| BackgroundGliomas are the most common and serious primary brain tumors with highly aggressive and neurodestructive properties and poor prognosis.At present,the treatment of glioma includes surgery,radiotherapy and chemotherapy.Simple surgical treatment does not prolong the survival period,and radiotherapy has been proved to prolong the median survival period of 6-8 months.Therefore,radiotherapy plays an important role in the treatment of brain tumors,but most of the patients receiving radiotherapy will still recur in the vicinity of the primary tumor.It is very important to explore the cellular and molecular mechanism of improving the therapeutic effect of glioma.Epigenetic silencing of tumor suppressor genes can affect signal transduction,adhesion and invasion,cell cycle,angiogenesis,DNA repair and apoptosis of tumor cells.DNA methylation is a common form of epigenetic silencing,in which DNA methyltransferase(DNMT)is the key enzyme responsible for the establishment and maintenance of DNA methylation.UHRF1(ubiquitin-like wih PHD and ring finger domains1)has recently been shown to play an important role in the maintenance of methylation,which can induce the recruitment of DNMT and protein deacetylase to DNA by combining the role of methylation of DNA.There is growing evidence that UHRF1 is an important oncogene.It was found that UHRF1 regulates gene expression and chromatin modification by interacting with DNMT 1 and histone deacetylase 1.Although UHRF1 plays an important role in DNA methylation,the role of UHRF1 in glioblastoma is rarely reported,and the specific expression pattern is still unclear.Therefore,this study firstly used real-time quantitative PCR(QPCR)to detect the UHRF1 mRNA level in glioblastoma tissue,and further analyze the relationship between the UHRF1 expression and the common clinicopathological parameters of glioma,and the effect of the expression of UHRF1 on the proliferation and apoptosis of glioma U251 cells by plasmid transfection is observed.These results provide a theoretical basis for the pathogenesis of glioma and target therapy.Objective1.To determine whether the expression of UHRF1 in brain glioma tissue is abnormal and the specific expression pattern.2.To analyze the correlation between the mRNA level of UHRF1 and the clinicopathological parameters of glioma,and further determine whether UHRF1 is involved in the occurrence and development of glioma.3.The effect of silencing UHRF1 expression in vitro on the proliferation and apoptosis of glioma cells.MethodsForty-four cases of glioma and 37 normal brain tissues were collected from January 2014 to December 2016 in our hospital.The UHRF1 mRNA levels in the above tissues were detected by real-time fluorescent quantitative PCR(QPCR).The level of UHRF1 mRNA in glioma tissues and the main clinical pathological parameters(age,sex and pathology of brain glioma)were analyzed.The relationship between type,differentiation,pathological grade,peritumoral edema and depth of infiltration were analyzed.The efficacy of tissue UHRF1 mRNA to differentiate glioma from normal brain tissue and different clinicopathological parameters of brain glioma was analyzed by the receiver operating characteristic(ROC).The plasmid LV-UHRF1-shRNA and negative control plasmid LV-scramble-shRNA were transfected into U251 cells(LV-UHRF1-shRNA group and LV-scramble-shRNA group)using lipid Attractene reagent,respectively.Cells only transfected liposomes were used as the control group.The UHRF1 mRNA level was detected by real-time fluorescent quantitative PCR(QPCR)at 48 h after transfection.MTT method was used to detect the proliferation of 24,48 and 72 h in each group.BrdU staining was used to detect the positive rate of BrdU after transfection of 12,24,48,72 h,and Annexin V-FITC/PI double staining was used to detect the apoptotic rate at 48 and 72 h after transfection via flow cytometry.Western blotting was used to detect the levels of apoptosis related proteins(Bax,Caspase-3 and Bcl-2)at 72 h after transfection.Results1.The level of UHRF1 mRNA in 37 normal brain tissues and 44 glioma tissues was detected by QPCR.The results showed that the level of UHRF1 mRNA in glioma tissues was 2.668±1.856,higher than 1.217±0.741 in normal brain tissues(P<0.05).The area under curve(AUC)of UHRF1 mRNA was 0.735(95%CI:0.628-0.843,P<0.001).When the cut-off value was 2.895,the highest Jordan index was 0.386 with the sensitivity of 38.64%and the specificity of 100.00%.2.The level of UHRF1 mRNA was not correlated with age,sex,pathological type and peritumoral edema(P>0.05),but with differentiation,pathological grade and depth of invasion(P<0.05).The level of UHRF1 mRNA in poorly differentiated tissues was 3.526 ± 1.736,higher than 2.075±1.726 in high and moderately differentiated tissues.The level of UHRF1 mRNA in pathological stages ? and ?was 3.182 ± 1.759,higher than 1.993 ± 1.803 in stage ? and ?.The level of UHRF1 mRNA in tissues with depth of invasion>1/2 was 3.562 ± 1.903,higher than 2.106 ±1.618 in tissues with depth of invasion<1/2.The difference was statistically significant(P<0.05).3.Using the average value of UHRF1 mRNA in glioma tissue(2.668)as the threshold,44 patients were divided into low expression group(25 cases)and high expression group(19 cases).This study analyzed the relationship between UHRF1 mRNA level and age,sex,pathological type,differentiation degree,pathological grade,peritumoral edema and depth of invasion.The results showed that the distribution of UHRF1 mRNA level was not related to age,sex,pathological type and peritumoral edema(P>0.05),but related to differentiation degree,pathological grade and depth of invasion(P>0.05).The high expression rate of UHRF1 was 66.67%(12/18)in poorly differentiated tissues,higher than 26.92%(7/26)in high and moderately differentiated tissues.The high expression rate of UHRF1 was 56.00%(14/25)in pathological stage III and IV,higher than 26.32%(5/19)in stage ? and ?.The high expression rate of UHRF1 was 64.71%(11/17)in dtissues with depth of invasion>1/2,higher than 29.63%(8/27)in tissues with depth of invasion ? 1/2.The above differences were statistically significant(P<0.05).4.The results of QPCR showed that the levels of UHRF1 in the control group,LV-scramble,shRNA group and LV-UHRF1-shRNA group were 1.098±0.136,1.127±0.193 and 0.309 ± 0.073),respectively.The levels of UHRF1 in the LV-UHRF1-shRNA group were lower than those in the control group and LV-scramble-shRNA group(P<0.05).The difference was not statistically significant(P>0.05).5.MTT assay was used to detect the proliferation of U251 cells in three groups at 0,24,48 and 72 h after transfection.The results showed that the proliferation rate of U251 cells in LV-UHRF1-shRNA group was significantly lower than that in control group and LV-scramble-shRNA group(P<0.05).There was no significant difference in the proliferation rate between the control group and the LV-scramble-shRNA group at the time point above(P>0.05).6.BrdU positive rate of U251 cells was detected by BrdU staining at 12,24,48 and 72 h after transfection.The results showed that the positive rate of BrdU in LV-UHRF1-shRNA group was significantly lower than that in control group and LV-scramble-shRNA group at the above time points(P<0.05).There was no significant difference in the positive rate between control group and LV-scramble-shRNA group(P>0.05).7.Annexin V-FITC/PI double-staining flow cytometry was used to detect the apoptosis rate of U251 cells at 72 h after transfection.The results showed that the apoptotic rates of control group,LV-scramble-shRNA group and LV-UHRF1-shRNA group were(7.73 ± 0.66)%,(7.86 ± 0.59)%and(25.71 ± 1.87)%,respectively.The apoptotic rate of LV-UHRF1-shRNA group was higher than that of control group and LV-scramble-shRNA group.There was no significant difference in the apoptotic rate between the control group and LV-scramble-shRNA group(P>0.05).8.Western blotting was used to detect the expression of apoptosis-related proteins in U251 cells of the three groups at 72 h after infection.The results showed that the levels of Bax and Caspase-3 in LV-UHRF 1-shRNA group were higher than those in control group and LV-scramble-shRNA group,while the level of Bcl-2 was lower(P<0.05).There was no significant difference in the level of related protein between the control group and LV-scramble-shRNA group(P>0.05).Conclusions1.The level of UHRF1 in brain gliomas is abnormally elevated,and it is effective in the diagnosis of brain gliomas.2.The elevated expression of UHRF1 is involved in the occurrence and development of glioma.3.Silencing UHRF1 expression can inhibit the proliferation of glioma and induce apoptosis. |
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