The Clinical Significance And The Related Regulatory Mechanism Of HOXA4 And HOXB1 Expression In Human Glioma

Gliomas are the most frequent primary malignant tumors of the adult central nervous system(CNS), and are characterized by high morbidity and poor survival.Despite advances in the diagnosis and appropriate systemic therapies for glioma,including surgery, radiotherapy, and chemotherapy, there has been very little improvement in the clinical outcomes of patients with the high-grade glioma, the average survival period of the patients with glioblastoma was only 12-14.6 months,and the 5-year survival rate of them was less than 3%. Accumulating research has shown that several biological and molecular factors are involved in the development,progression, and metastasis of glioma. Therefore, it is essential to identify novel molecular markers that can efficiently predict its prognosis and provide targets for molecular therapies.It is well known that the expression products of the HOX genes have DNA-binding activity and act as transcription factors. HOX genes are divided into HOXA, HOXB, HOXC, HOXD 4 clusters, and are split into 39 genes according to the location of the chromosome and the genetic similarity between clusters.Functioning as both positive and negative regulatory factors, HOX proteins not only control the proliferation and differentiation of embryonic cells, but also have important functions in the differentiation of adult cells. The dysregulation of HOX gene expression leads to abnormal morphological structures during the growth of individuals, and can even cause tumorigenesis, including glioma, by altering the expression of apoptosis-related proteins and/or signaling pathways. HOXA4 or HOXB1, as a member of the HOX family genes, plays an important role in the pathogenesis of different tumors, but the expression and regulation mechanism of them are still unclear. This research will be through two-part experiment to explore the expression, clinical significance, biological behaviour and related regulatory mechanism of HOXA4 and HOXB1 in human glioma.Part I HOXA4 as oncogene in glioma promotes proliferation and inhibits apoptosis via Wnt/β-catenin pathwayObjective:To explore the expression level of HOXA4 and the relationship between HOXA4 and prognosis of patients with glioma, to further analysis the related regulatory mechanism of HOXA4 via studying the effect on malignant biological behavior of glioma cell. Methods:(1) Bioinformatics analysis(GEO and CGGA database) was performed to preliminary investigate the expression levels of HOXA4 in human glioma. The q RT-PCR and immunohistochemical analyses were performed on 8 non-neoplastic brain tissues and 40 glioma tissues to examine the expression of HOXA4 m RNA and protein. HOXA4 expression was detected in glioma cell lines(A172, U251 and U87) and astrocyte cell line(HA1800) by q RT-PCR and Western bolt.(2) c2 test was performed to analysis the relationship between HOXA4 and clinical pathological characteristics of glioma patients. The Kaplan–Meier life table method was performed to analyze the relationship between HOXA4 and survival rate of glioma patients. Prognostic factors were examined with Cox regression models.(3) MTT assay, colony formation assay, Transwell assay,scratch wound assay and Flow cytometry assay were performed to analyze the effect of HOXA4 on glioma cell proliferation, invasion, apoptosis and cycle in vitro.BALB/c nude mice subcutaneously tumor-burdened model was performed to validate the effect of HOXA4 on the growth of glioma in vivo.(4) Western bolt and IHC analysis of several Wnt/β-catenin pathway related proteins(β-catenin, DVL2,Gsk3β, Cyclin D1 and P53) was performed to investigate the signaling pathway of HOXA4-related in vitro and in vivo. Results:(1) The expression of HOXA4 acted as oncogene in glioma is up-regulated, and positively correlated with the malignant degree of glioma.(2) HOXA4 expression level is closely related to the glioma patient’s survival rate and prognosis.(3) HOXA4 promotes glioma cell proliferation and invasion, suppresses apoptosis.(4) Western bolt and IHC analysis shows that the expression of the β-catenin, DVL2 and Cyclin D1 proteins are markedly decreased in the HOXA4 knockdown group, whereas Gsk3β and P53 expression is elevated in vitro and in vivo. Conclusion : The expression level of HOXA4 is significantly increased in human glioma and positively correlated with the malignant degree. The overexpression of HOXA4 predicts shorter OS and PFS in glioma patients, and HOXA4 expression is an independent predictor of prognosis. HOXA4 promotes glioma cell proliferation and invasion and inhibits apoptosis via Wnt/β-catenin pathway.Part II HOXB1 is a tumor suppressor gene regulated by mi R-3175 in gliomaObjective : To explore the expression level of HOXB1 and the relationship between HOXB1 and prognosis of patients with glioma, to further analysis the targeting regulatory mechanism of HOXB1 and mi R-3175 by studying the effect on malignant biological behavior of glioma cell. Methods:(1) Bioinformatics analysis(GEO and CGGA database) was performed to preliminary investigate the expression levels of HOXB1 in human glioma. The q RT-PCR and immunohistochemical analyses were performed on 8 non-neoplastic brain tissues and 40 glioma tissues to examine the expression of HOXB1 m RNA and protein. HOXB1 expression was detected in glioma cell lines(A172, U251 and U87) and astrocyte cell line(HA1800)by q RT-PCR and Western bolt.(2) c2 test was performed to analysis the relationship between HOXB1 and clinical pathological characteristics of glioma patients. The Kaplan–Meier life table method was performed to analyze the relationship between HOXB1 and survival rate of glioma patients. The relationship between HOXB1 and prognosis was examined with Cox regression models.(3) MTT assay, Transwell assay, scratch wound assay and Flow cytometry assay were performed to analyze the effect of HOXB1 on glioma cell proliferation, invasion and apoptosis in vitro.(4)Bioinformatics analysis was applied to predict the HOXB1 gene 3′-UTR targeted mi R-3175. The q RT-PCR was performed to examine the expression of mi R-3175 in glioma. MTT assay, Transwell assay, scratch wound assay and Flow cytometry assay were performed to analyze the effect of mi R-3175 on glioma cell proliferation,invasion and apoptosis in vitro.(5) The Dual Luciferase Reporter Gene System was applied to detect the role of mi R-3175 on the HOXB1 gene 3′-UTR in the human glioma cell. The q RT-PCR and Western blot were performed to analyze the regulation of HOXB1 expression by mi R-3175 in the glioma cell lines(A172, U251 and U87). Spearman’s rank correlation analysis was performed to explore the relationship between HOXB1 and mi R-3175 in human glioma tissues.(6) MTT assay was performed to analyze the regulatory effect of mi R-3175 on cell proliferation induced by low HOXB1 expression. Flow cytometry assay was performed to determine the regulatory effect of mi R-3175 on cell apoptosis induced by low HOXB1 expression. Western bolt analysis of several downstream apoptosis-related proteins(procaspase-3, p53 and cytochrome C) was performed to preliminarily investigate the signaling pathway of HOXB1-induced apoptosis that functions in the pathogenesis of glioma. Results:(1) HOXB1 as a tumor suppressor gene in glioma is down-regulated expression, and negatively correlated with the malignant degree of glioma.(2) HOXB1 expression level is closely related to the glioma patient’s survival rate and prognosis.(3) HOXB1 inhibits glioma cell proliferation and invasion, promote apoptosis.(4) mi R-3175 expression is increased in glioma, and promotes glioma cell proliferation and invasion, prohibits apoptosis.(5) The Dual Luciferase Reporter Gene System reveals that mi R-3175 can significantly down-regulate the luciferase activity of the wild-type HOXB1 gene.The q RT-PCR and Western blot demonstrate that mi R-3175 mimics is negative regulator for HOXB1 expression, and mi R-3175 inhibitor is positive regulator.Spearman’s rank correlation analysis shows that the expression levels of HOXB1 and mi R-3175 in 40 glioma tissues are inversely correlated(R =-0.466).(6) MTT assay reveals that low-expression of mi R-3175 observably inhibits the glioma cell promoting proliferation induced by low HOXB1 expression. Flow cytometry assay reveals that low-expression of mi R-3175 significantly inhibits the glioma cell inhibiting apoptosis induced by low HOXB1 expression. Western bolt reveals that after oncogenicity induced by low HOXB1 expression is prevented by mi R-3175 inhibitor in glioma cells, the cytochrome C and p53 proteins are markedly increased,whereas procaspase-3 expression is decreased. Conclusion : The expression of HOXB1 is significantly decreased in human glioma, and inhibits glioma cell proliferation and invasion and promotes apoptosis. The low-expression of HOXB1 predicts shorter OS and PFS in glioma patients, and HOXB1 expression is an independent predictor of prognosis. The expression of mi R-3175 is significantly increased in human glioma, and promotes glioma cell proliferation and invasion and inhibits apoptosis. HOXB1 is a direct target of mi R-3175. Oncogenicity induced by low HOXB1 expression is prevented by mi R-3175 inhibitor in glioma cells.