| Objective: Malignant glioma is one of the most common primary tumors in the brain.It is one of the most deadly tumors in humans.Although some progress has been made over the last few decades of treatment,brain surgery is still The efficiency of the main and the poor is poor,and the 5-year survival rate in Europe and the United States is less than 3%on average.Exploring new treatments has always been the pursuit of researchers.In recent years,scientists have found that non-coding miRNAs play an important role in the regulation of life.This topic is to look for mi RNAs that differ in expression between glioma cells and normal neurons,and by overexpression and interference Study of miRNA,to determine its impact on glioma cell-related functions,and to identify its possible signaling pathways and regulatory target genes,which is the purpose of this study.Methods: Eight cases of glioblastoma specimens were collected(3 cases of brain glioma surgery),3 cases of normal brain tissue(brain contusion and brain tissue after brain injury).Normal neuronal NHA and glioma cells SNB19,U87 MG,LN444,LN18,U251 MG,U118MG.The purity and concentration of miR-106 a were detected by QPCR technique.To construct a stable cell line expressing miR-106 a,a fragment of 200 bp upstream and downstream with miR-106 a precursor was amplified from DNA template.Lentiviral vector was constructed and packaged into lentivirus and then infected with glioma cells.Cell lines expressing miR-106 a and miR-106a-shRNA were used for the experiment: 1)MTT experiment,inoculated cells to 96-well plate,were cultured for 1-4 days,each hole by adding MTT solution color,select the 490 nm wavelength for color,record the results and draw the growth curve;2)cell cycle detection,with PI staining nuclei,with a certain excitation wavelength excitation to produce fluorescence,fluorescence intensity and intracellular DNA content is proportional to the excitation of the fluorescence group strength is identified by the machine,you can read the cell development period The level of change;3)invasive test,37? incubator,cultured 12 h,24h,36 h,remove the transwell chamber,each group repeated three samples.One of the cells discarded the medium,washed with calcium-free PBS three times,4%paraformaldehyde fixed for 10 min,cotton swabs to remove the upper layer of non-migratory cells,PBS washed 3 times,crystal violet staining,observed under a microscope;The remaining 3 cells removed the lower layer of the cells with 0.25%trypsin digestion,calculated the number of migrated cells,or taken several visual field counts to take the mean.4)The expression of Sanil,RUNX2,CD44,SOX9,OCT4 and ALP genes in the cells was detected.RNA was extracted from each group and the PCR reaction was carried out;5)the protein in each group was extracted,the protein concentration and purity were detected,and the standard curve was drawn.The contents of CyclinD1,p-pRb,pRb and MMP2 were detected by WB method;6)chromatin immunoprecipitation detection,extraction of nuclear DNA and ultrasonic treatment,the MMP2,Nanog,C-myc antibody incubation,dissociation of DNA and QPCR method for DNA content detection;7)the use of kit to detect the relative ratio of TOP/FOP,to detect ?-catenin into the nuclear level;8)luciferase detection of miR-106 a and APC mutual regulation,while the detection of APC-3 'mutant.Results: QPCR was detected in 3 cases of normal brain tissue and 8 cases of glioma tissues.The expression of 8 cases was higher than that of normal tissues(P<0.01).The levels of SNB19,U87 MG,LN444,LN18,U251 MG and U118 MG in glioma cells were statistically significant(F=0.233,P<0.01).MTT results suggest that the cells that attenuated miR-106 a were slower than controls and overexpressing mi R-106 a cells,whereas the cells that proliferated miR-106 a proliferated rapidly and grew from the number of cells that proliferated on day 4 The inhibition of miR-106 a reached a base of 2.8,while the control and overexpression of miR-106 a were 4.0 and 6.0,respectively.The results of cell cycle test showed that the control group G1/G0=60.46%,S=32.85%,G2/M=6.69%.Overexpression group G1/G0=32.29%,S=57.63%,G2/M=6.08%.The expression of G1/G0=73.71%,S=19.04%,G2/M=7.25%,and the S phase of overexpression group was significantly higher than that of the control group,and the cell proliferation was accelerated by overexpression of miR-106 a.The number of positive cells in the miR-106 a group was 3.53±0.13,which was significantly higher than that in the control group(T=-33.71,P<0.01),and the number of positive cells in the miR-106 a group was significantly higher than that in the control group significance.The positive rate of mi R-106 a cells was 0.21±0.05,which was less than that of the control group(T=27.37,P<0.01),which was statistically significant.The TOP/FOP results showed that the activity value of overexpressing miR-106 a group was 3.5±0.056,T=-74.66,P < 0.01.The inhibitory effect on miR-106 a group was 0.23±0.019,T=78.45,P<0.01.The results of CHIP showed that the expression of MMP2,Nanog and c-MyC was significant(P<0.01)compared with control group,overexpression and inhibition of mi R-106 a group.Compared with the control group,the expression of miR-106 a was 0.279±0.021(T=59.467,P<0.01),while that of the control group was 1.832±0.076(T=-18.961,P<0.01),While the mutant group was not statistically significant relative to the control group.Conclusion: Our experiments demonstrate that miR-106 a affects APC gene by targeting and affects the proliferation of human glioma cells through the WNT signaling pathway,which provides miR-106 a for the treatment of human glioma New ideas.All the statistics used by the statistical software SPSS 13.0 for data analysis.The results are expressed as mean±standard deviation((?|-)±s s).The two samples were compared with the T test,P<0.05 that the difference was significant. |
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