| Research purpose:To detect whether mi R-154 has expression function abnormality in glioma,to verify this and give proper interference at cytological level,to observe parameter changes of theglioma cell,and to clarify the biological effect of miR-154 in glioma.Materials and methods:In this study,88 intracerebralglioma patients with definite pathological diagnosis after operation were selected,in which 53 cases suffered high-level gliomas(WHO III &IV)and 35 cases suffered low-level gliomas(WHO I & II);normal brain tissues of control group came from 23 patients with severe craniocerebral injury.The differences between two groups of patients in gender composition and average age presented no statistical significance(P > 0.05),so there was comparability.The loss level of miR-154 gene and expression level of miR-154 in normal brain tissues,low-level gliomas and high-level gliomas were detected through Western blotting analysis and qPCR,and a comparative analysis was made via statistical methods to see the differences among three groups.Later,sensitivity test on Temozolomide treatment was conducted for glioma cell line U-138 MG after treatment with miR-154.U-138 MG was divided into three groups which were blank control group(only normal saline was added),negative group(miR-154 precursor was added)and experimental group(miR-154 mimic was added).A series of tests reflecting the growth activity of glioma cells was adopted to compare the biological activity changes of glioma cells before and after treatment with miR-154,and a comparative analysis was conducted via statistical methods.Results:First,our study found that the expression of miR-154 in surgery of patients with clinical glioma was significantly lower than that in normal brain tissues(P <0.05),among which lower levels(WHO I & II)and high level gliomas were reduced by 52.4%(P<0.05 and 79.9%(P<0.01)respectively,compared to normal brain tissues.By the comparison between gliomas tissues,it was found that the expression of mi R-154 in high level gliomas was significantly lower than that in low level gliomas,existing an obvious difference(P<0.05).However,it was observed in the low level and high level glioma chromosomal assays that the proportions of miR-154 gene loss in the two groups were 82.5% and 92.7%,respectively,no significant difference(P> 0.05).Thus,we detected the methylation ofmiR-154 gene near the transcription initiation(-14 to 0 bp).The level of methylation in the transcription initiation of high-grade glioma was found to be as high as 40±21.9%,while that of low-grade gliomaswas only 22.2 ± 24.8%(P <0.05).Then,we conducted a series of tests on the interaction between CCND2 and TLR-2in the tumor tissue of glioma patients.First,the method qPCR was used to evaluate the expression of CCND2 and TLR-2 in glioma cell line U-138 MG.When the effective concentrations of miR-154 mimic were 74.89 ±77.25 pM and 22,147 ±86.534 pM,respectively and the inhibitory rate of mRNA in CCND2 and TLR-2 is 50%(IC50),there was significant statistic difference between the two groups(P <0.01).In addition,statistical studies showed that when the effective concentration of miR-154 was 100 pM,mRNA in CCND2 and TLR-2 was inhibited(P <0.01).Based on the expression of miR-154 in normal brain tissue,after the treatment on cell line U-138 MG using miR-154 of 100 pM concentration,it was found that the protein expressions of CCND2 and TLR-2 were decreased by 53.7%(P <0.01)and 0.9%(P<0.05)compared with negative control group.Later,a series of experiments was conducted to test the growth activity of glioma cells in order to compare the differences in glioma cells before and after treatment with miR-154.Firstly,we observed the changes in sensitivity of cell line U-138 MG on temozolomide treatment: sensitivity of experimental cells transfected by mi R-154 to temozolomide was significantly increased,and the median lethal concentration of LC50 was reduced from 472.8 Um in the blank control group to 209.6 uM(P <0.01).Secondly,we compared the growth rate of U-138 MG before and after transfection.In the cell scratches assay,although miR-154 transfected experimental group had no significant difference with the blank group on the 2nd day(D2: P> 0.05),the growth rates on day 5and day 14 showed significant decrease compared with the blank control group(D5:P<0.05,D14: P<0.01).Then,we again compared the ability of miR-154 transfected experimental group cells to migrate: we found that the ability of miR-154 transfected experimental group to migrate reached 188±16 cell / mm2,significantly lower than that in the blank control Group of 225±28 cells / mm2(P <0.05).Finally,we compared the protein expression related to growth(EGFRvlll)and death(p53)in the tumor cells of two groups.It was found that in the experimental group of miR-154 transfection,relative expression volumes of U-138 MG cells EGFRvlll and p53 were 70% ± 7%(P <0.05)and 160% ±13%(P <0.05)of those in control group,respectivelyConclusions:It is finally discovered in the research that: 1)the expression level of miR-154 is closely related to pathological degree of gliomas,as 2)the major reason for the significant decrease of miR-154 expression level in glioma lies in the loss of miR-154 gene and excessive methylation in the transcription initiation of miR-154 gene.3)After miR-154 is supplemented,the malignant grade of glioma cells is restrained greatly.The results suggest that as a cancer suppressor gene,miR-154 can suppress tumors in glioma,so miR-154 can be treated as an effective spot of target therapy glioma,providing richer choices for glioma treatment in the future. |
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