The Effect And Mechanism Of Targeting PBK On The Proliferation And Apoptosis Of Medulloblastoma

ProposeThis study aimed to explore the role of the key gene PBK in the development of medulloblastoma and the potential of PBK as a therapeutic target for medulloblastoma.MethodsBioinformatics analysis revealed the abnormal expression of the key gene PBK in different subtypes of medulloblastoma and its effect on the prognosis of each medulloblastoma.In vitro experiments,RT-qPCR and Western blot experiments were used to detect the primary expression of the hub gene PBK in medulloblastoma cell lines.EdU proliferation assay and Cell counting kit-8(CCK-8)proliferation assay were used to demonstrate the effect of PBK inhibitor HI-TOPK032 on the proliferation of medulloblastoma cell lines.The effect of PBK targeting inhibitor HITOPK032 on apoptosis of medulloblastoma cell lines was detected by Annexin VAPC/7-AAD double staining apoptosis kit.Western Blot was used to explore the downstream mechanism of PBK inhibitor HI-TOPK032 inhibiting the proliferation and inducing apoptosis of medulloblastoma cells.The PBK knockdown virus was used to construct stably transfected Daoy and D341 cell lines,and the transfection efficiency was verified by qPCR and Western blot experiments.The above in vitro functional experiments were used to verify the changes in the proliferation ability and apoptosis level of Daoy and D341 cells after PBK knockdown.In vivo experiments,the stereotaxic instrument and the microinjector were used to establish orthotopic models of human medulloblastoma,and a small animal in vivo optical imaging system was used to dectect the growth of medulloblastoma in the orthotopic xenograft mouse model.ResultsThe results of bioinformatics analysis showed that high expression of PBK was significantly associated with poor prognosis in non-WNT subtype medulloblastoma.EdU proliferation experiments,Cell counting kit-8(CCK-8)proliferation experiments proved that targeting PBK can inhibit the proliferation of medulloblastoma cells.The Annexin V-APC/7-AAD double staining apoptosis kit was used to detect cell apoptosis,and the results showed that targeting PBK could induce the apoptosis of medulloblastoma cells.The results of Western blot indicated that targeting PBK with the PBK inhibitor HI-TOPK-032 would reduce the phosphorylation levels of PBK downstream signaling molecules ERK1/2 and Akt and activate Cleaved-caspase3,thereby inhibiting the proliferation and inducing the apoptosis of Daoy and D341 cells.The results of EDU proliferation assay and CCK-8 proliferation assay showed that there was no significant difference in the proliferation ability of Daoy and D341 cells after knockdown of PBK.The results of apoptosis assay showed that the apoptosis level of D341 cells was significantly increased after PBK knockdown,while there was no significant difference in Daoy cell apoptosis level after PBK knockdown.The unexpected results of functional assays involved in PBK knockdown Daoy and D341 may be due to the low efficiency of PBK knockdown and the short experimental period in vitro.The results of optical imaging experiments showed that compared with the idling control Daoy,the proliferation ability of Daoy after the knockdown of PBK was significantly inhibited in the orthotopic mouse models.ConclusionTargeting PBK can inhibit the proliferation and induce apoptosis of medulloblastoma cells,and PBK may be a potential prognostic marker for medulloblastoma and an effective target for targeted therapy.