Expression of MAD2L1 in Lung Adenocarcinoma and Its Effect on Immune Microenvironment / 肿瘤防治研究

Objective To investigate the expression of MAD2L1 in lung adenocarcinoma and its effect on the prognosis and immune microenvironment of patients. Methods The difference of MAD2L1 expression in lung adenocarcinoma tissue and normal lung tissue was analyzed by TCGA and GEO database. Survival analysis was carried out to evaluate the prognostic significance of MAD2L1 gene expression in lung adenocarcinoma patients. StarBase database was used to construct miRNA-MAD2L1 regulatory network of lung adenocarcinoma. The relation between the expression of MAD2L1 and immune cell infiltration in lung adenocarcinoma was analyzed by TIMER database. Results The expression of MAD2L1 was up-regulated in lung adenocarcinoma, and the high expression of MAD2L1 was significantly correlated with pathological stage and lymph node metastasis of lung adenocarcinoma. The patients with high expression of MAD2L1 had a poor prognosis. miR-101-3p/MAD2L1 axis was identified as the most potential upstream regulation pathway of MAD2L1 in lung adenocarcinoma. The expression level of MAD2L1 was significantly correlated with tumor immune cell infiltration and immune checkpoint expression. Conclusions MAD2L1 is highly expressed in lung adenocarcinoma, which is related to poor prognosis and tumor immune infiltration. MAD2L1 can be used as a potential target for the treatment of lung adenocarcinoma.

Effects of SP600125 on Proliferation and Invasion of Human Cervical Cancer HeLa Cells / 肿瘤防治研究

Objective To investigate the effect of SP600125 on the proliferation, cell cycle, apoptosis and invasion of human cervical cancer HeLa cells. Methods CCK-8 method was used to detect the proliferation of HeLa cells treated with different concentrations of SP600125 at different time points. The 20 μmol/L of SP600125 was determined for subsequent experiments. Cell proliferation ability was detected using plate clone formation assay; nuclear morphology was observed by DAPI staining; cell cycle and apoptosis were measured by flow cytometry; cell migration and invasion were detected by cell scratch and Transwell methods; the mRNA and protein levels of p53, Mad2L1 and CDC20 were measured by qRT-PCR and Western blot after SP600125 treatment at different time points. Results Compared with control group (0.1%DMSO), cells proliferative activity were reduced by 10, 20, 30, 40 and 50 μmol/L SP600125 treatment for 24h. Compared with control group, the rate of apoptosis was significantly increased in SP600125 treatment groups, and the cell proportion in G2/M phase increased (P < 0.001), while the cell proportion in G0/G1 phases cells was reduced after SP600125 treatment for 24h and 48h (P < 0.001), and the clonal number, migration and invasion ability of HeLa cells also decreased significantly (P < 0.001). qRT-PCR and Western blot results showed a significant decrease in Mad2L1 mRNA and protein expression (P < 0.05) and a significant increase in p53 and CDC20 mRNA and protein expression (P < 0.01). Conclusion SP600125 can induce cell cycle arrest of cervical cancer HeLa cells in G2/M phase by upregulating p53 and CDC20 and downregulating Mad2L1 expression, and promote cell apoptosis to inhibit cell proliferation, migration and invasion.

The clinical research about early diagnosis of small cell lung cancer through mad2 de-tection / 中国肿瘤临床

Objective:To explore combined detection of mad2 with anti-nuclear mitotic spindle apparatus antibody(MSA)and anti-centromere antibody(ACA)and their clinical value for the diagnosis of small cell lung cancer(SCLC).Methods:One hundred and twen-ty SCLC patients,110 non-small cell lung cancer(NSCLC)patients,and 115 pulmonary nodule(PN)patients were enrolled in this study. The expression of mad2 was analyzed by qt-PCR.MSA and ACA were detected by indirect immunofluorescence(IIF)staining.Results:mad2 was overexpressed in SCLC and NSCLC samples(P<0.05).There were significant differences between the results obtained for SCLC and NSCLC samples by qt-PCR(P<0.05).AUC in ROC curve for mad2 expression was 0.799 with an intermediate diagnostic value. In the correlative analysis,the odds ratio of MSA and ACA was 6.94 and 5.60,respectively.In the correlation analysis,Kappa value of mad2 with MSA was 0.49,and Kappa value of mad2 with ACA was 0.42.In the parallel analysis,the sensitivity and specificity was 83.31% and 79.34%,respectively,while the Youden Index was 0.62.Moreover,in the serial analysis,the sensitivity and specificity was 65.32% and 93.35%,respectively,and the Youden Index was 0.59.Conclusions:In comparison with the NSCLC and PN samples,mad2 was overexpressed in SCLC samples.Therefore,mad2 ought to play a critical role in the pathology of SCLC.The combined expression of mad2 with MSA and ACA may contribute to enhancing the sensitivity and specificity of detection;this expression may allow early diag-nosis and clinical diagnosis of SLCC and may be a promising treatment for SCLC.

Mouse MAD2L1 gene editing by CRISPR/Cas9 and analysis of its off-target effect / 中国实验动物学报

Objective To establish a method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9, and analyze its off-target effect. Methods The gene editing site for MAD2L1 gene was designed by CHOP?CHOP, and the Cas9?MAD2L1 vector was constructed based on the designed editing site. Cas9?MAD2L1 was then transfected into NIH/3T3 cells and screened with puromycin, followed by observing GFP expression using fluorescence microscopy. The genomic DNA from transfected cells was extracted and a partial fragment of MAD2L1 gene was amplified by PCR. T7E1 analy?sis and Sangger sequencing were used for gene editing and off?target analysis. Results After Cas9?MAD2L1 transfection and puromycin screening, a large number of GFP?expressing cells were observed under the fluorescence microscope. Combined the PCR result with TE71 analysis, the amplified 228 bp PCR products can be digested into 166 bp and 62 bp fragments. The se?quencing result showed that the second exon of MAD2L1 gene was successfully edited, and the off?target effect was undetected in our system. Conclusions The method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9 is successfully established, and off?target effect of MAD2L1 gene is not detected.

MAD2 Expression in Ovarian Carcinoma: Different Expression Patterns and Levels among Various Types of Ovarian Carcinoma and Its Prognostic Significance in High-Grade Serous Carcinoma

BACKGROUND: Mitotic arrest deficiency protein 2 (MAD2) is a key component of spindle assembly checkpoint function, which mediates cell apoptosis through microtubule kinetics. Aberrant expression of MAD2 is believed to be associated with the development of chromosome instability. MAD2 also has a signihicant role in cellular drug resistance to taxane chemotherapeutic agents. METHODS: Expression of MAD2 and p53 was investigated using immunohistochemistry in 85 cases of ovarian carcinomas. Clinicopathological data including progression-free survival were analyzed. RESULTS: A significant (p=.035) association was observed between the grade of serous carcinoma and the expression level of MAD2. While low-grade serous carcinoma showed a low-level expression of MAD2, high-grade serous carcinoma showed a high-level expression of MAD2. We also determined that low-level expression of MAD2 was associated with reduced progression-free survival (PFS) (p=.016) in high-grade serous carcinoma. CONCLUSIONS: MAD2 expression in ovarian carcinoma is related to the grade of serous carcinoma and PFS of high-grade serous carcinoma. Expression level of MAD2 detected by immunohistochemistry may serve as an indicator in predicting the response of microtubule-interfering chemotherapeutic agents.

Effect of Mad2 on Paclitaxel-induced Cell Death in Ovarian Cancer Cells / 华中科技大学学报（医学）（英德文版）

In this article, the status of spindle assembly checkpoint and the alteration of its major component, Mad2 protein level were examined in A2780 and SKOV3 ovarian cancer cell lines. Recombinant eukaryotic expression plasmid pEGFP-Mad2 was transfected into paclitaxel-resistant SKOV3 cells and Mad2 protein was knocked down by Mad2-specific siRNA in paclitaxel-sensitive A2780 cells. Then the expression level of Mad2 gene was detected by Western blotting. Flow cytometry revealed that SKOV3 cells were not fully arrested in G2/M phase in contrast to A2780 cells in the presence of paclitaxel. However, paclitaxel sensitivity assay showed that sensitivity to paclitaxel was reversed after the transfection in both cell lines in terms of number of cells arrested at G2/M phase and the expression of Bcl-2 was significantly changed. These results suggest that weakened spindle checkpoint with reduced expression of Mad2 is associated with resistance to paclitaxel in ovarian cells and Bcl-2 may be involved in this process.

Mutation analysis of p31(comet) gene, a negative regulator of Mad2, in human hepatocellular carcinoma

Failure of mitotic checkpoint machinery leads to the chromosomal missegregation and nuclear endoreduplication, thereby driving the emergence of aneuploidy and tetraploidy population. Although abnormal nuclear ploidy and the resulting impairment of mitotic checkpoint function are typical physiological event leading to human hepatocellular carcinoma, any mutational change of mitotic checkpoint regulators has not yet been discovered. Therefore, we investigated the mutation of p31(comet), a recently identified mitotic checkpoint regulator, in human hepatocellular carcinoma. Of 51 human hepatocellular carcinoma tissue and 6 cell lines tested, five samples exhibited nucleotide sequence variations dispersed on four sites within the entire coding sequence. Among these sites with sequence substitutions, three were found to be missense mutation accompanied with amino acid change but one was a silent mutation. Of these sequence substitutions, two were present in both tumor and non-tumor liver tissues, suggesting the possibility of polymorphism. The present findings indicate that p31(comet) does not have an impact on the formation of aneuploidy and tetraploidy found in human hepatocellular carcinoma.

Expression of MAD2 and tumor suppressor gene in the colorectal cancer and clinical significance / 中国癌症杂志

Background and purpose:MAD2 is one of the mitotic checkpoints and it is closely associated with tumors.Our aim was to study the expression of gene MAD2 and tumor suppressor gene in colorectal cancer and to demonstrate the relationship between the expression of gene MAD2 and tumor suppressor gene in colorectal cancer,adenoma and normal tissue and to evaluate their clinical significance.Methods:Immunohistochemistry method and real-time fluorescence quantitative PCR were used to analyze the expression of gene MAD2 in colorectal cancer,adenoma and normal tissue.PCR amplifi cation and DNA sequencing were performed to detect the mutations of gene MAD2.p53,p27,p21 and p16 were determined in colorectal cancer and normal tissue by immunohistochemistry methods.Results:The expression of MAD2 in colorectal cancer was obviously higher than in adenoma and normal tissue(66.7% vs 39.6% vs 22.9%) ,there were signifi cant differences among colorectal cancers,adenoma and normal tissue(P

Correlation between Gene Expression of MDR1, Cyclin B, MAD2 and BAX in Childhood Acute Lymphocytic Leukemia / 대한소아혈액종양학회지

PURPOSE: Although outcome of the children with ALL has been improved remarkably with the multidrug chemotherapy and supportive therapy, relapse is still important cause of treatment failure. One of the mechanism related to the relapse has been reported to be multidurg resistance (MDR). To investigate the relation between the expression of MDR related MDR1 gene and cell cycle and apoptosis related genes in children with ALL, this study was conducted. METHODS: The samples were collected from 9 children with ALL (5: at presentation, 4: at relapse) diagnosed at the pediatric department of Yeungnam University Hospital. From the mononuclear cells isolated from the peripheral blood or bone marrow, mRNA was extracted and analysed by RT-PCR. Using actin as a control, relative levels of mRNA of MDR1 gene, cell cycle control protein cyclin B and MAD2 and apoptosis related BAX gene were analysed. RESULTS: The expression of MDR1 gene at the presentation and the relapse were variable and showed high correlation (Pearson correlation: 0.826) with the expression of BAX gene but low correlation with the expression of cyclin B and MAD2 gene. CONCLUSION: These results suggest that mechanism involved in relapse of ALL include mechanisms other than MDR1 gene. High correlation between the expression of MDR1 gene and BAX gene suggests that high level of BAX expression increases probability of relapse but small sample size of this study precludes definite conclusion and further study is needed.

MicroRNA depresse the expression of MAD2 gene in trophoblastic cells / 重庆医科大学学报

Objective:To discuss the mechanism of down-regulated expression of MAD2 gene in trophoblastic cells by microRNA.Methods:Endogenous MAD2 mRNA level was compared by using quantitative real-time PCR before and after transfected with microRNA plasmid;and the Mad2 protein level was compared by western blot.Results:Ratio of MAD2 mRNA/GAPDH mRNA of control group was 0.1780?0.0688,and after transfected with microRNA recombinant plasmid was 0.1778?0.0689 and 0.1778?0.0670respectively.Statistical study of samples showed they have no instinct different;otherwise the protein level in one of the experimental groups was significantly decreased after transfected with microRNA plamid.Conclusion:This study established microRNA can down-regulate gene expression in mammalian cells at translation level,it is a good foundation to study on the control of gene expression.

Construction of the plasmid expressing gfp and MAD2 gene controlling separation of cell and its expression in HepG2 cell / 重庆医科大学学报

Objective: To establish an in vitro screening system for testing effects of MAD2 gene of embryonic cell genome.Methods: In order to construct the plasmid expressing MAD2-GFP fusion plasmid,the target gene fragment was obtained by RT-PCR amplification and inserted into pEGFP-N1 reporter vector.The structure of constructed plasmid was confirmed by electrophoresis analysis and DNA sequencing.The function of construct was confirmed by lipofectamine-mediated transient expression in HepG2 cells.Results: DNA sequencing showed that the inserted fragment of the constructed plasmid was the same as the template MAD2 genome.The HepG2 cells transfected with the constructed plasmid could express reporter gene of gfp.Conclusions: the plasmid expressing gfp gene controlled by MAD2 gene is successfully constructed and an in vitro testing system for evaluating founction of MAD2 gene in separation of cell is established.