| ObjectiveGlioma is the most common primary intracranial malignant tumor,and its treatment plan is still dominated by surgical resection,postoperative radiotherapy combined with temozolomide combined with adjuvant therapy.However,the recurrence of most gliomas is still inevitable.Although dozens of domestic and international drugs have entered the phase ii or iii clinical trials of recurrent gliomas,the drugs that can simultaneously improve the survival time and asymptomatic survival period have not been found.Apatinib,as a small-molecule anti-angiogenesis targeted drug,highly selectively competes for the atp-binding site of intracellular vegfr-2 and blocks the downstream signal transduction,which was first approved in gastric cancer and the effect was clinically recognized.Apatinib has also shown promise in clinical trials of patients with recurrent glioma.However,the mechanism of action of apatinib against glioma is not fully understood.At present,there are no reports on the inhibitory effect of apatinib on the proliferation,migration and invasion ability of glioma cells and the related mechanisms.The purpose of this study was to investigate the inhibitory effect of apatinib on proliferation,migration and invasion of glioma cells and the related mechanisms.Methods1.MTT assay was used to detect the effect of apatinib on the proliferation of N14069 cells and N14042 cells from patients,and the half-inhibitory concentration of apatinib on the proliferation of glioma cells was determined(ic-50).2.The effect of 250umol/l apatinib on the cell cycle of N14042 cells and N14069 cells was investigated by pi-facs experiment.3.Conduct the Matrigel chamber test to investigate the inhibitory effect of 250umol/l apatinib on the migration and invasion ability of N14042 cells and N14069 cells.4.After 72 hours of administration,total RNA was extracted from the administration group and the control group by Trizol method,and the differential genes in the administration group and the control group were searched by whole transcriptome microarray bioinformatics analysis.Protein was extracted from the cells of the drug administration group and the control group by lysis method,and the differential proteins of the two groups were searched by the relative quantitative proteomics method with multiple labels of equal weight isotopes.Through combined bioinformatic polymerization analysis,RNA and protein were clearly down-regulated at the same time,and this positive gene was probably the driver gene that promoted the proliferation of glioma cells.5.Western-blot and rt-pcr were used to verify the difference in the expression of the positive gene between the administration group and the control group.6.Design 3 gene sequences specifically binding to THBS1 gene fragments,identify and sequence them by PCR,and synthesize RNA interfering lentivirus vectors.After the titer was determined,three lentiviruses were transfected into N14069 and N14042 cells and the control group,respectively.The expression and knockout efficiency of THBS1 in N14069 and N14042 cells before and after transfection were detected by rt-pcr and fluorescence microscopy.Lentivirus with the highest knockdown efficiency was selected and the effects of down-regulation of THBS1 on proliferation and invasion function of glioma cells were investigated by cell cycle assay,MTT assay and invasion assay.7.Lentivirus containing THBS1 gene fragment was transfected into N14042 cells with virus enhancement solution,and blank and negative control groups were set up.The transfection efficiency of glioma cells before and after transfection was detected by fluorescence microscopy.Apatinib was cultured at 250umol/l for 36 h,and the effects of upregulation of THBS1 on proliferation and invasion of glioma cells were investigated by cell cycle assay,MTT assay,and invasion assay.Results1.MTT assay showed that apatinib had an inhibitory effect on the proliferation of N14069 and N14042 cells in vitro,with ic-50 of 239.4umol/l and 442.1ummol/l,respectively.After administration:at 96h and 120h,apatinib significantly inhibited the growth of N14042 and N14069 cells more than the control group,and the difference was statistically significant(P<0.01).The results of cell cycle experiment showed that the percentage of G1 cells in N14069 cells increased from 61.66%to 68.32%(P<0.01).The percentage of G1 cells in N14042 cells increased from 76.45%to 85.48%(P<0.01).However,in N14069 cells,the percentage of s-phase cells decreased from 15.22%to 8.45%(P<0.01).The percentage of s-phase cells in N14042 cells decreased from 8.51%to 5.4%(P<0.01).The invasion experiment results showed that compared with N14069 and N14042 cells in the control group,the number of N14069 and N14042 cells in the administration group invaded and invaded the basement membrane of the ventricle was significantly reduced(P<0.01).2.Whole transcriptome microarray bioinformatics analysis showed that apatinib down-regulated 380 genes in N14069 cells and 649 genes in 14042 cells,and 155 of these genes were overlapped.The isoweight isotope multi-label relative quantitative proteomics showed that there were 559 down-regulated proteins in the drug delivery group compared with the control group.The gene chip was selected to down-regulate up to 10 genes,which were combined with the upstream genes of the proteins down-regulated by proteomics,and a total of 9 genes were found to be duplicated,among which THBS1 was speculated to be the driving gene of apatinib acting on glioma cells due to the highest down-regulation.3.Western-blot results showed that the expression of THBS1 protein in N14069 cells and N14042 cells in the administration group was significantly lower than that in the control group(P<0.01).4.Rt-pcr showed the expression abundance in N14069 cells,and the ratio between the control group and the administration group was 1.004:0.187(P<0.01),and 1.003:0.060(P<0.01)in N14042 cells.5.Transfection of 3 lentiviruses with shrna-thbsl gene fragment into N14069 and N14042 cells showed that the expression level of THBS1 in glioma cells transfected with the third virus was higher than that of the control group by rt-pcr and fluorescence microscopy,and THBS1 gene knockdown efficiency reached 60.2%in N14069 cells(P<0.05).THBS1 gene knockout efficiency reached 48.2%in N14042 cells(P<0.05).6.After the down-regulation of THBS1 gene,MTT assay showed that cell proliferation decreased in the interference group(P<0.01).Cell cycle experiment results showed that the percentage of G1 cells in N14069 cells increased from 51.28%to 54.25%(P<0.01),and the percentage of S cells decreased from 14.11%to 13.29%(P<0.01).The percentage of G1 cells in N14042 cells increased from 58.68%to 62.95%(P<0.01),and the percentage of S cells decreased from 20.5%to 17.69%(P<0.01).The invasion experiment results showed that compared with N14069 and N14042 cells in the control group,the number of N14069 and N14042 cells in the interference group invaded and invaded the basement membrane of the ventricle was significantly reduced(P<0.01).7.After THBS1 gene overexpression,MTT assay showed that after apatinib culture,cells in the overexpression group reversed apatinib inhibition and increased proliferation compared with the negative control group(P<0.01).Cell cycle experiment results showed that:after apatinib culture,the overexpression group reversed the effect of apatinib,compared with the negative control group,the percentage of G1 cells in N14042 cells increased from 62.09%to 64.86%(P<0.01),and the percentage of S cells decreased from 11.09%to 9.55%(P<0.01).The percentage of G2/M cells decreased from 26.82%to 25.58%(P<0.05).The invasion experiment results showed that after apatinib culture,compared with N14042 cells in the negative control group,there was no statistical difference in the number of N14042 cells in the overexpression group invading through the invaded ventricular floor membrane(P>0.05).Conclusion(1)apatinib inhibits the proliferation and invasion of N14069 cells and N14042 cells,and blocks the progress of cell cycle.(2)the comparison of whole transcriptome microarray and proteomics microarray between the administration group and the control group suggested that apatinib may affect the proliferation capacity of N14069 cells and N14042 cells by down-regulating THBS1 gene,and significantly down-regulate the protein and mRNA expression of THBS1 gene in the administration group through the verification of western-blot and rt-pcr.(3)N14042 and N14069 cells after thbsl-shrna interference,cell proliferation assay,cell cycle assay and invasion assay showed that the proliferation and invasion ability of N14042 and N14069 cells were significantly inhibited after THBS1 expression was down-regulated.(4)after THBS1 overexpression,the effect of apatinib was reversed,and the proliferation ability of N14042 cells was enhanced,affecting the cell cycle.The cell invasion experiment showed that the overexpression of THBS1 did not significantly reverse the effect of apatinib,and there was no difference in the invasion ability of N14042 cells.(5)apatinib inhibits the proliferation and cell cycle of glioma cells by regulating THBS1 gene,thus affecting the growth of glioma. |
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