The Mechanism Of HOXA5 Gene Amplification To Promote The Malignant Progression And Drug Resistance Of Glioblastoma

Glioblastoma,the most lethal tumor of the central nervous system,lacks effective clinical treatment strategies,with the average patient survival time being only about 12 months.The difficulty in precise treatment of glioblastoma is lacking of effective biomarkers to evaluate tumor progression and patient prognosis.In addition,there are rare therapeutic strategies targeting glioma stem cells,which are believed to be the root of invasive growth,drug resistance,and recurrence of gliomas.Clinically,glioblastoma patients often show resistance to the first-line chemotherapeutic drug temozolomide,which results in a limited therapeutic effect.Therefore,identification of effective biomarkers to be associated with the self-renewal ability of glioma stem cells and to predict the prognosis of glioblastoma patients,and exploring combination therapy strategy of these biomarkers and temozolomide will provide new perspective for the clinical diagnosis and treatment of glioblastoma.HOX is a class of genes that determines the basic structure and orientation of an organism.There are 4 HOX families in the human body.HOXA families were found to be highly expressed in pediatric glioma drug-resistant cells and to be an indicator of drug resistance pathway activation.HOXA5,one of the HOXA family genes located on chromosome 7,plays an important role in inducing embryonic development and regulating the differentiation of adult stem cells.We previously found that HOXA5 could regulate the stemness of glioma stem cells.However,the role of HOXA5 glioma progression and the corresponding regulatory mechanism need to be further clarified.In this study,HOXA5 was screened as a key prognostic factor through overlapping the genes upregulated in glioma stem cells and GBMs relative to low grade gliomas.Then,the expression and amplification of HOXA5 in gliomas were analyzed by immunohistochemistry,next-generation sequencing and fluorescence in situ hybridization to determine its relationship with the prognosis of glioma patients.The role and mechanism of HOXA5 in regulating self-renewal ability and invasion ability of glioma stem cells were also investigated.Finally,the role and underlying mechanism of HOXA5 in promoting temozolomide resistance was investigated by database analysis,as well as in vitro and in vivo assay.The main conclusions and results are as follows:Part Ⅰ: Clinicopathological significance of HOXA5 gene amplification and expression in gliomaThrough analysis of the differential expression genes in gliomas with different WHO grade,HOXA5 was identified as an effective indicator.The expression characteristics and manifestations of HOXA5 in glioma and the relationship between HOXA5 and prognosis of patients were also analyzed.The main methods,results and conclusions are as follows:1.HOXA5 amplification is a prevalent molecular event in GBMs,with clinical significance for molecular diagnosis and prognostic determination.(1)HOXA5 is an important marker for the grades and prognosis of glioma patients: By analyzing the database of gliomas,we screened out HOXA5 as a key molecule of the grade of glioma,especially the positive correlation between its amplification and gene expression,suggesting the important role of HOXA5 gene amplification in the malignant behavior of gliomas.(2)HOXA5 amplified in glioblastoma: HOXA5 gene amplification was found to be a specific event in tumor cells through fluorescence in situ hybridization and secondgeneration sequencing analysis.(3)The positive correlation between HOXA5 gene amplification and glioma grades:Further analysis of glioma database also showed that the ratio of HOXA5 gene amplification gradually increased with grades,and the amplification rates differentially presented in different tissue types and subtypes.(4)The clinical significance of HOXA5 gene amplification for molecular diagnosis and prognostic determination: By analyzing the samples of glioma patients from Southwestern Hospital,TCGA and Rembrandt database,we found a worse prognosis of glioblastoma patients with HOXA5 gene amplification.2.The important clinicopathological significance of HOXA5 expression caused by gene amplification(1)HOXA5 gene amplification leads to an increase of its expression: Fluorescence in situ hybridization,immunohistochemistry and Western blot experiments results showed that HOXA5 gene amplification is positively correlated with its expression.Further analysis of database also showed that the cases with high HOXA5 expression show an enrichment of gene amplification.(2)The increase of HOXA5 expression is positively correlated with the grades of glioma and can be used as an independent prognostic factor: By analyzing the TCGA and Gravendeel databases,we found enhanced HOXA5 expression was negatively correlated with the overall survival of GBM and LGG patients.Multivariate survival analysis also confirmed that HOXA5 expression is an independent prognostic factor of glioma.The above results confirm that HOXA5 amplification and subsequent gene overexpression are correlated with increased glioma malignancy and poor prognosis,and may be a potential diagnostic and therapeutic target for malignant glioma.Part Ⅱ: Regulation and mechanism of HOXA5 on the self-renewal and invasion ability of glioma stem cellsHOXA5 amplification is an important factor to determine the malignant of gliomas,but its role and mechanism in regulating the malignant behavior of glioma are still unclear.In addition,the previous research of our group also found that the high expression of HOXA5 may be related with the regulation of glioma stem cells.Therefore,we preliminarily explore the regulation and mechanism of HOXA5 on glioma cells in this section.The main methods,results and conclusions are as follows:1.HOXA5 gene amplification and high expression are important characteristics of glioma stem cells(1)High expression of HOXA5 is an important feature of glioma stem cells: We first found that the HOXA5 high expression group showed an enrichment of glioma stem cell gene characteristics through GSEA analysis,further analysis revealed that HOXA5 expression was positively correlated and co-localized with glioma stem cell markers.(2)HOXA5 gene amplification is an important feature of glioma stem cells: Through the detection of HOXA5 probe,we found the amplification rate of HOXA5 gene in glioma stem cells is higher than that in differentiated glioma cells.The fluorescence in situ hybridization and immunofluorescence double staining experiments also confirmed that HOXA5 gene amplified cells showed positive expression of glioma stem cell markers.2.HOXA5 regulates the self-renewal and invasion ability of glioma stem cells:(1)Silencing HOXA5 significantly inhibits the self-renewal ability of glioma stem cells:In vitro limiting dilution analyses and tumorsphere formation assay showed that the tumorsphere formation ability of GSCs was robustly disrupted by HOXA5 inhibition.(2)Silencing HOXA5 significantly inhibits the growth of transplanted tumors in mice:Orthotopic transplantation tumor experiments in mice showed that silencing HOXA5 potently inhibited GSC-derived tumor growth.(3)Silencing HOXA5 attenuates GSC invasiveness: Through microscopic observation,we found that reduced glioma cells infiltrated into the adjacent normal brain tissues after HOXA5 disruption.In vitro transwell experiment and detection of invasion ability markers also confirmed this conclusion.3.The mechanism of HOXA5 on regulating self-renewal and invasion ability of glioma stem cells(1)HOXA5 transcriptionally regulate PTPRZ1 and the clinicopathological significance of the combined analysis: Our previous research found that HOXA5 binds to the PTPRZ1 promoter region and positively regulates its expression.Further studies found the decreased proportion of PTPRZ1 positive glioma stem cells after silencing HOXA5 expression.Immunohistochemical staining analysis of glioma samples also confirmed that HOXA5 and PTPRZ1 expression were positively correlated,and combination of their expressions showed a more precise predictable of prognosis.(2)HOXA5 regulates the self-renewal ability and invasion ability of glioma stem cells through PTPRZ1: Through the detection of stem cell markers,limiting dilution assay and tumorsphere formation assay,we found that knocking down the expression of PTPRZ1 can partially restore the self-renewal ability of glioma cells caused by overexpression of HOXA5.Transwell experiment and detection of invasive ability marker also showed the same results.Furthermore,a series of functional experiments after mutation of the HOXA5 binding site on the PTPRZ1 promoter region also confirmed that HOXA5 regulates the self-renewal and invasion ability of glioma stem cells by binding to the PTPRZ1 promoter.The above results confirm the important role of HOXA5 amplification in regulating the self-renewal and invasion ability of glioma stem cells,and its mechanism is transcriptionally activating PTPRZ1.The combined expression of the two factors also showed a more predictable effect for prognosis of glioma patients.Part Ⅲ : The phenome and mechanism of HOXA5 promoting temozolomide resistance of glioma cellsThe results of previous studies have shown that HOXA family is highly expressed in glioma resistant cells,and the relationship of HOXA5 and glioma stem cells further suggests the effect of HOXA5 on regulating temozolomide resistance of glioma cells.Therefore,we preliminarily explore the effect and mechanism of HOXA5 on temozolomide resistance of glioma cells in this section.The main methods,results and conclusions are as follows:1.HOXA5 promotes temozolomide resistance of glioma cells and the clinical significance(1)The expression level of HOXA5 in glioma cells is positively correlated with temozolomide resistance: We found that HOXA5 expression is positively correlated with IC50 value of temozolomide,and HOXA5 is highly expressed in glioma resistant cells.After silencing the expression of HOXA5,the genes expression of apoptosis signal pathway increased,the IC50 value of glioma cells decreased,and the proportion of apoptotic cells increased,suggesting that HOXA5 plays an important role in maintaining the temozolomide resistance of glioma cells.(2)The clinicopathological significance of HOXA5 in TMZ treatment: By analyzing the TCGA database,we found that HOXA5 can be used as an important factor to predict the therapeutic effect of temozolomide.(3)The combination of HOXA5 and TMZ can effectively inhibit tumor growth and prolong the survival of mice: NOCD-SCID mouse orthotopic xenograft tumor treatment experiments show that the combination therapy of silencing HOXA5 with temozolomide can effectively inhibit tumor growth and prolong the survival of mice.2.The mechanism of HOXA5 promoting temozolomide resistance in glioma cells(1)Bioinformatics analysis results showed that the expression of HOXA5 and EZH2 is positively corelated: Database analysis showed that HOXA5 and EZH2 may interact with each other,and further immunofluorescence staining and immunohistochemical staining also showed that HOXA5 and EZH2 are co-localized,and positively corelated.(2)HOXA5 binds with EZH2 CXC subunits to regulate temozolomide resistance in glioma cells: Co-IP experiment results showed that HOXA5 interacts with EZH2.The expression of EZH2 and downstream molecules H3k27me3 decreases after silencing HOXA5.Through detection of apoptosis signaling pathway factors and the proportion of apoptotic cells,we found that knocking down EZH2 or using of EZH2 inhibitors can partially restore the effect of temozolomide treatment caused by overexpression of HOXA5.Through truncation analysis of the full-length fragment of EZH2,the CXC subunit was found to be combined with HOXA5.(3)HOXA5-EZH2 pathway cause DNA methylation of the drug sensitive genes: By knocking down HOXA5 expression with the Ch IP-seq analysis,we screened for the key downstream molecule of EZH2-H3k27me3 mediated DNA methylation and the gene expression silencing related with temozolomide resistance.The above results clarify the role of HOXA5 in regulating temozolomide resistance of glioma cells,and its mechanism is regulating the expression of downstream gene H3k27me3 by combined with the EZH2 CXC subunit.In summary,our results confirmed the relationship between HOXA5 gene amplification and its expression for the first time.The clinical diagnosis,treatment and prognosis of glioma were also found to be corelated with the gene amplification.Then a series of experiments showed that HOXA5 play an important role in maintaining the phenotype of glioma stem cells and temozolomide resistance.The mechanism of HOXA5 regulating the self-renewal ability and invasion ability of glioma cells is binding to the PTPRZ1 promoter region and it can also bind with the EZH2 CXC subunit to activate the expression of its downstream H3k27me3 which leading to temozolomide resistance.The results of this study provide new ideas for targeting glioma stem cells and temozolomide resistance.