Research About The Expression And Function Of The Long Non-coding RNA HOXD-AS2 In Glioma

Glioma is the most common primary malignant tumor of the brain,in which the highest degree of malignancy is the glioblastoma(multiforme Glioblastoma,GBM).In standard therapy,the median survival time of patients with GBM was only 15 months.Although the comprehensive treatment model of surgery,local radiotherapy and systemic chemotherapy has been widely used,the prognosis of GBM patients results are still difficult to make people satisfied,the vast majority of glioma patients will still relapse within a short time after operation.In order to improve the treatment of patients with glioma,gene and molecular level to grasp the mechanism of tumorigenesis has become a pressing matter of the moment.In recent years,a lot of lncRNAs in transcript of the human genome was found;and studies show that they play an important biological regulation function in the development of a variety of human neoplasms,including liver cancer,gastric cancer,renal cell carcinoma,rectal cancer,glioma and so on;The abnormal expression of some lncRNAs are closely related with recurrence and prognosis of glioma patients;In the research field of lncRNAs,its function and mechanism are still poorly understood by people;Fully understanding how lncRNAs works in the occurrence and development of glioma and the underlying molecular regulation mechanism will help us find new glioma therapeutic targets and molecular diagnosis markers.We constructed the complete gene expression profile microarray database before,using gene expression microarray technology,through detecting the gene expression differences between 5 cases of normal brain tissue samples and 5 cases of glioblastoma tissue samples.Then through the bioinformatics analysis,a number of lncRNAs which expressed differentially and may play a key role in the occurrence and development in glioma was screened out.Homebox D cluster antisense RNA 2(HOXD-AS2)is one of them.HOXD-AS2 is located on human chromosome 2q31.1,with the full length of 692 bp,which is the antisense strand of Homeobox D gene.Microarray data indicated that the expression of the gene was significantly higher than that in normal brain tissues.This study focused on the exploration of the long noncoding gene Homebox D cluster antisense RNA 2(HOXD-AS2).First,we verified the reliability of microarray data by detecting the expression of HOXD-AS2 gene and the other five randomly selected genes in the 5 cases of glioblastoma tissue samples and 5 cases of normal brain tissue samples through reverse transcription polymerase chain reaction(RT-PCR)to see if it coincides with the gene expression profile microarray data establised before.It was found that the results of RT-PCR were in good agreement with the data of gene chip,and the expression of HOXD-AS2 in glioblastoma was significantly higher than that in normal brain tissue.And then we further expand the sample,by detecting the expression of HOXD-AS2 gene in 45 cases of low grade gliomas and 57 cases of high grade glioma and to observe if its expression is in correlation with the pathological grades and other clinical features of gliomas,and to analyze the relationship between the expression level of HOXD-AS2 gene and the clinical prognosis in patients.Finally,the effect of HOXD-AS2 gene expression on the growth,proliferation,apoptosis and invasion of human glioma cell line U251 was observed by small interfering RNA technique in glioma cell line U251.Part I The expression of long noncoding RNA HOXD-AS2 in glioma and its relationship with clinical prognosisObjective: To study the expression of HOXD-AS2 gene in normal brain tissue and different grade glioma tissues,and analyze its relationship with the prognosis of the patients.Methods: The expression of HOXD-AS2 in 5 cases of glioblastoma tissue samples and 5 cases of normal brain tissue samples was detected by real-time PCR.And then we expanded the sample size,detected HOXD-AS2 gene expression in 8 cases of grade I,37 cases of grade II,24 cases of grade III,33 cases of grade IV glioma clinical samples,to observe its relationship with the pathological grades of glioma;at the same time we take some clinical indications of patients,for example age,gender,tumor size,surgical resection into account,to observe the correlation between HOXD-AS2 gene expression(+,+ +,+ + +)and the clinical indications.And according to the expression level of HOXD-AS2 gene,a total of 87 patients with complete clinical information in the the I-IV grade glioma were divided into high expression group and low expression group,the survival time of patients were followed-up,the relationship between the expression level of HOXD-AS2 and the survival of patients were analysised through Kapian-Meier survival curve method.A total of 40 patients with tumors of grade I and II will be included in the low grade malignant tumor group,A total of 47 cases of tumor of grade III and IV into high grade malignant tumor group,the same method above was used for survival analysis in low grade malignant tumor group(I + II)and high grade tumor group(III,IV grade),the relationship between the expression level of HOXD-AS2 and the survival of patients were further observed within the two subgroups.Results: The result of the Real-time PCR showed that the relative expression values of HOXD-AS2/GAPDH in normal brain tissue and the glioma of grade I,II,III,IV were 0.0073 + 0.0046,0.0519 + 0.0514,1.3477 + 2.2276,1.9717 + 2.9306 and 1.8884 + 3.1063,the difference of gene expression between normal brain tissue and all grades of glioma was statistically significant(p < 0.05);The relative HOXD-AS2/GAPDH expression values in normal brain tissue,low-grade gliomas(grade I+II)and high grade gliomas(grade III+IV)were 0.00733 + 0.00460,1.142 + 2.060 and 1.924 + 3.039,the level of the differences were statistically significant(p<0.05)between normal brain tissue and low grade glioma group,between normal brain tissue and high grade glioma group,and between low grade and high grade glioma.Chi square test and the Fisher's exact probability test was used to observe the correlation between the expression level of HOXD-AS2 gene and patient's age,gender,tumor size,operation resection extent,finding the result that the HOXD-AS2 expression levels is closely related to tumor pathological grade(P = 0.003).With K-M method and log rank test,the survival analysis shows the median overall survival time of HOXD-AS2 high expression group was significantly shorter than that in the low HOXD-AS2 expression group(respectively for 16 months and 42 months,P = 0.007),and median overall survival time differences in low grade tumor group(I + II)is more obvious.Conclusion: The expression level of HOXD-AS2 gene in gliomawas significantly increased compared with normal brain tissue,and the expression level is related to the grade of the tumor,the expression of high grade gliomas was higher than that of low grade gliomas.The high expression of HOXD-AS2 gene is a risk factor of poor prognosis of patients with glioma.Part II Effect of knocking down the expression of HOXD-AS2 on the function of brain glioma cell line U251Objective: To study the Effect of knocking down the expression of HOXD-AS2 on the function of brain glioma cell line U251 through siRNA.Methods: We specific knockdown HOXD-AS2 expression in U251 Cell Lines by siRNA technology,using Lipofectamin3000 as transfection carrier,and observe its influence on the proliferation,apoptosis,cell cycle and migration and invasion ability of U251 cells.In the control group,untreated U251 cells + Lipofectamin3000 vector were used as Mock control(Mock,M),U251 cells + Lipofectamin3000 vector + nonspecific oligonucleotid as negative control(NC).According to different genetic loci of HOXD-AS2,the experimental group was designed and synthesized 3 specific siRNA,Lipofectamin3000 was used as the transfection carrier,and the expression of HOXD-AS2 in U251 cell line was specifically knocked down.PCR Real-time technique was used to detect the expression level of HOXD-AS2 in the transfected 24 h and 48 h,and the highest interference efficiency was detected.The siRNA sequence was used as interference,CCK8,edu cell proliferation assay was used to detected the ablity of cell proliferation,flow cytometry was used to detect the cell cycle distribution and apoptosis,cell scratch assaywas used to detect the migration ability and Transwell assay to detect cell invasion ability.Results: After the interference of the expression of HOXD-AS2 in forty-eighth hours in U251 cells,CCK8 assay and EDU cell proliferation assay showed that the cell proliferation ability of the interferenced group was lower than that in group NC(P < 0.05);after the interference in forty-eighth hours,flow cytometry showed that in the experimental group the number of U251 cells in the proliferative phase(S +G2)decreased in the cell cycle(P < 0.05)compared with the NC group,cell cycle arrest in G1 phase;flow cytometry showed no significant change was found in cell apoptosis ratio(P>0.05);scratch experiments suggested that inhibition of HOXD-AS2 expression after 36 hours and 72 hours,the migration ability of U251 cells decreased significantly(P < 0.05);The invasion ablity detected by Transwell assy had no significant changes(P > 0.05).Conclusion: knocking down the expression of HOXD-AS2 in glioma cell line U251 induce G1 phase arrest of the cell cycle and reduce the ability of cell proliferation,and decrease cell migration ability significantly,but the invasion ability was not significantly affected.