Objective:To explore the effects and potential mechanisms of chemokine-like factor-like MARVEL transmembrane domain-containing Protein 3 (CMTM3) on the proliferation and migration of glioblastoma (GBM) cells.Methods:Using CMTM3 expression data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases, we analyzed the differential expression of CMTM3 in GBM tissues and its impact on the prognosis of GBM patients. Immunohistochemical staining and protein content determination of CMTM3 was performed on GBM and adjacent non-cancerous tissue samples from 11 GBM patients who underwent surgical treatment at the Affiliated Hospital of Guizhou Medical University between November 3, 2022 and March 15, 2023. Additionally, the expression of CMTM3 was validated in GBM cell lines U87, U251, LN229, and the human astrocyte (NHA) cell line using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analyses. Stable cell lines with silenced and overexpressed CMTM3 (sh-CMTM3 group and OE-CMTM3 group) were constructed using U251 cells. The effect of CMTM3 expression on cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was employed to examine the impact of CMTM3 expression on the cell cycle. Transwell assays were conducted to evaluate the influence of CMTM3 expression on cell migration. Bioinformatics analysis, Western blotting, NF-κB activation-nuclear translocation assays, and the NF-κB pathway inhibitor pyrrolidine dithiocarbamate ammonium (PDTC) were used to validate the effect of CMTM3 on the NF-κB pathway. Finally, a subcutaneous tumorigenesis assay in nude mice was performed to observe the impact of CMTM3 expression on the in vivo growth of U251 cells. Results:Bioinformatics analysis revealed that CMTM3 is highly expressed in GBM tissues. Patients with a high CMTM3 expression had lower overall survival (OS) and disease-free survival (DFS) rates compared with those with a low CMTM3 expression (with P values of 0.010 and 0.032, respectively). Among the 11 GBM pathological specimens, 10 samples exhibited higher CMTM3 protein expression levels in the cancerous tissue compared with the adjacent non-cancerous tissue. The average CMTM3 protein expression in these samples was 0.44±0.09, significantly higher than that in the adjacent non-cancerous tissues (0.12±0.02, P＜0.001). In one sample, the difference in CMTM3 protein expression between the cancerous and adjacent non-cancerous tissues was not statistically significant ( P=0.750).The RT-qPCR results showed that the mRNA expression level of CMTM3 in NHA cells was 1.0±0.1, whereas in GBM cells U87, LN229, and U251, the levels were 2.1±0.3, 3.4±0.5, and 3.7±0.8, respectively, all significantly higher than that in NHA cells (all P＜0.01). Western blot results demonstrated that the protein expression levels of CMTM3 in GBM cells U87, LN229, and U251 were 1.5±0.2, 1.8±0.2, and 1.9±0.1, respectively, also higher than that in NHA cells (0.7±0.2, all P＜0.01), with the highest level observed in U251 cells. The CCK-8 assay, Flow cytometry, and Transwell migration experiments indicated that cell viability was inhibited in the sh-CMTM3 group, with an increase in the proportion of cells in the G 0/G 1 phase ( P＜0.01) and a decrease in the S phase ( P＜0.01), and the number of migrated cells was 233.6±35.5, lower than that in the sh-NC group ( P＜0.001). Conversely, the OE-CMTM3 group showed enhanced cell viability, a reduction in the proportion of cells in the G 0/G 1 phase ( P＜0.01), and an increase in the S phase ( P＜0.01), and the number of migrated cells was 1212.0±20.8, higher than that in the OE-NC group ( P＜0.001). However, in the OE-CMTM3+PDTC group, cell viability, cell cycle distribution (G 1, S, and G 2 phases), and cell migration numbers showed no significant changes (all P＞0.05). Western blot analysis and NF-κB activation-nuclear translocation assay results indicated that in the sh-CMTM3 group, the p-p65/p65 ratio was 0.51±0.04 and the p-IκBα/IκBα ratio was 0.39±0.03, both lower than those in the sh-NC group (both P＜0.01). The cytoplasmic staining rate was (49.29±1.98)%, higher than that in the sh-NC group ( P＜0.01). In the OE-CMTM3 group, the p-p65/p65 ratio was 2.27±0.10 and the p-IκBα/IκBα ratio was 2.14±0.15, both higher than those in the OE-NC group (both P＜0.01). The cytoplasmic staining rate was (18.96±1.44)%, lower than that in the OE-NC group ( P＜0.01). In the OE-CMTM3+PDTC group, there were no significant differences in the p-p65/p65 ratio, p-IκBα/IκBα ratio, and cytoplasmic staining rate compared with the OE-NC group (all P＞0.05). The subcutaneous tumorigenesis assay in nude mice showed that the tumor volume in the sh-CMTM3 group was (408.9±96.2) mm3, smaller than that in the sh-NC group ( P=0.003). The tumor volume in the OE-CMTM3 group was (1 514.5±251.5) mm3, larger than that in the OE-NC group ( P=0.005). Conclusions:In GBM, CMTM3 is highly expressed and negatively correlated with both OS and DFS of patients. CMTM3 regulates the proliferation and migration abilities of U251 cells through the NF-κB signaling pathway.

Objective:To explore the effects and potential mechanisms of chemokine-like factor-like MARVEL transmembrane domain-containing Protein 3 (CMTM3) on the proliferation and migration of glioblastoma (GBM) cells.Methods:Using CMTM3 expression data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases, we analyzed the differential expression of CMTM3 in GBM tissues and its impact on the prognosis of GBM patients. Immunohistochemical staining and protein content determination of CMTM3 was performed on GBM and adjacent non-cancerous tissue samples from 11 GBM patients who underwent surgical treatment at the Affiliated Hospital of Guizhou Medical University between November 3, 2022 and March 15, 2023. Additionally, the expression of CMTM3 was validated in GBM cell lines U87, U251, LN229, and the human astrocyte (NHA) cell line using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analyses. Stable cell lines with silenced and overexpressed CMTM3 (sh-CMTM3 group and OE-CMTM3 group) were constructed using U251 cells. The effect of CMTM3 expression on cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was employed to examine the impact of CMTM3 expression on the cell cycle. Transwell assays were conducted to evaluate the influence of CMTM3 expression on cell migration. Bioinformatics analysis, Western blotting, NF-κB activation-nuclear translocation assays, and the NF-κB pathway inhibitor pyrrolidine dithiocarbamate ammonium (PDTC) were used to validate the effect of CMTM3 on the NF-κB pathway. Finally, a subcutaneous tumorigenesis assay in nude mice was performed to observe the impact of CMTM3 expression on the in vivo growth of U251 cells. Results:Bioinformatics analysis revealed that CMTM3 is highly expressed in GBM tissues. Patients with a high CMTM3 expression had lower overall survival (OS) and disease-free survival (DFS) rates compared with those with a low CMTM3 expression (with P values of 0.010 and 0.032, respectively). Among the 11 GBM pathological specimens, 10 samples exhibited higher CMTM3 protein expression levels in the cancerous tissue compared with the adjacent non-cancerous tissue. The average CMTM3 protein expression in these samples was 0.44±0.09, significantly higher than that in the adjacent non-cancerous tissues (0.12±0.02, P＜0.001). In one sample, the difference in CMTM3 protein expression between the cancerous and adjacent non-cancerous tissues was not statistically significant ( P=0.750).The RT-qPCR results showed that the mRNA expression level of CMTM3 in NHA cells was 1.0±0.1, whereas in GBM cells U87, LN229, and U251, the levels were 2.1±0.3, 3.4±0.5, and 3.7±0.8, respectively, all significantly higher than that in NHA cells (all P＜0.01). Western blot results demonstrated that the protein expression levels of CMTM3 in GBM cells U87, LN229, and U251 were 1.5±0.2, 1.8±0.2, and 1.9±0.1, respectively, also higher than that in NHA cells (0.7±0.2, all P＜0.01), with the highest level observed in U251 cells. The CCK-8 assay, Flow cytometry, and Transwell migration experiments indicated that cell viability was inhibited in the sh-CMTM3 group, with an increase in the proportion of cells in the G 0/G 1 phase ( P＜0.01) and a decrease in the S phase ( P＜0.01), and the number of migrated cells was 233.6±35.5, lower than that in the sh-NC group ( P＜0.001). Conversely, the OE-CMTM3 group showed enhanced cell viability, a reduction in the proportion of cells in the G 0/G 1 phase ( P＜0.01), and an increase in the S phase ( P＜0.01), and the number of migrated cells was 1212.0±20.8, higher than that in the OE-NC group ( P＜0.001). However, in the OE-CMTM3+PDTC group, cell viability, cell cycle distribution (G 1, S, and G 2 phases), and cell migration numbers showed no significant changes (all P＞0.05). Western blot analysis and NF-κB activation-nuclear translocation assay results indicated that in the sh-CMTM3 group, the p-p65/p65 ratio was 0.51±0.04 and the p-IκBα/IκBα ratio was 0.39±0.03, both lower than those in the sh-NC group (both P＜0.01). The cytoplasmic staining rate was (49.29±1.98)%, higher than that in the sh-NC group ( P＜0.01). In the OE-CMTM3 group, the p-p65/p65 ratio was 2.27±0.10 and the p-IκBα/IκBα ratio was 2.14±0.15, both higher than those in the OE-NC group (both P＜0.01). The cytoplasmic staining rate was (18.96±1.44)%, lower than that in the OE-NC group ( P＜0.01). In the OE-CMTM3+PDTC group, there were no significant differences in the p-p65/p65 ratio, p-IκBα/IκBα ratio, and cytoplasmic staining rate compared with the OE-NC group (all P＞0.05). The subcutaneous tumorigenesis assay in nude mice showed that the tumor volume in the sh-CMTM3 group was (408.9±96.2) mm3, smaller than that in the sh-NC group ( P=0.003). The tumor volume in the OE-CMTM3 group was (1 514.5±251.5) mm3, larger than that in the OE-NC group ( P=0.005). Conclusions:In GBM, CMTM3 is highly expressed and negatively correlated with both OS and DFS of patients. CMTM3 regulates the proliferation and migration abilities of U251 cells through the NF-κB signaling pathway.

Asthenozoospermia is a condition of deficient motility of spermatozoa, and about 27.8% of male infertility results from asthenozoospermia. Its etiology is complex and its pathogenesis is still unclear, and exploring its pathogenesis can help to find effective prevention and treatment strategies. In recent years, with the development of proteomic technologies (two-dimensional electrophoresis, yeast two-hybrid technology, mass spectrum, etc.), many researchers have focused their researches on the study of asthenozoospermia based on proteomics, revealing the differential proteins of asthenozoospermia and the biological pathways that underlie them, providing numerous theoretical support for the mechanistic study of asthenozoospermia. In this review, we focus on the relevant techniques of proteomics and summarize the many achievements made in understanding the differential protein expression, etiology, and mechanism of asthenozoospermia, which will hopefully provide new insights into the diagnosis and treatment of asthenozoospermia. Protein-protein interactions and pathways and metabolic abnormalities provide a promising direction to study sperm motility in asthenozoospermia.

Asthenozoospermia is a condition of deficient motility of spermatozoa, and about 27.8% of male infertility results from asthenozoospermia. Its etiology is complex and its pathogenesis is still unclear, and exploring its pathogenesis can help to find effective prevention and treatment strategies. In recent years, with the development of proteomic technologies (two-dimensional electrophoresis, yeast two-hybrid technology, mass spectrum, etc.), many researchers have focused their researches on the study of asthenozoospermia based on proteomics, revealing the differential proteins of asthenozoospermia and the biological pathways that underlie them, providing numerous theoretical support for the mechanistic study of asthenozoospermia. In this review, we focus on the relevant techniques of proteomics and summarize the many achievements made in understanding the differential protein expression, etiology, and mechanism of asthenozoospermia, which will hopefully provide new insights into the diagnosis and treatment of asthenozoospermia. Protein-protein interactions and pathways and metabolic abnormalities provide a promising direction to study sperm motility in asthenozoospermia.

Objective To detect the mutations of Von Hippel-Lindau (VHL) gene via analyzing the prevalence of family members of VHL syndrome,clinical diagnosis and treatment,and gene analysis of patients with hemangioblastoma.methods All members of the VHL syndrome family members improved all relevant tests and plotted the family map.5 ml peripheral blood was extracted for gene sequencing,and the sequencing Result s were compared with the reported mutations of VHL gene in NCBI database.Result s(1)Analysis of clinical data of four members of the family:Ⅰ-2,Ⅱ-1,Ⅱ-5 suffering from central nervous system hemangioblastoma, Ⅱ-3 with pancreatic,retinopathy and pheochromocytoma,and Ⅱ-5 also combined with kidney,pancreatic lesions.The second generation of patients in the family have been treated surgically.(2)Gene sequencing Result s showed that all subjects in the test had the same mutation:exon2 109 sequence ATATCACACTGCCA was deleted and termination codon UGA appeared in exon 502.Conclusion Through the mutations of the VHL syndrome family,it is found that the family mutation type is a new mutation.For patients with central nervous system hemangioblastoma-based should be suspected of the disease and improve the family history survey.Once the diagnosis of familial VHL syndrome patients are confirmed,it is necessary to inform the other members of the family for clinical screening,and carry out genetic testing to reduce the harm of the disease to the greatest extent.

Objective To establish nude mouse model with human brain glioma SHG-44 and understand its growing characteristics in vivo.Methods The 4-week-old male mice were randomly divided into high density cell suspension inoculation group(n=10),low density cell suspension group(n=10),the tumor tissue mass vaccination group(n=10)and the blank control group with normal saline injection(n=10).The SHG-44 human brain glioma cell suspension was injected into the subcutaneous of the nude mice' s armpit.The tumor tissue was cut into 1 mm3 after tumor tissue growth and formation,and re-inoculated into the subcutaneous of the new nude mice' s armpits.Apart from daily observation,the long and short diameters of tumor were recorded every 5 days after graft.All the mice were sacrificed at 60 days and the tumor tissues were harvested for pathological examination.Results With a longer incubation period and slower growth rate,the tumor formation rate in high density cell suspension inoculation group and low density cell suspension group was lower compared with that in the tumor tissue mass vaccination group.Around day 20,grafted tumor appeared remarkably big((41.51 ±6.42)mm3) with good morphology.On day 50,the tumor derived from group the tumor tissue mass vaccination group((565.69± 123.36)mm3) showed a bigger size in comparison with that from high density cell suspension inoculation group((203.85±104.63) mm3) and low density cell suspension group ((153.02± 31.76) mm3,all P＜0.05).The tumors in three groups were well defined with a rich vascularity and no apparent invasion was observed.The positive expression of GFAP and S-100 in a large body of tumor cells was observed under optical microscope.Conclusion With a shorter incubation period and faster growth,the mouse tumor models established with tissue pieces from the tumor-bearing mice are much better compared to those with cell suspension.

Objective The derivative of Gefitinib was used to treat glioma cells in vitro to explore a more effective new drug for the clinical treatment of astrocytoma. Methods (1) Fifteen kinds of gefitinib derivatives, gefitinib and temozolomide were used to treat glioma cells, and the effect of 0, 10, 15, 20, 25 and 30 μmol/L of each kind of drug on cell proliferation was detected by by MTT assay , respectively. (2) To calculate the concentration of IC50 , then select lower IC50 of derivativs combinate gefitinib and temozolomide with 10, 20 and 30 μmol/L to treat cells, then the apoptosis of cells were detected by flow cytometry. Expression of p-EGFR was detected by western–blot assay. Results (1) NO.LPY-5,9,11, but not other derivatives of Gefitinib could effectively inhibit the growth of cells. (2) IC50 of NO.LPY-9 was less than that of the 5th drug, and both of them were lower than those of gefitinib and temozolomide; NO. LPY-11 was excluded. (3) The cell apoptosis of No. LPY-9 was higher than that of gefitinib and temozolomide , respectively. However, No.LPY-9-induced cell apoptosis was significantly higher than that of No. LPY-5-induced cell. (4) Levels of p-EGFR expression in No.LPY-9 and gefitini-induced cells were significantly lower than that in the negative control group. Conclusion No.LPY-9 has asignificant inhibitory effect on glioma cells in vitro , resulting from the inhibition of the ERFR-mediated signaling pathways and induction of cell apoptosis.