were selected as transcriptome sequencing interfering with REEP2.(4)The transcriptome sequencing enriched 112 common differentially expressed genes,55 of which were up-regulated and 57 of which were down-regulated.Bioinformatics analysis showed that the differentially expressed genes participated in551 biological processes,106 cell components,73 molecular functions and 3 possible KEGG signaling pathways.with statistical significance(p < 0.05).Differential genes,with EGFR and microtubule aggregation factor TPX2 as the core nodes of the molecular interaction network,are mainly involved in many biological processes and signaling pathways related to glioma,such as cell-to-cell junction,glutathione signaling,cell surface receptors,kinase cell signaling pathways,lipid and amino acid metabolism.RT-q PCR validation showed that TPX2 m RNA was significantly down-regulated,which was the main research object in the next experiment.(5)TPX2 protein expression level of glioma was significantly higher than that in normal brain tissue by Western blotting.Compared with normal astrocyte NHA,LN229 and U251 glioma cell lines showed higher levels of TPX2 protein expression(p < 0.05);by interfering with TPX2 protein expression in glioma cells by si-TPX2,MTT cell growth curve showed that down-regulation of TPX2 inhibited glioma cell proliferation.Western blotting analysis showed that down-regulation of TPX2 could promote the expression of Bcl-2,Bax and Caspase-3 in glioma cells.(6)Star Base bioinformatics combined with luciferase reporter gene analysis showed that the expression level of wild-type TPX2 was down-regulated in the treatment group of over-expressing miR-361-5p.When circ POSTN was overexpressed,the expression level of TPX2 protein was up-regulated.RT-q PCR detected the expression level of glioma cells and tissues,and found that TPX2 was negatively correlated with the expression of miR-361-5p and TPX2 was positive with circ POSTN.Relatively,there was a negative correlation between miR-361-5p and circ POSTN,and the difference was statistically significant(p < 0.05).(7)With the down-regulation of circ POSTN,compared with the si-NC group,the glucose utilization rate and ATP consumption capacity of the two glioma cells decreased,the intracellular lactate accumulation,the activity of lactate dehydrogenase in the cells decreased,and the ROS of reactive oxygen species decreased.When the expression of miR-361-5p was antagonized,the levels of glycolytic metabolites recovered accordingly,with a statistically significant difference(p < 0.05).Interference with circ POSTN by Western blot analysis resulted in decreased expression of HK2 and LDHA proteins and marked inhibition of glycolysis and metabolism in glioma cells by decreasing TPX2 expression.(8)The subcutaneous tumorigenesis model of nude mice was constructed by shcirc POSTN interference with LN229 cell line.The results showed that the growth rate of tumors in sh-circ POSTN group was slower and the volume and weight of tumors were smaller than those in sh-NC group.In addition,the tumors were constructed by RT-q PCR and Western PCR.Compared with sh-NC group,the down-regulation of circ POSTN expression in tumor tissue caused an increase in the expression of miR-361-5p and a decrease in the expression of TPX2 protein in tumor tissue(p < 0.05).Conclusions:The REEP2 and EGFR proteins share a certain degree of co-localized expression effect,and its high expression may indicate the pathological grading of gliomas.Transcription group studies showed that when the expression of REEP2 in gliomas was down-regulated,the differential genes with the core molecule interactions of EGFR,TPX2,etc.were down-regulated accordingly.Moreover,circ POSTN can absorb miR-361-5p to regulate the expression of TPX2 in gliomas,and inhibit the circ POSTN/miR-361-5p/TPX2 signal axis can effectively promote apoptosis of gliomas and inhibit the level of key substances of glycolysis and metabolism,thus inhibiting the malignant proliferation of gliomas in vivo and in vitro and playing a role similar to carcinogenic genes in the genesis and development of gliomas. |
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