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Objective:To investigate the role of microchromosome maintenance protein 5 (MCM5) in promoting malignant progression and its mechanism in glioblastoma.Methods:(1) Three freshly excised brain tissues from non-tumor patients and 3 grading IV glioblastoma tissues were collected in our hospital from September 2020 to September 2021; mass spectrometry and quantitative proteomic analysis were performed to screen differentially expressed proteins for functional enrichment analysis. (2) The target protein MCM5, which was highly expressed in glioblastoma, was screened on the basis of proteomics, and its expression in glioblastoma was verified using The Cancer Genome Atlas (TCGA) database and further validated at the mRNA level in the collected clinical specimens. (3) U251 cells were divided into negative control group, knockdown group-1 (si-1 group) and knockdown group-2 (si-2 group) by siNRA transfection. The regulation role of MCM5 in malignant phenotype of glioblastoma was detected by CCK-8 assay, clone formation assay, 5-ethynyl-2'-deoxyuridine (EdU)staining, Transwell invasion assay and flow cytometry. (4) The transcriptome data of glioma patients from TCGA database were used to explore the possible molecular mechanisms of MCM5 regulation in the malignant process of glioblastoma by gene set enrichment analysis (GSEA) algorithm.Results:(1) In clinical samples of glioblastoma, 322 up-regulated proteins and 94 down-regulated proteins were screened out; MCM5 was highly expressed in these 3 glioblastoma samples. (2) Based on TCGA database, results of 163 patients with glioblastoma and 207 patients with non-tumor brain tissues showed that MCM5 expression was statistically up-regulated in glioblastoma ( t=3.340, P<0.001). Real-time quantitative PCR results of 3 glioblastoma tissues and 3 non-tumor brain tissues clinically collected in our hospital also indicated that significantly increased MCM5 expression in glioblastoma was noted as compared with that in the non-tumor brain tissues ( t=3.876, P<0.001). (3) As compared with the negative control group, the si-1 and si-2 groups had significantly decreased MCM5 mRNA and protein expressions, significantly lower proliferation rate 5 d after inoculation, statistically decreased number of cell clones, significantly decreased proportion of EdU positive cells, and significantly increased proportion of cells at G1 phase, and significantly impaired migration ability ( P<0.05). (4) GSEA analysis showed that mRNA in MCM5 high expression group was enriched in DNA damage repair gene, E2F target gene, MYC target gene, epithelial-mesenchymal transformation (EMT), nterleukin 6-Janus kinase-signal transduction and transcription activation factor 3 (IL6-JAK-STAT3), interferon, KRAS, NOTCH, transforming growth factor-β (TGF-β), Wnt/β-Catenin and other characteristic genes. Conclusion:MCM5 is highly expressed in glioblastoma, and MCM5 regulates the malignant progression of glioblastoma through multiple mechanisms including E2F, MYC, EMT, and Wnt/β-Catenin.
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Objective:To observe the safety and efficacy of one-stage bilateral temporal lobe lesions resection in patients with severe bilateral radiation-induced temporal lobe injury after radiotherapy for nasopharyngeal carcinoma.Methods:Clinical data of 12 patients with severe bilateral radiation-induced temporal lobe injury after radiotherapy for nasopharyngeal carcinoma underwent one-stage bilateral temporal lobe lesions resection in our hospital from January 2013 to December 2017 were retrospectively analyzed. Karnofsky Performance Scale (KPS) scores were used as predictors of surgical outcomes before and two weeks after surgery. Imaging changes of radiation-induced brain injury lesions after surgery before and 3 months after surgery, surgical-related complications, and death were observed.Results:All 12 patients (100%) had improved postoperative KPS scores, which showed significant difference as compared with the preoperative KPS scores ( P<0.05). Two patients had pulmonary infection after operation, one patient had poor wound healing, and one patient had intracranial infection; all of them recovered after expectant treatment. None of the 12 patients had new neurological symptoms after surgery. During the follow-up period, the postoperative cranial MR imaging showed that the lesions were completely removed, and the leukoencephalopathy was alleviated or completely subsided. Conclusion:One-stage bilateral temporal lobe lesions resection is feasible for bilateral radiation-induced temporal lobe injury after radiotherapy for nasopharyngeal carcinoma.
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Objective To investigate the effect of micro RNA (miR)-125a-3p on proliferation and apoptosis of glioma cells and its role in MAPK signaling pathway. Methods (1) The miR-microarray data from the Cancer Genome Atlas (TCGA, https:// cancergenome.nih.gov/) database were downloaded, and the miR-125a-3p expressions in 565 gliomas tissues and 10 normal brain tissues were compared. (2) Clinical collection of 30 glioma specimens surgically resected in our hospital from April 2015 to April 2018, was performed, including 7 of low-grade glioma and 23 of high-grade glioma;8 normal brain tissues needed craniocerebral trauma excision were collected at the same time period;reverse transcription (RT)-real-time quantitative (q) PCR was used to detect the miR-125a-3p expressions in glioma tissues and normal brain tissues. (3) The normal brain glial cells HA1800 and glioma cells (U251, U138, U87, U373, and T98G) were routinely cultured in vitro; RT-qPCR was used to detect the miR-125a-3p expression in normal brain glial cells and glioma cell lines. (4) The cultured glioma cell lines U251 and U373 at logarithmic phase were divided into miR-125a-3p group and negative control group;and miR-125a-3p mimic or nonsense sequence were transfected using LipofectamineTM 2000;72 h after transfection, the miR-125a-3p expression was detected by RT-qPCR; the proliferation rate was detected by clone formation after transfection; the apoptosis rate was detected by flow cytometry 72 h after transfection; the cleaved-caspase-3, cleaved-caspase-9, cleaved-caspase-7, P38 and P-P38/MAPK protein expressions were detected by Western blotting. Results (1) In TCGA database, the miR-125a-3p expression in glioma brain tissues was statistically lower as compared with that in normal brain tissues (P<0.05). (2) The miR-125a-3p expressions in clinically collected normal brain tissues, low-grade glioma specimens and high-grade glioma specimens were decreased successively, enjoying statistically significant differences (P<0.05). (3) As compared with normal glial cells, the miR-125a-3p expressions in glioma cell lines were significantly lower (P<0.05), of which, U251 and U373 enjoyed the most obvious decrement. (4) As compared with the blank control group, the miR-125a-3p group had significantly increased miR-125a-3p expression, significantly decreased colony forming efficiency, significantly increased proliferation rate, significantly increased expressions of apoptosis-related proteins cleaved-caspase-3, cleaved-caspase-9, and cleaved-caspase-7, and statistically increased phosphorylated-P38/MAPK expressions (P<0.05). Conclusion The miR-125a-3p expression is low in glioma tissues and cells; miR-125a-3p over-expression can inhibit the proliferation of glioma cells and promote apoptosis through MAPK signaling pathway, which may provide a new potential target for treatment of glioma.
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Objective To investigate the expression and biological value of heme oxygenase I (HO-1) in gliomas. Methods Fifty-six patients with gliomas, admitted to and accepted surgery in our hospital from January 2010 to January 2015, were chosen in our study; WHO grade I was noted in 4 patients, grade Ⅱ in 16, grade Ⅲ in 10, and grade IV in 26 patients; patients with grade I and Ⅱ were as low-grade glioma group and patients with grade Ⅲ and IV were as high-grade glioma group. The HO-1 expression in the two groups was detected by immunohistochemistry. R-langrage survival tool was used to analyze the relation between HO-1 expression and prognosis of 1107 patients with gliomas selected from The Cancer Genome Atlas (TCGA) database. Results Significant differences of HO-1 expression were observed in different grades of gliomas (P<0.05), and HO-1 high-expression rate in the high-grade gliomas (75%) was significantly higher than that in the low-grade group (30%, P<0.05). HO-1 protein expression level in the high-grade gliomas was significantly higher than that in the low-grade group (P<0.05). Moreover, area under the curve (AUC) of the receiver operating characteristic curve was suggested that the HO-1 could be an ideal determine factor (AUC=0.747, P=0.002). Log rank analysis indicated that the accumulate survival rate in patients with low HO-1 expression was significantly higher than that in patients with high HO-1 expression (P<0.05). TCGA database analysis showed that simultaneous survival rate in patients with low HO-1 expression was significantly higher than that in patients with high HO-1 expression (P<0.05). Conclusion Expression of HO-1 is correlated with the malignant degrees and prognoses of gliomas, and it has potential to be a novel biological marker for the diagnosis and treatment of gliomas; furthermore, HO-1 could also be a target for the study and treatment of gliomas.
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Objective To investigate the sorting nexin 10 (SNX10) expression in glioma tissues and its relationship with prognosis of the patients.Methods Thirty glioma specimens,collected from surgery and conformed by pathology in our hospital from January 2007 to December 2012,were used in our study,and in them,9 were WHO grade Ⅰ and Ⅱ and 21 were WHO grade Ⅲ and Ⅳ;and 30 nonneoplastic tissue specimens collected during decompression were used as control group.Immunohistochemical staining using polyclonal SNX10 antibody was performed on paraffin embedded specimens.The staining intensity was stratified as absent (-),weak (+),moderate (++) and strong (4+++).The relationships of SNX10 expression with several clinic pathologic indicators and prognosis were analyzed.Results High SNX10 expression was noted in 12 specimens and low SNX10 expression in 18 specimens of the glioma group.High SNX10 expression was noted in 25 specimens and low SNX10 expression in 5 specimens of the control group;the high SNX10 expression rate in glioma tissues was significantly lower than that in non-neoplastic brain tissues (P＜0.05);the high SNX10 expression rate in high-grade glioma tissues was significantly lower than that in low-grade glioma tissues (P＜0.05).The median survival time ofglioma patients with high SNX10 expression was 22.50±8.27 months,and that of glioma patients with low SNX10 expression was 15.50±0.99 months.The survival rate of glioma patients with low SNX10 expression was significantly lower than that of glioma patients with high SNX10 expression (34％ vs.65％,P＜0.05).By Cox multi-factor risk scale model,the expression level of SNX10 and grading of tumors were identified as the independent risk factors of patient's post-operative death;following the decreased SNX10 expression,the risk of postoperative death increased 1.983 times (95％ confidence interval=1.602-2.314,P＜0.05).Conclusions Decreased SNX10 expression is associated with occurrence and development of gliomas,and has a significant effect on patients' post-operative survival time.Decreased SNX10 expression level may be an important index of poor prognosis in glioma patients.
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Objectives To study the effect of VIM in Enterovirus 71 (EV71) infection of (human brain microvascular endothelial cells (HBMEC) and elaborating the mechanism of EV71 infection in the nervous system. Methods Knocked out the VIM by CRISPR technology , the differences in EV71 absorption , replication , release between wild VIM and VIM knocked-out (VIM-KO) HBMEC were detected by fluorescence quantitative PCR. Results 4 ℃ absorption experiment conformed that EV71 adsorption in VIM- KO is 40% less than in the normal HBMEC. After EV71 infect HBMEC for 48 h (48 h p. i.), the quantitative PCR result showed intracellular viral RNA in VIM-KO was only 1/12 of that in the normal HBMEC. Also the extracellular viral RNA was quantified, and the number of cells in VIM-KO had been reduced 1.4 times compared with the normal HBMEC. Conclusions Once VIM knocking out, EV71 attachment has been obviously reduced. Meanwhile, the level of viral RNA replication and release are decreased compared with the normal HBMEC. VIM may be an attachment receptor of EV71 in HBMEC , when the virus invades HBMEC with the binding of VIM. Moreover , VIM plays an important role in the replication and release of EV71.
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Objective To explore the changes of cell proliferation and differentiation of neural stem cells induced by fasudil treatment,and to primarily study the mechanism in C17.2 mice.Methods C17.2 cells were cultured in vitro; 5,25,50 and 100 μmol/L fasudil were given to the cells,respectively,for 24 h,and cells in the blank control group were given the same volume of culture medium.The changes of cell morphology were observed under a phase-contrast microscope; cell viability and cell necrosis rate were determined by MTT assay and lactate dehydrogenase (LDH) assay,respectively.Western blotting was applied to detect the expression levels of neural markers (nestin,glial fibrillary acidic protein [GFAP],double cortisol [DCX],microtubule-associated protein-2 [MAP-2]),and Notch Ⅰ and Hes 1 proteins in the notch signaling in cells from the 100 μmol/L fasudil treatment group and blank control group.Immunofluorescence staining was used to detect the nestin and GFAP expressions in the C 17.2 cells.Results As compared with that in the blank control group,the cell viability in the 50 and 100 μmol/L fasudil treatment groups was significantly decreased; that in the 100 μmol/L fasudil treatment group was significantly lower than that in 50 μmol/L fasudil treatment group (P＜0.05); LDH assay showed no significant difference of cell necrosis among the five groups (P＜0.05).Western blotting indicated that 100 μmol/L fasudil treatment group had significantly decreased nestin expression,significantly elevated DCX,MAP-2 and GFAP expressions,and statistically decreased expression levels of Notch 1 and Hes 1 as compared with blank control group (P＜0.05).Immunofluorescence staining indicated that the percentage of nestin positive cells was markedly decreased,the percentage of GFAP positive cells was significantly increased in the 100 μmol/L fasudil treatment group as compared with those in the blank control group (P＜0.05).Conclusion Fasudil treatment could inhibit the proliferation of C17.2 cells and promote them differentiate into neuronal and glial cells via decreasing the expression level of Notch signaling.
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AIM:TostudythechangeofradiosensitivityofU251cellsaftertreatedwithsodiumdichloroacetate ( DCA) and further to explore the possible mechanism .METHODS: The U251 cells were divided into 4 groups: control group, DCA group, X-ray irradiation without DCA pretreatment ( IR) group and X-ray irradiation with DCA pretreatment ( DIR) group.MTT assay was applied to determine the cell viability .The intracellular reactive oxygen species ( ROS) were detected by DHE fluorescence .The expression level of Bcl-2 was assessed by Western blot .The percentage of apoptosis of cells was determined by flow cytometry .RESULTS:No difference between control group and DCA group in cell viability (P>0.05) was observed.However, the cell viability of both IR group and DIR group was markedly reduced compared with control group ( P<0.05).Furthermore, the viability of DIR group was significantly decreased compared with IR group ( P<0.05 ) .The percentage of ROS positive cells was obviously increased in DIR group compared with IR group (P<0.05).The expression level of Bcl-2 was sharply decreased in DIR group (P<0.05) and the percentage of apoptosis of cells was significantly elevated ( P<0.05) in DIR group compared with IR group .CONCLUSION:The better antitu-mor effect was obtained by improving the radiosensitivity through pretreating the cells with DCA , and the possible mecha-nism was down-regulation of the Bcl-2 expression by developing the intracellular ROS .