The Mechanism Of NPC2 Mediated IGFBP3 Regulating GBM Proliferation And Appoposis

Objective: Glioblastoma is one of the most common and fatal intracranial malignant tumors in human.Conventional treatment includes surgery,radiotherapy and chemotherapy,and TTF,but the prognosis of patients is poor,and the median survival time is only 15 months.In the brain,abnormal cholesterol signaling is closely related to the tumor genotype of glioma and the tissue environment in which tumor cells live,especially glioblastoma(GBM),which often has serious cholesterol metabolism disorder.NPC2(NPC intracellular cholesterol transporter 2)is an intracellular cholesterol transporter protein,which is abnormally expressed in a variety of tumors and is involved in lipid metabolism and innate immunity of tumors.However,its role in glioma is still unclear.In this paper,the expression differences of NPC2 in different patients were analyzed by bioinformatics and immunohistochemistry,the effect of NPC2 silencing on glioma was studied by cell proliferation and apoptosis experiments,and the transcriptional regulation mechanism of P53 signaling pathway and P53 transcription factor on IGFBP3 promoter was clarified by RNA-seq and Dual-luciferase experiments.Finally,it was confirmed that NPC2 may be an important regulatory factor of GBM,and the NPC2-p53-IGFBP3 axis may be a potential therapeutic target.Methods:1 The expression of NPC2 in GBM patients was queried by GEO,CGGA(China Glioma Genome Atlas Database),TCGA(Cancer Genome Atlas),the expression of NPC2 in GBM patients and normal people,the expression of patients with different levels of GBM,and the patients-related survival curve were queried.Thus,the relationship between NPC2 expression and prognosis of patients can be further explored;Real-time fluorescence quantitative PCR(RT-q PCR)was used to detect the m RNA expression of NPC2 in normal brain tissue and glioma samples.Immunohistochemistry(IHC)is used to detect differences of NPC2 protein expression levels in normal brain tissue and glioma samples;2 The protein and m RNA expression levels of NPC2 in NHA and glioma cell lines were detected by WB and PCR.We Selected the cell lines for the next experiments;U87 and LN229 cell lines were transfected with lentivirus which could knocked down NPC2 stably,and negative control lentivirus sh-NC was transfected at the same time.In order to verify knockout efficiency,we used RT-q PCR and Western blotting to detect the difference of NPC2 expression between sh-NPC2(experimental group)and sh-NC(control group),and screened out the virus sequences with the highest knockout efficiency;CCK-8and ANXIN V were used to detect the difference of cell proliferation and apoptosis between the experimental group and the control group;Glioma cells(sh NPC2 and sh NC)were injected into the intracranial of nude mice.Tumor volume was compared by HE staining,and the difference in survival time of nude mice was counted to detect the effect of NPC2 knockout on nude mice;3 RNA-seq(RNA transcriptome sequencing)was used to determine the changes of m RNA levels after NPC2 deletion,and the differential genes were enriched and analyzed to find downstream pathways.Dual-luciferase assay was used to detect the binding of transcription factor P53 to promoter IGFBP3.4 The protein and m RNA expression levels of P53 and IGFBP3 were detected by WB and PCR.The effect of NPC2 and P53 knockout on cell proliferation and apoptosis was detected.Results:1 According to GEO data,GBM expression level of NPC2 was significantly higher than that of normal brain tissue.According to CGGA and TCGA data,the expression level of NPC2 was negatively correlated with patient survival rate.The higher the expression level of NPC2,the worse the prognosis of patients with glioma.The pathological grade of glioma was positively correlated with the expression of NPC2;The results of RT-q PCR and IHC showed that the m RNA and protein expression levels of NPC2 in glioma specimens were higher than those in normal brain tissues.The results suggested that the pathological grade of glioma determined the expression level of NPC2.The higher the WHO grade of glioma,the higher the expression level of NPC2,and the two were positively correlated;2 The results of RT-q PCR and Western blotting showed that the expression levels of NPC2 in U87 and LN229 cells were higher than those in NHA(human astrocytes);The results of RT-q PCR and Western blotting showed that the expression of NPC2 in U87 and LN229 was significantly inhibited after transfection with lentivirus;Cell proliferation and apoptosis experiments showed that the proliferation ability of U87 and LN229 glioma cells decreasing and the apoptotic ratio increased after NPC2 knockdown.HE staining showed that the intracranial tumor volume was significantly reduced in the experimental group,and the survival curve showed that the survival time of the experimental group was prolonged.3 Compared with the control group,the gene expression in the experimental group increased by 1166 and decreased by 1015.Enrichment analysis showed that the differential genes were mainly enriched in P53 signaling pathway,and the P value was the minimum.Dual-luciferase showed that P53 promoter could activate IGFBP3 promoter,which was 517% higher than the control group.4 RT-q PCR and WB results showed that the expression levels of P53 and IGFBP3 were increased in both protein level and m RNA level after NPC2knockout;After knockout of NPC2 and P53,the proliferation capacity of the cells recovered and the apoptosis ratio decreased.Conclusion:1 The high expression of NPC2 has important significance in indicating poor prognosis of glioma.2 NPC2 regulates the proliferation and apoptosis of glioma.3 NPC2 regulates GBM cell proliferation and apoptosis by activating P53-IGFBP3 pathway.4 P53 silencing significantly reduced NPC2-dependent tumor growth and apoptosis inhibition.