| Glioma is a common brain malignant tumor in adults.The median survival time of patients is only 15-23 months,and the mortality rate is very high.In recent years,the disease onset age is gradually getting younger,and it has become one of the main diseases endangering human life and health.Glioma cells are characterized by invasive growth,invading deeply into the brain.There is no obvious tumor boundary,and it is hard to eradicate the tumor by radiotherapy,chemotherapy or surgery.The professionals believe that the occurrence and development of tumor is caused by somatic alienation induced by some mutations of key genes in molecular signaling pathways,but the main signaling pathways and regulatory mechanisms of glioma are still poorly understood.The in-depth development of high throughput transcriptome sequencing(RNA-seq)technology can quickly and comprehensively obtain the transcriptome information of target cells or tissues,which helps researchers to understand and explore the major genes involved from an overall regulatory network point of view.In this study,the whole transcriptome sequencing of glioma tissues and paracancerous tissues with consistent pathological segmentation was carried out,and the key genes and expression patterns that mediated the occurrence and development of gliomas were excavated through bioinformatics and molecular biological analysis,and their molecular regulation pathways were constructed,and the biological function was verified at the cellular and animal levels to elucidate the molecular mechanism of proliferation and invasion of gliomas.It aims to provide new ideas for the early diagnosis of glioma and the development of targeted drugs to improve the treatment strategy of glioma.The main findings are as follows:1.The whole transcriptome data information of glioma tissue and paracancerous tissue was obtained.3088 m RNAs,106 lnc RNAs,82 circ RNAs and 15 mi RNAs were differentially expressed by sequencing analysis,and 33 differential genes were obtained,including BOC,MAP4K4,SPOCK3,GPRASP1,KCNIP4,AK5,ZEB2,POU2F1,RAB,etc.Differentially expressed m RNA was significantly enriched in 19 signaling pathways,including Hh signaling pathway,glioma signaling pathway,pancreatic cancer signaling pathway,axon-oriented and other related pathways.These pathways are closely related to physiological and biochemical processes such as immune response,amino acid metabolism,cell proliferation,and protein transport.2.A key candidate gene BOC that regulates the development of glioma was discovered.By constructing the mi RNA-nc RNA-m RNA interaction network,it is found that BOC are at key nodes of multivariate networks,are highly expressed in glioma tissues,and have potential targeted binding sites with mi RNAs such as has-mi R-4763,has-mi R-6848,has-mi R-6860.These mi RNAs have transcripts of genes such as POU2F1 and TP53,which are closely related to tumorigenesis,suggesting that BOC may be involved in the development of gliomas.3.The expression pattern of BOC in different pathological subtypes,molecular subtypes and IDH mutations of glioma was clarified.BOC is in the Hh signaling pathway and its expression pattern is highly consistent with that of the effector gene SMO of this pathway.Both genes expression level are higher in the tumor core compared to the edge,and also higher in the mesenchymal gliomas than other subtypes(p<0.05).The expression of SHH,BOC,SMO,GLI,and HHIP in the Hh pathway of the IDH wild group was higher than that of the IDH mutation group(p<0.05).4.At the cellular level,the effect of BOC expression changes on glioma cell proliferation and invasion was determined.q RT-PCR testing showed that the expression level of BOC in tumor tissue in patients with glioma was higher than that of the parenchyma(p<0.05).The CCK8 assay of the U251 and A172 cell lines with knocking down BOC(KD-BOC)showed that the proliferation of cells reduced after KD-BOC transfection for 24 h,and the OD450 value decreased markedly after 96h(p<0.01);the BOC over-expressing(OE-BOC)U251 and A172 cell lines showed that the OD450 values of OE-BOC were significantly increased after transfection for 24 h,48h and 72h(p<0.01).Migration experiments showed that the migration capacity of KD-BOC cells was remarkably reduced,while that of OE-BOC cells was significantly enhanced(p<0.01).Invasion experiments showed that the invasion ability of KD-BOC cells was distinct weakened,while the invasion ability of OE-BOC cells was greatly improved(p<0.01).5.A molecular signaling pathway for BOC to regulate proliferation and invasion of gliomas was established.The KEGG clustering of the difference genes between the KD-BOC group and the control group were mainly localized in the axon-oriented,Hh signaling pathway,and glioma signaling pathway.The downregulation of BOC in KD-BOC cells has led to the downregulation of the expression of a number of key oncogenes in the signaling pathways such as Hh,RAS,MAPK and APOPTOSIS,including SMO,MAPK,RAS,ERK,RTK,P21 Ras,Gas1,PDGFR,etc.while the tumor suppressor genes PTEN and PTCH1 have been upregulated.q RT-PCR test confirmed that after U251 glioma cells knocked down BOC,the expression levels of SMO,HRAS,and MRAS m RNA decreased markedly(p<0.05),indicating that BOC promoted the proliferation and invasion of glioma cells by activating the expression of the Hh pathway effector protein gene SMO and the RAS signaling pathway key genes HRAS and MRAS.6.Knock-down BOC downregulated the expression of key proteins in the Hh pathway and RAS pathway,thereby inhibiting the glioma growth in nude mice.The tumor volume of the three groups,Control,KD-BOC and Vector was measured at different times after establishment of glioma model by injection of glioma cells subcutaneously in nude mice,and the tumor growth rate in the KD-BOC group began to slow down after 16 thday,and the tumor volume was remarkably reduced at 28 th day(p<0.01).There was no difference between control and vector groups(p>0.05).q RT-PCR detection showed that compared with the Control and Vector groups,the expression of EGFR,HRAS,MRAS,PIK3,SMO,and Gli m RNA in the KD-BOC group decreased prominently,while the expression of the tumor suppressor gene PTEN increased significantly(p<0.01).The WB results also showed that the expression of EGFR,MRAS,SMO and Gli proteins in the KD-BOC group decreased markedly(p<0.01),HRAS and PIK3 protein expression decreased(p<0.05),while PTEN protein expression increased significantly(p<0.01).It was confirmed that downregulating BOC exerts an inhibitory effect on tumors by inhibiting the expression of its downstream related proteins.In summary,through the transcriptome sequencing analysis,the key candidate gene BOC was found,and its key role in the development of glioma investigated.The BOC-SMO/RAS regulatory pathway that promoted the malignant progression of glioma was established,and the regulatory mechanism of BOC on the proliferation,invasion and migration of glioma was elucidated at the molecular,cellular and in vivo levels.The results of this study lay an important foundation for in-depth disclosure of the pathogenesis of glioma,the discovery of new drug targets,and even the strategy of improving treatment. |
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