The Experimental Study On The Expression Of ING4 And CTGF In Human Glioma Tumor And Their Correlation

Glioma is the most frequently occurring malignant tumor in central nervous system and and is associated with a poor prognosis, especially for high grade gliomas. With the development of molecular biology, the molecular mechanisms of glioma, the relevant signal transduction, invasion of the malignant process have made some research, which is closely related to genes with glioma, molecular function is gradually taken seriously. Present study suggests that the molecular etiology of glioma and other tumors is similar, which is closely linked to the activation of oncogenes and the absence of tumor suppressor gene. The study of incidence and the process of development of glioma found that many important molecules, genes and proteins, which play different roles and have a clear distinction and contact.Connective tissue growth factor (connective tissue growth factor, CTGF) is a member of the CCN family, because Cyr61, CTGF and nov is the first member of the family, so called CCN family.They have been detected in multiple tissues in a variety of animals including human beings, we speculated that the gene family originated in the same gene 4 million years ago. CCN family of translation products are secreted cysteine-rich peptides, as an important member of the CCN family, CTGF are closely related with cell proliferation, differentiation, embryonic growth and development, wound healing, wound healing, atherosclerosis, Liver, lung, kidney fibrosis and tumor development etc.Further understanding the role of CTGF has great theoretical significance to diagnosis and treatment of glioma from the molecular level.In recent years, brain tumor gene therapy to obtain rapid development, and gradually becoming more mature, and clinical studies have confirmed that brain tumor gene therapy is a novel and promising therapy. The principle of gene therapy is to use gene transfer technology gene into target cells, to obtain a specific function, and then execute or mediated killing and inhibition of tumor, or to protect normal cells from chemotherapy and radiation therapy of serious Injury. ING4 (Inhibitor of growth famility, member4, ING4) is a recent discovery of a new tumor suppressor gene, are widely involved in tumor occurrence and development of multiple processes, including the growth of tumor blood vessels, cell hypoxia adaptation, Inhibition of tumor cell invasion and metastasis as well as participate in cell cycle regulation and so on. ING4 is one member of growth inhibitory factor (inhibitor of growth, ING) family, which was discovered and cloned by Shiseki when they were to find similar sequences of ING1 and ING2 in 2003. Many present experimental studies have shown that ING4 gene may have blocked cell cycle progression, inhibition of angiogenesis and promote apoptosis.Growth inhibitory factor 4 (Inhibitor of growth famility, member4, ING4) as an important tumor growth inhibitory factor, by itself, the suppression of tumor-specific research provides a new means of gene therapy for glioma. Recognizing the role of ING4 on glioma cells and influence has important significance.to intervention glioma in a more effective way.We use Real-time quantitative PCR to detect growth inhibitory factor 4 (ING4) and connective tissue growth factor (CTGF) mRNA expression in 30 cases of human glioma, Combined with immunohistochemistry to observe the features of the two expression under the microscope, and to explore the role in glioma genesis and development and their relevance.ObjectiveWe use Real-time fluorescence quantitative PCR method to detected ING4 mRNA and CTGF mRNA in30 cases of human gliomas and 10cases of normal brain tissue. Combined with immunohistochemistry to observe the features of the two expression under the microscope, and investigate the relationship between the expression of CTGF mRNA、ING4 mRNA and the pathological grade in human gliomas,and analysis their correlation.MethordsThe 30 cases of glioma specimens taken for pathological type and grade According to WHO classification of tumors of central nervous system (2000) standard:Ⅰ5 cases,Ⅱ,12 cases,Ⅲ4 cases,Ⅳ9 cases. Which statesⅠ,Ⅱlevel for the low-level group, a total of 17 patients,Ⅲ,Ⅳ-class high-level group, a total of 13 cases. Normal control group,10 patients needed decompression surgery from the patients with severe head injury, take the vacuum removal of non-functional areas of brain tissue as control specimens. The specimens were taken were divided into two, one specimen in 10% formalin fixed reserve soaking liquid, paraffin embedded sections. Another was immediately placed in liquid nitrogen after collection, transfer to-80℃ultra-low temperature freezer spare for long-term cryopreservation. And all patients were not receiving chemotherapy and radiotherapy or other biological therapy. Will add about 100mg tissue grinder, add 1ml of TRLzol Rengent (invitrogen, USA) grinding 20-30min; until you see the large organization. The liquid into sterilized centrifuge tube, add chloroform 250μl, upside down, mix well and let stand for 5min,4℃centrifugation,13000 r/min, 10min.500μl supernatant in another sterilized tube (EP), add the same amount of isopropanol, mix into the refrigerator at 4℃removed after 15min,4℃centrifugation,13000 r/min, 10min; dumped out of the top of the liquid, remain in the bottom of the tube is the white precipitate RNA. Adding 75% ethanol 1ml,4℃centrifugation,7500 r/min,5min. Adding an appropriate amount of deionized water to dissolve. Reverse transcribed cDNA:RT kit was purchased from (invitrogen, USA). In 1.5ml Eppendorf centrifuge tube (Eppendorf tube) was added RNA 2μg, oligo (dt) 18 primer and DEPC-H2O1μl to 12μl, mixing, shaking 3-5s. Placed 70℃,5min. Heating the mixture at 65℃for 5min, and rapidly cooled on ice, add 5x buffer (pH7.2-7.5) 4μl,0.1M DTT 2μl, RNase inhibitor 1μ1.37℃for 2min, at room temperature by adding M-MLV reverse transcriptase (M-MLV Reverse Transcriptase) 1μl,37℃incubation 50min, 70℃heat 15min. Real-time fluorescence quantitative PCR detection:real-time fluorescence quantitative PCR Detection System (SLAN, Shanghai, HONGSHI) detection. Toβ-actin (β-Actin gene) expression as internal control. PCR reaction volume of 25μl, containing cDNA,10×Buffer (pH7.2-7.5),2.5μl; SYBR Green mix (TOYOBO, Japan),12.5μl, ddH2O,8μl; primer (5pmol/μl),2μl. reaction conditions were:95℃60s, and then 95℃15s, annealing 15Ss,72℃45s,40 cycles.β-Actin of RT-PCR reaction system and conditions with CTGF and ING4. To simplify the calculation method of the 2-ΔΔCt. For example, ING4 gene expression in the calculation of the amount of time to ING4 specimens with a threshold cycle (Cycle threshold) Ct threshold value that is within the reaction tube fluorescent signal reaches the set threshold number of cycles experienced byβ-Actin Ct value set it asΔCt. The specimens 0.5ΔCt is the expression of normal brain tissue and the mean ratio of ING4, ING4 gene expression level (relative to normal brain tissue, expressed as a percentage.) The same method to calculate the level of CTGF gene expression. Statistical analysis of data with SPSS 13.0 software, measurement data through the normal test and homogeneity of variance test to x±s said. ANOVA further action is different from the comparison between the use of pairwise Bonferroni method. Correlation between the two expression analysis using Spearman rank correlation. The P<0.05 is statistically significant difference.Paraffin-embedded specimens according to the following treatments: conventional biopsy dewaxing, dehydration; 3% H2O2 were incubated for 10 minutes with deionized water, distilled water; the slices immersed in 0.01M citrate (PH6.0), microwave heated to boiling off electricity, every 10 minutes, repeated twice for hot fixes antigen; dropping 5% BSA blocking solution at room temperature for 20 minutes; dropping biotinylated rabbit anti-human CTGF polyclonal antibody, ING4 antibody,50uml/Zhang, placed in incubated overnight at 4℃after the PBS wash 2min×3 times; dropping biotinylated goat anti-rabbit IgG, at room temperature for 20 minutes, PBS (PH7.2-7.6) wash; dropping reagents SABC, at room temperature for 20 minutes, PBS Wash for 5 minutes×4 times; DAB color:Take lml of distilled water, add kit A, B, C and 1 drop of reagent, mix, add to the slices at room temperature, color 18 minutes; stained with hematoxylin mild; running water to fully rinse, stained, dehydrated, transparent, were mounted. Then observe the expression characteristics of CTGF and ING4 under the microscope.Results1. ING4 mRNA in the normal expression level of (103.1±20.2)%, low-level group (Ⅰ-Ⅱgrade) was (49.9±10.4)%, high-level groups (Ⅲ-Ⅳgrade) was (16.7±6.89)%; in the normal expression of CTGF mRNA Was (103.0±31.8)%, low-level group (Ⅰ-Ⅱgrade) was(281.1±68.6)%, high-level group (Ⅲ-Ⅳgrade) was (672.4±99.7)%.2. The comparison of ING4 in the normal group, low-level group, high-level expression of group, ING4 expression in the 3 groups comparison, the differences were statistically significant ((P=0.000<0.01); 3 group, the mean failure ING4 expression is equal ((P=0.000<0.01).3 groups of ING4 expression of the pairwise comparison, statistics showed:normal group, the expression of ING4 than low-level group ((P=0.000<0.01), low-level expression of ING4 group than the high-level group ((P=0.000<0.01), ING4 expression in the normal group was significantly higher than the high-level group ((P=0.000<0.01), in which the expression of the normal group, the highest level of ING4, followed by low grade, high-level group, the lowest expression of ING4. That, with increased ING4 expression of tumor grade has gradually decreased.3. The comparison of CTGF in the normal group, CTGF expression in the 3 groups comparison, the differences were statistically significant ((P=0.000<0.01); 3 group, the mean failure CTGF expression is equal ((P=0.000<0.01). CTGF further expression of 3 groups were compared pairwise, statistical results showed that:the high-level group, the expression of CTGF was higher than the low-level group ((P=0.000<0.01), low-level expression of CTGF group than normal group ((P=0.000<0.01), high-level group, the expression of CTGF was significantly higher than the normal group ((P=0.000<0.01), in which high-level group, the highest level of CTGF expression, followed by low-level group, the normal group, the lowest expression of CTGF. With the increase in the level of CTGF expression in the tumor is gradually increasing trend.4. According to the expression levels of ING4 and CTGF in 30 glioma samples, the Spearman rank correlation analysis showed that both in the glioma was negatively correlated (γ=-0.723, P=0.000< 0.01).5. Immunohistochemistry showed that ING4 gene was expressed in the cytoplasm and nucleus, and the expression is rich in normal brain tissue but reduced in glioma tissues; and expression of CTGF in normal brain tissue is decreased but abundant in high grade gliomas.Conclusions①CTGF mRNA in human gliomas was significantly higher than in normal brain tissue, and the tumor grade increased, expression levels increase accordingly, and ING4 mRNA in human gliomas, the expression level was significantly lower than normal brain Organizations, and with higher grade and lower levels of the tumor. Tip of glioma CTGF and ING4 occurrence and development.②The expression level of CTGF mRNA and ING4 mRNA in human glioma, was negatively correlated. The results show that both can be synergistic, interaction in the development process of human gliomas, and both detection can be used as a reference to determine the extent of malignant glioma. CTGF and ING4 is closely related to occurrence and development of glioma.