Mechanism Of Comb Ined MTOR And Its Bypass Activation Pathway Blockade On Glioma Growth

OBJECTIVE:To study up-regulated genes and signaling pathways in glioma cells after knockdown of mTOR gene.Based on this,study the possibility of simultaneously blocking the mTOR signaling pathway and other signaling pathways with blockers of related signaling pathways at the glioma cellular level.Investigate the possibility of combined targeted therapy in glioma patients.METHODS: First selectedfive glioma cell lines including U87,U251,T98,LN229,U373 MG cells.Q RT-PCR was used to detect the m RNA expression in five human glioma cell lines.Then we used Western-Blotting to check the expression of mTOR protein.Then selected U87 and U251 stably transfected with sh RNA and dividedthem into blank control group,mTORsh RNA1 group and m TO Rsh RNA2 group.Western Blotting was used to double check whether m TO R being effectively blocked.Then high-throughput sequencing was used to perform deep coverage of exome sequencing with Illumina GA2000 and Hi Seq platforms to detects upregulated genes and signaling pathways in glioma cell lines after knockdown of the mTOR gene.Next,find the relevant literature,find the corresponding gene and pathway inhibitors for the differential genes selected above,and confirm the inhibition of glioma cell proliferation by cell dosing experiments.RESULTS:In primary glioblastoma,the expression of mTOR m RNA and m TO R protein was significantly higher in U87 and U251 cell lines compared to T98,LN229,and U373 MG cell lines.After successful establishment of glioma cell line stably transfected with sh RNA,RNA was extracted from glioma cell lines stably transfected with sh RN A,and high-throughput sequencing was used to screen out 24,528 new transcripts and 1906 new genes.The next step is to select the top 12 up-regulated genes with high log2 FC and FDR value that can be queried in the signaling pathways.Drug sensitivity assays screen out pathway inhibitors and confirm the inhibition of glioma cell proliferation by pathway inhibitors by CCK8 assay.CONCLUSION:Through the study of this subject,It was confirmed that some genes and signaling pathways of human glioma cells were up-regulated after knockdown,and the inhibitor of up-regulation signaling pathway was used to inhibit the proliferation of human glioma cells,which proved that the combined blocking of mTOR and its bypass pathway effectivelyinhibiting glioma cell proliferation was preliminarily demonstrated.