| Glioma originated from glial cells,which is the most common malignant tumor in the central nervous system.Glioma is characterized by high invasiveness,high recurrence rate and high fatality rate.Because glioma and normal brain tissue lack of obvious boundaries,it is difficult to be completely removed by surgery,and those which grow in the important parts of the brain,for example brain stem,can’t even undergo surgery.Glioma is not sensitive to radiotherapy and chemotherapy,thus clinical treatment effect is poor.The mechanism of occurrence and development in glioma has not yet been fully elucidated,and to explore the pathogenesis of glioma is particularly important for the development of effective treatment.GAB2（Grb2 related binding protein 2）belongs to an evolutionary reserve protein family,in addition to GAB1,GAB2 and GAB3 in mammals,this protein family consists of three kinds of mammalian paralogues:Drosophila melanogaster homolog daughter of sevenless,Caenorhabditis elegans homolog suppressor of clear and Nematostella-Gab（Ne-Gab）.The GABs family members reveal 40 □ 50%sequence homology,but each one exhibits unique cellular functions.GAB1 and GAB2 is widely expressed in various human tissues and organs,and high expressed in brain,kidney,lung,heart,testicles,ovaries and so on.GAB2 is located on chromosomal band 1lq14.1,and amplification of 11q13□14.1 is frequently found in human malignant tumors.In recent years,a series of studies and experiments have confirmed that GAB2 is associated with the cell migration and invasion of human malignant tumors and plays an important role in the occurrence and development of human malignant tumors.Some scholars have found that GAB2 has high expression in ovarian tumors and GAB2 can regulate migration by activating the PI3K pathway in ovarian cancer.Besides,in the animal model experiment,the cell migration ability was markedly reduced in the silencing GAB2 mouse model,which decreased the proportion of lung metastasis in mouse breast cancer model.Vitro experiments had confirmed that GAB2 promoted the migration of glial cells,and the migration of cells decreased after GAB2 expression was reduced.The identification of GAB2 as a potential oncogene has been reported in the studies of breast cancer,ovarian cancer,leukemia,melanoma and gastric cancer.Recently,high expression of GAB2 in glioma cells has been reported,and the reason of GAB2 overexpression in glioma and how to reduce its expression in glioma tissue has not been fully elucidated.MicroRNAs are a class of non-coding single-stranded RNA molecules with short chains,which generally include 18 to 25 nucleotides,and belong to the non-coding and evolutionary reserve RNA,microRNAs have important roles in regulating genes,and can regulate the target microRNAs by combining 3 ’untranslated region（3’UTR）,adjust transcription and post-transcriptional genes,including inhibition of translation and the degradation of the target mRNAs,even tiny changes in the expression of microRNAs can have significant effect on the expression of hundreds of microRNAs.MicroRNAs have increasingly been identified as a key regulatory factor in many biological processes.Among the numerous microRNAs,miR-197,transcribed from genome chromosome 1 p13.3 area,has an obvious and abnormal regulation in many diseases,such as lung cancer,breast cancer,ovarian cancer,hepatocellular carcinoma,colorectal cancer,thyroid cancer,prostate cancer,thyroid follicular carcinoma and so on.In addition,more and more evidences show that miR-197 interacts with specific RNA species and plays a vital role in cell proliferation,differentiation,apoptosis,migration,and drug resistance.Recently,the regulation of microRNAs on the phenotype of human glioma has been reported.For example,the lower expression of microRNA-21 leads to growth disorders of glioma in vivo and manifests the synergistic cytotoxic effect with the neural precursor cells in gliomas,which carries the secretory type of tumor necrosis factor related apoptosis inducing ligand in the human brain glioma cells.MicroRNA-34 in brain tumors and glioma stem cells acts as a tumor suppressor,miR-181a increases human malignant glioma U87MG cells sensitivity to radiation therapy by targeting B cell lymphoma-2 genes.In spite of the abnormal expression of miR-197 in many tumors,the expression level and function in glioma are still poorly understood.More and more researchs have confirmed that the expression of GAB2 is related to glioma WHO grade and the invasion of glioma.The higher the WHO grade,the higher the expression of GAB2.However,the expression of miR-197 in glioma is still unknown.Whether miR-197 and GAB2 have interaction in the occurrence,development and invasion of glioma is still seldom reported.Illuminating the relationship between miR-197 and GAB2 in the occurrence,development and invasion of glioma is very helpful for us to put insight into the mechanism of pathogenesis and development of glioma,which can be applied as a useful marker of judging prognosis of glioma,and a new therapeutic target of glioma invasion intervention.ObjectIn this study,the expression levels of miR-197 and GAB2 in glioma tissues were first tested,then the cellular functions of miR-197 and GAB2 were detected in the Human A172 glioma cell lines,and the correlation and interaction between miR-197 and GAB2 were investigated,and the potential interaction sites of miR-197 and GAB2 were further examined.1.In This study,we selected some glioma specimens and the corresponding adjacent normal brain tissues,detected the expression levels of miR-197 and GAB2 respectively,and verified the difference of expression levels between glioma tissues and the corresponding adjacent normal brain tissues.2.The glioma A172 cells were cultured and passed on,then were transfected with pre□miR□197 and GAB2 plasmids respectively,and the corresponding control was set.The cellular functions of miR-197 and GAB2 in the A172 cells were detected.3.To predict and detect the possible interaction sites of miR-197 and GAB2,and to explore the feasibility of new targets for glioma therapy,are helpful to provide a possible novel treatment for glioma.Methods1.Some tumor specimens and adjacent normal brain tissues were selected.The A172 cells were cultured and passed on,then transferred respectively with pre□miR□ 197 and GAB2□ expressing plasmids;the control miR and empty vector plasmids transfection of A172 cells were respectively set as controlled group.The efficiency of the transfection of pre-miR-197 was verified by RT-qPCR and Western blot analysis was used to verify the efficiency of GAB2-expressing plasmid transfection.2.The expression of miR-197 was detected in glioma tissues and its corresponding adjacent normal brain tissue samples by In situ hybridization,the difference between the two groups were testified.3.The A172 cells transfected by pre□miR□197 and control miR were used to verify the effect of miR-197 on the proliferation and colony formation of A172 cells respectively by Cell counting assay and Colony formation assay;Cell cycle analysis was carried out to demonstrate the difference between the two groups of cells.The effects of miR-197 on DNA synthesis were confirmed by BrdU incorporation assay.Western blot analysis was used to further confirm the effect of miR-197 on the expression levels of the proliferating markers.4.The expression of GAB2 was detected in glioma tissues and its corresponding adjacent normal brain tissue samples by Western blot analysis,the difference between the two groups were testified.The expression level of GAB2 in glioma tissue sample sections was detected by immunohistochemistry,then we investigated the correlation between the expression level and glioma WHO grade.A172 cells were transfected with various doses of GAB2□ expressing plasmids（1nM、10nM、40nM）,the A172 relative quantity at different time points were determined using an MTT assay.GAB2 was speculated whether it was relative to cell proliferation and it was dose □ dependent.5.Correlation detection of miR-197 and GAB2（1）Western blot analysis was used to detect GAB2 protein expression level in the two groups of A172 cells transfected by pre□miR□197 and control miR.RT-qPCR was used to determine the expression level of GAB2 mRNA in A172 cells.Immunofluorescence analysis and mean fluorescence intensity were adopted to detect the immunofluorescence staining intensity of GAB2 antibodies.（2）Differences of expression level of miR-197 in the two groups of A172 cells transfected by GAB2□ expressing plasmids and empty vector plasmids was determined by RT-qPCR,Northern blot and microRNA microarray.（3）MTT assay and BrdU analysis were used to detect the difference of proliferation in A172 cells co-transfected.The two groups of A172 cells transfected by GAB2□ expressing plasmids or empty vector plasmids,were transfected by pre□ miR□ 197 respectively;The cells were named GAB2/pre-miR-197 and Vector/pre-miR-197 group after transfection,MTT assay and BrdU analysis were used to detect the difference of proliferation in the two groups of A172 cells.The two groups of A172 cells transfected by pre□miR□ 197 or control miR,were transfected by GAB2□ expressing plasmids.After transfection,two groups of A172 cells were named pre-miR-197/GAB2 and control-miR/GAB2 group.MTT assay and BrdU analysis were used to detect the difference of proliferation in the two groups of A172 cells.6.To explore the target site of miR-197 in GAB2（1）TargetScan（http://www.targetscan.org/vert61/）was used to predict the possible binding sites of miR-97 in GAB2 mRNA.（2）The luciferase reporter assay:According to the predicted binding site,the luciferase reporter plasmid（Wild-type-GAB2-WT-luc）containing the directional sequence of the possible binding sites of miR-197 in GAB2 mRNA was constructed,and then introduced into the two groups of A172 cells transfected by pre miR-197 and control miR.The luciferase reporter assay was carried out after transfection,which was used to testfied whether miR-197 affected the luciferase activity.If miR-197 could inhibit the activity of certain plasmids luciferase,GAB2 wild-type luciferase gene containing the corresponding site was set as a template,a Quik Change Mutagenesis kit was used to make the part bases of the corresponding site mutate,then GAB2 mutant luciferase report plasmids（GAB2-MUT-luc）encompassing mutation binding site was constructed.And then the A172 cells above were transfected by the mutant luciferase report plasmids,whether miR-197 could inhibit the mutant luciferase activity of the plasmids in the two groups was veryfied,thus we can infer the specific target site of miR-197 in GAB2 mRNA.（3）GAB2 plasmids lack of corresponding binding sites were introduced into the A172 cells transfected by pre-miR-197 or control miR,Western blot analysis was used for detecting the differences of GAB2 expression in the two groups of cells.7.Statistical analysis:Each experiment was repeated at least three times.All results are expressed as the mean±standard deviation.Studentrs t-test or the x2 test were adopted to analyse the difference between the means.All statistical analyseswere implemented with SPSS 16.0 software（SPSS,Inc.）,and P<0.05 was ragarded as a statistically significant difference.Results1.RT-qPCR demonstrated that the expression level of miR-197 was significantly increased in the A172 cells transfected by pre-miR-197（P<0.01）,compared with the control group.Pre-miR-197 could steadily transfect A172 cells;Western blot analysis proved that GAB2-expressing plasmids increased the expression level of GAB2 protein in A172 cells as compared to that transfected by empty vector plasmids,and the GAB2-expressing plasmids could steadily transfect the A172 cells.2.It was demonstrated by In situ hybridization that the expression level of miR-197 in the tissues of glioma and adjacent normal brain tissues was significantly reduced compared with that of adjacent normal brain tissues（p<0.05）.3.Cell counting assay revealed that compared to control group and untreated group,cell activity ratios at each time point in A172 cells transfected with pre-miR-197 were obviously lower（P<0.01）.Cell cycle analysis showed that A172 cells transfected with pre-miR-197 manifested a lower phase ratio of S phase（p<0.05）;Colony formation assay displayed that there was a significant difference in more than 50 cell colony counts between two groups of A172 cells transfected with pre-miR-197 and control miR（P<0.01）,miR-197 significant inhibited colony formation in A172 cells.BrdU incorporation assay showed that DNA synthesis level was significantly different in 24 hours and 48 hours between the two groups of A172 cells transfected with pre-miR-197 and that with control miR（P<0.05）,the group transfected with pre-miR-197 was lower than the control group.4.Western blot analysis showed that the difference of expression level of GAB2 between the glioma tissues and adjacent normal brain tissues was significant（p<0.05）.The expression level of GAB2 was higher in most specimens of higher glioma tissues.Immunohistochemistry demonstrated the expression level of GAB2 in grade Ⅰ,Ⅱ,Ⅲ and IV was23.68%,38.46%,59.09%and 75%respectively,the expression level of GAB2 in glioma at all grades was statistically significant（x2=24.053,P<0.001）,and the higher the grade of glioma,the higher the expression level of GAB2 was:grade Ⅰ vs grade Ⅱ（P<0.05）;grade Ⅱ vs.Ⅲ（P<0.05）;grade Ⅲ vs.Ⅳ,（P<0.05）.The number of A172 cells transfected by different doses（1nM,10nM,40nM）of GAB2-expressing plasmids was markedly different in MTT assay,and the larger the dose,the greater the relative number of A172 cells was.5.Correlation of miR-197 and GAB2（1）In the two groups of A172 cells transfected with pre□miR□ 197 and control miR,Western blot analysis revealed that the expression level of GAB2 protein was significantly reduced in the pre-miR-197 group（p<0.05）.Immunofluorescence analysis and mean fluorescence intensity determination indicated that there was a significant difference in GAB2 antibody immunofluorescence staining intensity between the two groups（P<0.01）,the level of GAB2 in the pre□miR□197 group was significantly lower than that in the control group.RT-qPCR confirmed that there was no significant difference in the expression levels of GAB2 mRNA in the two groups（P>0.05）.（2）In the two groups of A172 cells transfected by GAB2□ expressing plasmids and empty vector plasmids,Northern blot analysis demonstrated that the difference of the level of miR-197 expression between the two groups was significant（p<0.05）,the level in the group transfected with GAB2-expressing plasmids was lower than that in the control group.RT-qPCR also demonstrated that the level of miR-197 expression in the group transfected with GAB2-expressing plasmids was lower than the control group（P<0.01）.MicroRNA microarray analysis showed that the expression level of miR-197 was significantly reduced in the group transfected with GAB2-expressing plasmids.（3）The results of MTT assay and BrdU analysis confirmed together that in the GAB2/Pre-miR-197 and empty-Vector/Pre-miR-197 groups of cells,there was a significant difference in proliferative activity of A172 cells（P<0.01）,and the group of GAB2/pre-miR-197 was markedly higher than that of empty-vector/pre-miR-197 group;no significant difference occured between the Pre-miR-197/GAB2 and control-miR/GAB2 groups（P>0.05）.6.The target site of miR-197 in GAB2（1）TargetScan algorithm predicted the three possible target sites of miR-197 in GAB2 mRNA:3885-3891,1151-1158,2044-2050.（2）Luciferase reporter assay verified that luciferase reporters were carried out with three targeting sequences,three fluorescein plasmids were constructed:Wild-type-GAB2-WT-luc-1（containing 3885-3891）,GAB2-WT-luc-2（containing 1151-1158）,GAB2-WT-luc-3（containing 2044-2050）.After three luciferase plasmids were introduced into the two groups of A172 cells transfected with pre[口miR□ 197 and control miR,GAB2-WT-luc-1 and GAB2-WT-luc-3 plasmids luciferase activity showed no significant difference in the two groups of cells（P>0.05）,while GAB2-WT-luc-2 plasmids luciferase activity demonstrated significant difference in the two groups（p<0.05）,the Pre-miR-197 group was much lower than the control group.GAB2-MUT-luc-2 plasmid（mutant type）luciferase activity showed no significant difference in the two groups（P>0.05）.（3）The two groups of A172 cells transfected with pre-miR-197 and control miR were then treated with GAB2-expressing plasmids lacking predicted 3’-UTR sites respectively,Western blot analysis proved that the expression of GAB2 showed no obvious difference in the two groups（P>0.05）.Conclusion1.Expression of miR-197 is downregulated in glioma cells,and miR-197 inhibits cell proliferation in A172 cells.2.In glioma cells,GAB2 expression is upregulated and is positively correlated with glioma grade.GAB2 in A172 cells promoted cell proliferation.3.The expression of miR-197 in the glioma tissue cells was significantly inhibited by GAB2.In the A172 cells,the expression of GAB2 was down-regulated by the overexpression of miR-197 caused by the transfection of the pre-miR-197.The overexpression of GAB2 caused by the transfection of GAB2-expression plasmids inhibited the expression of miR-197 and further promoted cell proliferation.4.MiR-197 suppressed the protein expression levels of GAB2 by targeting its mRNA 3’-UTR at position 1151-1158,but mRNA expression levels remained unchanged.5.In the treatment of glioma,GAB2 acts as target protein of miR-197,down-regulation of GAB2 can be achieved by restoring or increasing the expression of miR-197,which can inhibit the proliferation of tumor cells,reduce invasiveness of glioma cells and represents a promising new method of targeted therapy for glioma. |
