The Correlation Between The Expressions Of Hypoxia Inducible Factor-1α And Centromere Protein W In Glioma Tissues And Cells

glioma is the most common refractory primary malignant tumors in the central nervous system. it Originated in the glial cells, around half of intracranial tumors. Past investigations have shown that the occurrence and development of glioma and attacks is the result of a multi- stage, multiple genes involved in[2], but the specific mechanism is still unclear.centromere protein W,also as CUG2,was was an oncogene was found in 2007 by Soojin Lee. The study found that C ENP-W was comonly up-regulated in various human cancer tissues,especially in ovay, liver, lung, pancreas, and colon. It has the very complicated mechanism, CENP-W overexpression Promote cell proliferation, also induces cell apoptosis. Experiments show that C ENP- W overexpression can induce tumorigenesis, has an important relationship with the occurrence and development,migration process of various malignant tumors.hypoxia inducible factor-1α,HIF-1α, is a nuclear transcription factor induced under hypoxia environment,which can regulate the expressions of a variety of target genes.but it is easy to break down in the general environment. However, it has no reports about the expression correlation of C ENP- W with HIF-1α in the glioma.ObjectiveThis study took Ⅰ ~ Ⅳ level glioma tissues, adjacent normal brain tissue and U251 cells as experimental object, exploring the expression correlation of HIF-1α and C ENP-W in glioma tissues and cells,and there relationship with the glioma biology behavior, in order to provide some basis for the control of glioma.MethodsThe first part: Immunohistochemical staining was applied to detect the expression level of the HIF-1α and CENP-W in 41 glioma tissues and 3 adjacent tissues. We Statistical analy protein expression level and there relationship with clinical pathological characteristics of glioma,also the relationship between the HIF-1α and CENP-W.The second part: After using cobalt dichloride to inhibit HIF-1α proteindegradation in glioma U251 cells, the expressions of CENP-W m RN A and protein were detected by real-time fluorescene quantitative PCR and Western blotting, then Statistical analying the influences of the up-regulated of HIF-1α level on the expressions of CENP-W m RNA and protein.The third part: After transfection with the specific small interference RNA(si RNA) targeting HIF-1α gene to repress HIF-1α expression in U251 cells, the changes of CENP-W m RNA and protein levels were detected by real-time fluorescene quantitative PCR and Western bloting. After transfection with the specific small interference RNA(si RNA) targeting CENP-W gene to repress CENP-W expression in U251 cells, the changes of HIF-1α m RNA and protein levels were detected by real- time fluorescene quantitative PCR and Western bloting.ResultsImmunohistochemical detection results suggest C ENP- W is mainly expressed in the nucleus, have obvious heterogeneity. Its expression positive rate in adja cent tissues, a low grade(Ⅰ ~ Ⅱ) and high grade(Ⅲ ~ Ⅳ) glioma specimens in was 67%(2/3), 82%(14/17) and 92%(22/24).statistical analysis showed that there was no difference between each CENP- W expression rate(chi-square = 1.794, P = 1.794). But the immunohistochemical score in high grade groups(3.58 ± 1.06) is generally higher than low level group(2.88 ±1.50), scores of the adjacent tissues group(2.00 2.00 mm) is minimum. HIF-1α is mainly expressed in the nucleus, partly found in cytoplasm of high grade glioma. It has obvious heterogeneity; Its expression positive rate in adjacent tissues, a low grade(Ⅰ ~ Ⅱ) and high grade(Ⅲ ~ Ⅳ) glioma specimens in was 0%(0/3), 47%(8/17) and 79%(19/24). statistical analysis showed that there was significant between the positive rate(chi-square = 9.440, P = 9.440). In addition, C ENP- W and HIF-1α have not obvious relationship with the patients’ gender, age, and the glioma pathological types.Cobalt chloride simulated hypoxia experiment results showed that, when the glioma U251 cells were treated with 50 umol/L cobalt chloride with after 24 ~ 48 h, the cells grow well, had no obvious abnormal cell. Real-time fluorescent quantitative PCR results showed that the expression of CENP-W m RN A was significantly up-regulated after 24 h(t = 18.263, P < 0.01, FIG. 3 a). Western bloting resultsshowed the CENP- W protein expression levels also increased significantly(t = 7.813, P < 0.01).After transfection with the specific small interference RNA(si RNA) targeting HIF-1α gene to repress HIF-1α expression in U251 cells for 36 h, Real-time fluorescent quantitative PCR results showed that the expression of C ENP-W m RNA was significantly down-regulated((q=9.95,P<0.01;q=10.52,P<0.01)) compared with the control group and NC group. Western bloting results showed the CENP- W protein expression levels also decreased significantly(q=5.01,P<0.05;q=5.82,P<0.01).After transfection with the specific small interference RNA(si RNA) targeting CENP-W gene to repress CENP-W expression in U251 cells for 36 h, Real-time fluorescent quantitative PCR results showed that the expression of HIF-1α m RNA had no obvious difference(P>0.05) compared with the control group and NC group. Western bloting results showed the HIF-1α protein expression levels also showed insignificnce(q=2.95,q=2.64,P>0.05).Conclusions:1.Compared With the adjacent tissue, HIF – 1 α and CENP- W were all high expressed in gliomas, positively correlated with tumor grade.There was certain relevance between the expressions of the HIF-1α and CENP-W in gliomas, HIF- 1α can promote the expressions of CENP – W, contributing to occurrence and development of glioma.2.After HIF-1α protein degradation was inhibited by cobalt dichloride, the expression of CENP-W was significantly up-regulated in glioma U251 cells.3.The expression of CENP-W was down-regulated when the expression of HIF-1a was repressed by HIF-1α siRNA in U251 cells.4.But the expression level of HIF-1α had no change after the expression of CENP-W was repressed by CENP-W si RN A in U251 cells.5.The expressions of HIF- 1α and CENP- W are positively correlated with glioma pathological level. HIF- 1 α can regulate the expression of CENP-W.