| Background: Glioma is the most common primary intracranial neoplasm in adults.It is a heterogeneous,invasive neoplasm with poor prognosis.In the past few decades,the incidence of malignant brain tumors in the Chinese population has risen rapidly,especially in large cities.Many environmental and lifestyle factors are closely related to the susceptibility of glioma,such as occupation,ionizing radiation,mobile phone radiation,smoking and diet.At the same time,genetic factors play a more important role in the occurrence of glioma.Genome-wide association studies(GWAS)found that some abnormalities of gene loci were associated with glioma,such as 5p13.33(TERT),8q24.21(CCDC26),9p21.3(CDKN2A/B),11q23.3(PHLDB1)and 20q13.33(RTEL1).Pleckstrin homology-like domain family B member 1(PHLDB1)is a protein first recognized on the bioinformatics screen.In recent years,a large number of studies have analyzed the relationship between PHLDB1 gene polymorphism and many diseases including glioma.Among them,the relationship between rs498872 located in the 5’untranslated region of PHLDB1 gene and glioma was the most studied.In neuroblastoma,11q23.3 is usually deleted,suggesting that the gene plays an important role in brain tumors.This study will analyze the role of PHLDB1 gene in the occurrence and progression of glioma through population data and cell experiments,and provide theoretical basis for the diagnosis and treatment of glioma.Part I A meta-analysis of the association between polymorphism ofPHLDB1rs498872 and susceptibility to gliomaObjective: To better understand the impact of rs498872 polymorphism on glioma risk,we conducted a meta-analysis of all published case-control studies to explore the association between rs498872 polymorphism and glioma susceptibility.Methods:Pub Med,Web of Science and Embase Pub Med websites searched the included articles with the following search terms: "pleckstrin homology-like domain family B member 1","PHLDB1","11q23.3","rs498872","polymorphism","variation","mutation","genotype"."Allele" and "glioma".The starting time of retrieval is from database establishment to December 2018.We used the Newcastle-Ottawa scale(NOS)to assess the quality of qualified research.The Begger method was used to draw funnel maps to detect the publication offset of the included literature.Review Manager version 5.3.3 was used for statistical analysis.Results: 1.Twelve eligible studies were included in the analysis.Twelve case-control studies included 11 482 cases and 2 4782 controls.2.In dominant model(CC and CT+TT),CC genotype could significantly reduce the susceptibility of glioma(P<0.0001,OR=0.81,95% CI 0.76-0.85);in recessive model(TT and CC+CT),TT genotype could significantly increase the susceptibility of glioma(P<0.0001,OR=1.23,95% CI=1.13-1.34);codominant model(TT and CC)results CT genotype significantly increased the susceptibility of glioma(P < 0.0001,OR = 1.15,95% CI = 1.09-1.21).The results of allele C compared with T showed that allele C could decrease the susceptibility of glioma(P < 0.0001,OR = 0.86,95% CI = 0.84-0.90).Further subgroup analysis showed that the positive results in Asians and Caucasians were similar to those in the general analysis.3.In all comparisons,the significant correlation found in the aggregate analysis remains unchanged,which indicates that our findings are statistically stable and the overall results are relatively stable and reliable.4.The shape of funnel graph is symmetrical in every comparison,which indicates that there is no serious publication bias in the literature we included.Conclusion: Our meta-analysis shows that TT genotype can significantly increase the susceptibility to glioma.rs498872 polymorphism may be a potential genetic biomarker for glioma in Asian and white populations.Part II Clinical significance of PHLDB1rs498872 polymorphism inpatients with gliomaObjective: To analyze the relationship between rs498872 polymorphism and glioma progression and prognosis in population data,and to further analyze the interaction between rs498872 polymorphism and major environmental factors and other glioma-related polymorphism loci.Methods: From January 2012 to December 2017,400 patients with glioma were collected in a hospital.During the same period,480 healthy controls were collected in a hospital.During the period of pathological collection,blood samples from patients and healthy controls who were newly diagnosed with glioma were collected in the laboratory of a hospital every day and stored in a refrigerator at 4 C.In the HIS case inquiry system,we inquired about the size,location,staging and Ki-67 value of the tumors at the time of initial diagnosis.The main influencing factors of the subjects were sex,age,education level,smoking,drinking,type of work,family history of cancer,frequency of mobile phone use and radiotherapy.From the time of diagnosis,all the subjects were followed up regularly with an interval of 4 weeks.Polymerase Chain Reaction-Lipase Detection Reaction(PCRLDR)was used to detect gene polymorphism.Result: 1.rs498872 polymorphism genotype is divided into CC,CT and TT.There are more CC genotypes in case group and more TT genotypes in control group.The frequency of T allele in the case group was significantly higher than that in the control group(P < 0.05).Compared with CC genotype,TT genotype can significantly increase the risk of glioma(P < 0.05),TT + CT genotype can significantly increase the risk of glioma(P < 0.05).In allele analysis,allele T significantly increased the risk of glioma compared with allele C(P < 0.05).2.The distribution frequency of CC genotype was higher in glioma patients whose tumors were less than 5cm,stage II+III,Ki-67 value less than 10 and KPS score more than 80.TT genotype was higher in glioma patients whose tumors were more than 5cm,stage IV,Ki-67 value more than 10 and KPS score less than 70.3.The median survival time of patients with TT,CT and CC genotypes was 23.5,27.3 and 32.1 months,respectively.There were significant differences among the three groups(Chi-square value = 14.86,P = 0.001),TT genotype patients had the shortest survival time and CC genotype patients had the longest survival time.Stage IV,tumor size greater than 5 cm,Ki-67 > 10,KPS score less than 70,multiple lobes involved and TT genotype were independent risk factors for the prognosis of glioma.4.The 1-year recurrence rates of CC,CT and TT genotypes were 48(34.8%),54(39.1%)and 36(26.1%)respectively,with significant difference(Chi-square value = 6.18,P < 0.05).TT genotype significantly increased the risk of glioma recurrence within one year(P < 0.05).5.Family history,smoking,radiation therapy,occupational exposure less than 10 years and occupational exposure more than 10 years were independent risk factors for glioma(P < 0.05).There was no significant interaction between rs498872 and family history in the occurrence of glioma.There was significant positive interaction between rs498872 and smoking,radiotherapy and occupational exposure in the occurrence of glioma.6.Mutant homozygotes in XRCC1 Arg194 Trp,XRCC1 Arg399 Gln,ERCC1 C8092 A,ERCC2 Lys 751 Gln and MGMT Leu84 Phe loci all significantly increased the susceptibility of glioma.Rs498872,XRCC1 Arg194 Trp and MGMT Leu84 Phe loci have positive interaction in influencing the susceptibility of glioma.Conclusion: The heterozygotes and homozygotes of rs498872 polymorphism locus can significantly increase the susceptibility of glioma.The TT genotype of rs498872 polymorphism is an independent risk factor for the prognosis and recurrence of glioma.Smoking,history of radiation therapy,occupational exposure,XRCC1 Arg194 Trp,MGMT Leu84 Phe and rs498872 polymorphisms had positive interaction on the susceptibility of glioma.Part III: The effect of PHLDB1 protein expression on the biologicalfunction of glioma cellsObjective: To analyze the effect of abnormal expression of PHLDB1 protein on the proliferation,apoptosis and migration of glioma cells,and the expression of PI3K/Akt signaling pathway-related proteins in glioma cells.Methods: Human astrocyte HA,human glioma cell lines U251,U87 and SHG-44 were purchased from the Institute of Basic Medicine,Chinese Academy of Medical Sciences.U251 and SHG-44 cells were divided into blank group,no-load group,PHLDB1 inhibition group and PHLDB1 overexpression group,respectively.The cells in blank group were not treated.The empty plasmid,PHLDB1 inhibitory plasmid and PHLDB1 overexpression plasmid were transfected into the empty plasmid,PHLDB1 inhibitory plasmid and PHLDDB1 overexpression plasmid,respectively.CCK8 assay was used to detect the proliferation of cancer cells.Transwell assay was used to detect cell invasiveness.Apoptosis was detected by flow cytometry.Scratch test was used to detect cell migration.Western blot was used to detect the expression of PHLDB1 and p-Akt in cells.The expression of PHLDB1 in cells was detected by RT-PCR.Result: 1.The relative expression levels of PHLDB1 protein in human astrocyte HA,human glioma cell lines U251,U87 and SHG-44 were 0.87±0.24,1.78±0.35,1.83±0.21 and 2.04±0.32,respectively.The relative expression levels of PHLDB1 in human astrocyte HA,human glioma cell lines U251,U87 and SHG-44 were 1.08 ±0.15,2.04±0.51 and 1.97±0.35 and 2.41±0.55,respectively.The levels of PHLDB1 protein and m RNA in Nthyori3-1 cells were significantly lower than those in human glioma cell lines U251,U87 and SHG-44(P < 0.05).2.The relative expression levels of PHLDB1 protein and m RNA in U251 and SHG-44 cells were significantly different in blank group,blank group,PHLDB1 suppression group and PHLDB1 overexpression group(P < 0.05).The relative expression levels of PHLDB1 protein and m RNA in PHLDB1 inhibition group were significantly lower than those in blank group and blank group(P < 0.05);the relative expression levels of PHLDB1 protein and m RNA in PHLDB1 overexpression group were significantly higher than those in blank group and blank group(P < 0.05).3.In U251 cells,the cell proliferation multiples in blank group,blank group,PHLDB1 inhibitory group and PHLDB1 overexpression group were 4.23±1.02,3.94±1.12,1.81±0.58 and 7.56±2.01,respectively.In SHG-44 cells,the cell proliferation multiples in blank group,no-load group,PHLDB1 inhibition group and PHLDB1 overexpression group were 5.51±1.03,5.06±2.14,3.21±1.08 and 7.53±2.19,respectively.The cell proliferation factor of PHLDB1 inhibition group was significantly lower than that of blank group and blank group,and that of PHLDB1 overexpression group was significantly higher than that of blank group and blank group.4.In U251 cells,the number of perforating cells in blank group,blank group,PHLDB1 inhibition group and PHLDB1 overexpression group were 241±53.6,286±49.3,108±21.6 and 352 ±75.2,respectively.In SHG-44 cells,the number of perforating cells in blank group,no-load group,PHLDB1 inhibition group and PHLDB1 overexpression group were 305±53.2,296±41.8,207±63.4 and 396±53.1,respectively.The number of penetrating cells in PHLDB1 inhibition group was significantly lower than that in blank group and blank group,and the proliferation rate of PHLDB1 overexpression group was significantly higher than that in blank group and blank group.5.In U251 cells,the healing rates of blank group,blank group,PHLDB1 inhibition group and PHLDB1 overexpression group were 12.3±3.29,14.6±4.05,8.12±2.05 and 26.3±5.23,respectively.In SHG-44 cells,the healing rates of blank group,no-load group,PHLDB1 inhibition group and PHLDB1 overexpression group were 16.3±2.54,18.5±4.21,9.12±2.41 and 31.0±5.19,respectively.The cell healing rate of PHLDB1 inhibition group was significantly lower than that of blank group and blank group,and that of PHLDB1 overexpression group was significantly higher than that of blank group and blank group.6.In U251 cells,the apoptotic rates of blank group,no-load group,PHLDB1 inhibitor group and PHLDB1 overexpression group were 18.9±4.25,20.5±3.96,39.6±8.21 and 10.3±3.05,respectively.In SHG-44 cells,the apoptotic rates of blank group,no-load group,PHLDB1 inhibitory group and PHLDB1 overexpression group were 12.6±2.36,13.5±2.15,27.5±3.69 and 6.34±1.54,respectively.The apoptotic rate of PHLDB1-inhibited group was significantly higher than that of blank group and blank group.The apoptotic rate of PHLDB1-overexpressed group was significantly lower than that of blank group and blank group.7.In U251 glioma cells,the relative expression levels of pAkt protein were 1.12±0.21,1.05±0.23,0.54±0.15 and 2.18±0.41 in blank group,blank group,PHLDB1 suppressor group and PHLDB1 overexpression group,respectively.The relative expression levels of p-Akt protein in SHG-44 glioma cells were 0.93±0.24,1.14±0.30,0.48±0.11 and 1.91±0.38 in blank group,no-load group,PHLDB1 inhibitor group and PHLDB1 overexpression group,respectively.The relative expression level of p-Akt protein in PHLDB1 inhibitory group was significantly lower than that in blank group and blank group.The relative expression level of p-Akt protein in PHLDB1 overexpression group was significantly higher than that in blank group and blank group.Conclusion: PHLDB1 gene inhibition can inhibit the proliferation,migration and invasion of glioma cells,and promote the apoptosis of glioma cells.On the contrary,PHLDB1 gene inhibition can promote the proliferation,migration and invasion of glioma cells,and inhibit the apoptosis of glioma cells.Overexpression of PHLDB1 gene increased the expression level of p-Akt protein,suggesting that PHLDB1 can activate PI3K/Akt signaling pathway. |
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