| Meningiomas are one of the most common primary intracranial tumors.They come from arachnoid cap cells and attach closely to the dura mater,with the incidence being second only to glioma,accounting for 35% of all intracranial tumours.As defined by the 2016 WHO classification of central nervous system tumors,meningiomas are categorized into three grades:grade I(benign meningioma),grade II(atypical meningioma),and grade III(malignant meningioma).About 80% of cases are WHO grade I,10-18% are WHO grade II,2-4% are WHO grade III.Patients with benign meningiomas can expect a good prognosis following resection of the tumor completely.In addition,there are also some meningiomas that cannot be completely removed because of the location of the disease or the invasion of critical blood vessels and nerves.Despite comprehensive treatment including radiation,chemotherapy,and drug therapy,there is still a high risk of recurrence and a poor prognosis.There is a positive correlation between the recurrence rate of meningioma,the degree of malignancy,and the invasiveness of meningioma.WHO II and WHO III meningiomas are more aggressive than WHO I and have a worse prognosis.Thus,the in-depth investigation of the molecular mechanism of meningioma is very important for finding new drug targets,increasing the treatment outcome of meningioma,reducing the suffering of patients,and ultimately improving the prognosis.Until recently,the exact pathogenesis of meningioma has not been completely understood.As with most tumors,changes in the environment can lead to epigenetic instability,which may result in abnormal expression of some genes that may impact individuals with genetic susceptibility to tumor development,which may be the primary cause of tumor development.It was discovered in the study that single nucleotide gene polymorphisms play an important role in the susceptibility of many tumors and that they largely determine genetic differences between individuals by affecting gene transcription and protein expression levels.Grb2-associated binding protein 1(GAB1),a scaffold protein for protein-protein interactions,is closely related to the occurrence,development,and prognosis of tumors.Studies have shown that GAB1 is highly expressed in meningiomas,suggesting that its expression correlates with the aggressiveness of the tumor.The polymorphisms of GAB1 containing common loci are closely related to the susceptibility of various tumors,such as biliary tract cancer and lung cancer.Some common loci are also associated with benign diseases,such as hypertension.To date,no research report has been published examining the association between GAB1 polymorphisms and the risk of meningioma.MiRNA is a category of endogenous,short,single-stranded,noncoding RNAs,which play an important role in epigenetic regulation.miRNA can influence post-transcriptional protein expression by interacting with the3’UTR region of target genes,and play a critical role in the development of diseases as well as the occurrence of diseases.miR-153-5p is a newly discovered micro RNA that could play an important role in the occurrence and development of tumors,especially in the central nervous system.In the recent study,miR-153-5p could inhibit cell migration and invasion in glioblastoma by targeting SNAI2 and play as a tumor suppressor gene in glioblastoma.However,few studies concerning miR-153-5p and meningioma have been reported.Firstly,the correlation between GAB1 gene polymorphism and meningioma susceptibility was examined;subsequently,the expression and correlation of miR153-5p and target gene GAB1 in meningioma were examined;lastly,the possibility and mechanisms of GAB1 inhibiting proliferation and invasion of malignant meningioma by regulating the MEK/ERK pathway were investigated as a preliminary hypothesis.The purpose of this study was to investigate the role and mechanism of miR153-5p and GAB1 in meningioma.Thus,it may provide a theoretical basis for the pathogenesis and gene therapy of meningioma,and provide new biological detection indicators and targeted therapy for the diagnosis of meningioma.Part one Association of Single-Nucleotide Polymorphisms of GAB1 Gene with Susceptibility to meningiomaObjective: This study aimed to determine the correlation between GAB1 SNPs and meningioma susceptibility in a northern Chinese Han population.Methods: A total of 205 patients with meningioma and 297 healthy controls enrolled in the Second Affiliated Hospital of Hebei Medical University and Handan City Central Hospital from January 2019 to May2021 were included in this study.All selected subjects were recruited to analyze four single nucleotide polymorphisms(rs3805246,rs3828512,rs3805236,and rs1397529)under allele and genotype,followed by clinical characteristics stratified analysis.Results: We found there were statistically significant differences in the frequencies of genotypes and alleles of the rs3805236 polymorphism between patients and controls.The frequency minor allele frequency(MAF)for G allele in the meningioma group(30.2%)was significantly higher than that in the healthy control group(20.9%),suggesting that the minor allele G has a risk factor compared with the major allele A,and is a meningioma risk alleles(P=0.001,OR=1.675,95%CI 1.252-2.240).There was a significant difference in the association of GAB1 genotypes with meningioma using co-dominant,dominant,and recessive genetic models of inheritance.Similar in GAB1 rs1397529,MAF for C the allele in the meningioma group(11.2%)was significantly higher than that in the healthy control group(7.1%),suggesting that the minor allele C has a risk factor compared with the major allele A,and is a meningioma risk allele(P=0.001,OR=1.675,95%CI 1.252-2.240).In addition,because there were only two alleles at this site,the co-dominant model was used to analyze.Individuals with a heterozygous AC genotype were more likely to have the disease than those with a wild-type homozygous AA genotype(P=0.020,OR=1.737,95%CI1.090-2.768).Stratification analysis was performed between the GAB1 polymorphisms and clinical characteristics of meningioma.Meningioma patients with AC genotype have a significantly higher rate of dural invasion than patients with AA genotype(P=0.027),suggesting that the AC genotype may be associated with the aggressiveness of meningiomas.Summary: This study explored GAB1 gene intronic region rs3805236A>G and 3’UTR rs1397529 A>C polymorphisms increase the risk of meningioma in the northern Chinese Han population for the first time.GAB1rs1397529 A>C was associated with dural invasion in meningioma patients,and the minor allele C was a risk allele for meningioma dural invasion and might potentially increase meningioma aggressiveness.Part two Expression difference and clinical significance of miR-153-5p and GAB1 in meningioma tissuesObjective: The purpose of this study is to determine whether miR-153-5p,GAB1 mRNA,and GAB1 protein levels are related to the clinical characteristics of patients with meningioma.Methods:1.RT-qPCR and immunohistochemistry were used to detect the expression of miR-153-5p,GAB1 mRNA and GAB1 protein in normal dura mater and different grades of meningioma tissues.We then analyzed the expression of miR-153-5p and GAB1 mRNA and the clinicopathology feature relationship.2.Pearson correlation was used to analyze the correlation between the expression levels of miR-153-5p and GAB1 mRNA in meningioma tissues.3.ROC curves were drawn to evaluate the diagnostic performance of miR-153-5p and GAB1 in meningiomas.4.Stratified analysis of miR-153-5p,GAB1 mRNA,and protein expression differences according to different genotypesResults:1.The expression characteristics of miR-153-5p and GAB1 mRNA in normal dura mater and different grades of meningioma.The results of RT-qPCR experiments showed that the expression of miR-153-5p in meningioma tissue was significantly decreased(P < 0.05)and the mRNA expression of GAB1 was significantly increased compared with normal dura mater tissue(P < 0.01),the differences were statistically significant.Compared with WHO grade I meningioma tissues,the expression of miR-153-5p in meningioma tissue was significantly decreased,and the mRNA expression of GAB1 was significantly increased(all P <0.01).There was no significant difference in the expression levels of miR-153-5p or GAB1 mRNA between WHO II and WHO III meningioma tissues.2.Expression characteristics of GAB1 protein in normal dura mater and different grades of meningiomaImmunohistochemical results showed that GAB1 was mainly expressed in the cytoplasm.The number of cases with negative expression(-)of GAB1 in normal dura mater tissue was 3/10(30.00%),and those in WHO I meningioma tissue were 4/30(13.33%),while there were no negative expression(-)patients in WHO II and III meningiomas.The number of GAB1 protein expression(++)cases with WHO grade I-III were 17/30 cases(56.66%),10/15 cases(66.66%)and 1/5 cases(20.00%),GAB1 protein expression(+++)were 1/30(3.33%),2/15(13.33%)and 4/5(80.00%),respectively.Compared with normal dura mater,the number of GAB1-positive cells in meningioma tissue increased,and the difference was statistically significant(P<0.001).Among different grades of meningioma,the differences were also statistically significant(P<0.001),suggesting that with the increasing WHO grade of meningioma,the protein expression level of GAB1 was higher.3.Correlation of miR-153-5p and GAB1 mRNA expression levels in meningioma tissuesPearson correlation analysis showed that the relative expression of miR-153-5p in meningioma patients was moderately negatively correlated with the relative expression of GAB1 mRNA(r=-0.521,P<0.001).4.Diagnostic efficacy of miR-153-5p and GAB1 mRNA in meningiomaThe ROC curves were drawn and the results were as follows: The ROC curves of miR-153-5p and GAB1 mRNA were statistically significant in meningioma(P < 0.001),and both had higher diagnostic accuracy for meningioma.5.The relationship between the expression of miR-153-5p and GAB1 mRNA in meningioma and the clinical characteristics of patientsUsing the Mann-Whitney U test,the results showed that the dura mater invasion group had down-regulated miR-153-5p expression and up-regulated GAB1 mRNA expression compared with the dura mater non-invasion group,(all P < 0.05).There were also significant differences in tumor grade,the same as result 1.However,there was no significant difference in patients’ age,gender,and peritumoral edema between the two groups(P>0.05).Binary logistic regression was used to assess whether both are risk factors for dural invasion and tumor grade,both miR-153-5p downregulation and GAB1 upregulation were risk factors for dural invasion and tumor grade(all P <0.05).6.Stratified analysis results of miR-153-5p,GAB1 mRNA,and protein according to different genotypesUnder the optimal model(recessive model),stratified analysis was performed according to the genotypes of GAB1 4 SNPs(rs3805246,rs3828512,rs3805236,and rs1397529),respectively.miR-153-5p and GAB1 mRNA expression levels were compared in different genotype subgroups,and neither were statistically significant.Similarly,GAB1 protein expression level was compared in different genotype subgroups.We found there was a statistically significant in GAB1 protein expression at rs1397529(AC vs AA).The protein expression level of GAB1 is up-regulated in meningioma patients with AC genotype compared with patients with AA genotype(P < 0.05).There were no statistical differences in other sites(P>0.05).Summary: miR-153-5p and GAB1 mRNA are expressed in normal dura mater tissue and meningioma,while miR-153-5p expression is down-regulated and GAB1 mRNA expression is up-regulated.GAB1 protein expression was elevated in meningioma tissues,and which expression level was positively correlated with pathological grade.In contrast with the non-dural invasion group,miR-153-5p was down-regulated and GAB1 mRNA was up-regulated in the dural invasion group.Both down-regulation of miR-153-5p and up-regulation of GAB1 mRNA were risk factors for dural invasion and tumor grade.Among meningioma patients with up-regulated GAB1 protein expression,rs1397529(AC vs AA)had the AC genotype.We speculated that the rs1397529 risk genotype AC might affect the occurrence and development of meningioma by regulating GAB1 protein levels.This study might provide a theoretical basis for future gene modification therapies for meningiomas.Part three Micro RNA-153-5p inhibits proliferation and invasion via the MEK/ERK pathway by targeting GAB1 in malignant Meningioma CellsObjective: Given the negative correlation between miR-153-5p and GAB1 expression in meningioma tissues,the effects of miR-153-5p and GAB1 on the proliferation,invasion,and migration of malignant meningioma cells and the targeting relationship between them would be deeply studied in this part in vitro.Methods:1.The direct target sites were verified with dual-luciferase reporter gene assay.With the help of the online prediction software,Target Scan,we analyzed the binding sequence of miR-153-5p and GAB1 3’UTR,and then constructed a plasmid with it.Wild type GAB1 3’UTR(WT)and GAB1 3’UTR(hsa-miR-153-5p MUT)plasmids were constructed.H293 T cells were transfected with negative control,miR-153-5p mimics and plasmids.Modifications in gene expression are reflected in the change in luciferase activity,and may quantitatively reflect the inhibitory effect of miRNA on the target gene.Luciferase activity was measured by using the Luciferase Assay System(Promega).2.Malignant meningioma cell line IOMM-Lee was cultured in vitro and transiently transfected with miR-153-5p mimics,miR-153 inhibitor,GAB1 siRNA and GAB1 siRNA + miR-153 inhibitor.We analyzed cell proliferation,cell invasion,and migration using CCK-8,transwell assay,and wound-healing test.The effect on GAB1 expression was detected by RT-qPCR and Western blot to further verify the targeting relationship between miR-153-5p and GAB1.3.GAB1 siRNA,and GAB1 siRNA + miR-153-5p inhibitor were transfected into IOMM-Lee cells by RNA interference technology,and their effects on the proliferation and invasion of malignant meningioma cells were observed by CCK-8 assay,Transwell assay and cell scratch assay.And the effect of migration,and its effect on GAB1 and the expression levels of MEK/ERK pathway key factors MEK1,p-MEK1,ERK1/2,p-ERK1/2 were detected by Western blot.Results:1.GAB1 was identified as a direct functional target of miR-153-5p.We further analyzed that miR-153-5p could target the binding site of GAB13’UTR by applying the database in the online prediction software Target Scan(http://www.Target Scan.org),and found that miR-153-5p could Binds to four sites of the GAB1 3’UTR.Our previous study found that the gene polymorphism of GAB1 was associated with meningioma susceptibility,and SNP rs1397529 was located in the 3’UTR,which might be a functional SNP.miR-153-5p mimics did not suppress the fluorescence activity of wild-type and mutant GAB1 vectors.Nevertheless,when all four sites predicted to target miR-153-5p binding to the GAB1 3’UTR were mutated using complementary mutations,the results showed that miR-153-5p could regulate the expression of luciferase with GAB1 3’UTR wild type(P<0.0001).When the binding sites were mutated,this regulatory relationship disappears(P<0.0001).2.miR-153-5p inhibits malignant meningioma cells proliferation,migration and invasion by targeting GAB1Assays for determining cell proliferation,migration and invasion were performed using a CCK-8 assay,transwell experiment,and scratch test respectively.GAB1 silencing,GAB1+miR-153-5p co-silencing,and miR-153-5p overexpression both significantly inhibited cell proliferation,invasion,and migration,while miR-153-5 silencing significantly promoted cell proliferation,invasion,and migration.It has been demonstrated that overexpression of miR-153-5p could inhibit the proliferation,migration,and invasion of malignant meningioma cells.GAB1 silencing partially blocks the action of miR-153-5p silencing on proliferation,migration,and invasion of malignant meningioma cells.miR-153-5p may inhibit malignant meningioma cell proliferation,migration,and invasion by targeting GAB1.GAB1 silencing approach partially inhibited miR-153-5p’s effect on proliferation,migration and invasion of malignant meningioma cells.This suggested that miR-153-5p might inhibit proliferation,migration and invasion of malignant meningioma cells by targeting GAB1.3.miR-153-5p exerts its functions by inhibiting the GAB1-MEK/ERK axis.We examined the expression of the signature genes of the GAB1-MEK/ERK axis to further explore the mechanism of the action of miR-153-5p.Compared with the normal controls,overexpression of miR-153-5p resulted in a significant decrease in GAB1 mRNA and protein expression in malignant meningioma cells(all P < 0.05).Meanwhile,miR-153-5p inhibitor increased GAB1 mRNA expression,but did not reach statistical significance.Instead,miR-153-5p inhibitor significantly increased GAB1 protein expression(P < 0.05).Compared with normal control,there was an overexpression of miR-153-5p,a decrease in the expression of GAB1 protein,and a significant decrease in the levels of ERK1/2,MEK1,and p-ERK1/2 protein expressions(all P < 0.01).The p-MEK1 protein expression also decreased,and the result was not statistically significant.After miR-153-5p was silenced,the expression of GAB1 protein was increased,and MEK1,ERK1/2,p-MEK1,and p-ERK1/2 expressions were also up-regulated at the same time,but only the expression of ERK1/2 reached statistical significance(P < 0.05).The results suggested miR-153-5p might regulate MEK/ERK pathway activation by inhibiting GAB1 expression..Summary: miR-153-5p could suppress the expression of its target gene GAB1 and regulate the activity of MEK/ERK signaling pathways,thereby inhibit the proliferation,migration,and invasion of malignant meningioma cells. |
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