A Preliminary Study On The Role Of Notch1 In The Effect Of MicroRNA-146a On Human Glioma U251 Resistant Strains

Objective: 1. To explore the effect of micro RNA-146 a on human glioma U251 resistant strains. 2. To investigate the relationship between Notch1 gene and mi R-146 a in human glioma resistance.Methods: 1. U251 cell lines which steadily over-expressed NICD or no-loaded plvx were obtained by packaging lentivirus, transfection and screening. The expression of Notch1 in U251 cells was identified through the Western blotting and immunofluorescence staining. 2. Differentially expressed micro RNAs in each group cells were measured by micro RNA microarray. 3. The expression of mi RNA-146 a in each group cells was identified by RT-q PCR. 4. Using increasing concentrations of temozolomide(TMZ) mode of action for induction, the initial induction dose of TMZ 0.25μg/ml(0.2875μM), the highest induction dose of TMZ 20μg/ml(103.0μM), we got the stable TMZ resistant cell lines, named U251 TR. The inhibition rate of cells was detected by CCK-8 assay. The half inhibition concentration IC50 and resistance index(RF) were calculated. 5. RT-q PCR was used to detect the expression of mi RNA-146 a in U251 TR and control U251 cells. 6. Mi R-146a-mimics were transfected into U251 TR through the liposome-mediated way, named the U251TR-mimics, whose transfected efficiency was observed by fluorescence microscopy. The expressions of mi R-146 a in the U251 TR and U251TR-mimics were detected by RT-q PCR. The inhibition rate of the U251TR-mimics was measured using CCK-8 assay. The half inhibition concentration IC50 and resistance index(RF) were calculated. 7. The expression of Notch1 gene in the U251, U251 TR and U251TR-mimics cells was detected by Western blotting. 8. Bioinformatics prediction on mi RNA-146 a targeted regulating Notch1 was validated by the dual luciferase reporter assay and Western blotting.Results 1. Successfully obtained U251 cell lines which steadily over-expressed NICD or no-loaded plvx. 2. The results of micro RNA microarray showed that the expression of mi RNA-146 a in the over-expressed NICD U251 cells was significantly lower than the control group(P <0. 05). 3. The TMZ resistant cells U251 TR were established successfully, whose IC50(320.68μM) is 10.5 times higher than uninduced U251 cells’(30.54μM)(P <0. 05), the resistance index(RF) is about 11. 4. The results of RT-q PCR showed that the expressions of mi R-146 a in over-expressed NICD U251 cells and control cells are consistent with the micro RNA microarray; which also displayed that the relative expression of mi R-146 a in U251 TR is less than U251(P <0.05). 5. The transfected efficiency of mi R-146a-mimics in the U251TR-mimics group was above 90%; The IC50(160.79μM) of the U251TR-mimics was 5.26 times higher than the IC50(30.54μM) of U251 cells and the resistance index(RF) was about 5(P <0. 05). 6. The dual luciferase reporter assay and Western blotting showed that the role of mi R-146a-5p can obviously reduce the luciferase activity and down-regulate the expression of Notch1(P <0. 05).Conclusions: We successfully constructed TMZ resistant U251 TR cells, whose resistance index(RF) is about 10. The relative expression levels of mi R-146 a in U251 TR cells were lower than U251 cells; After mi R-146a-mimics were transfected into the U251 TR cells, the resistance index decreased. Dual luciferase reporter assay and Western blotting experiments have shown that mi R-146 a targeted regulating Notch1. mi R-146 a maybe play a role in the formation of human U251 glioma cells’ resistance to TMZ by targeting Notch1 signaling pathway.