| Background: Glioma is the most common primary malignant tumor of the central nervous system.Although great progress has been made in surgical treatment,radiotherapy and chemotherapy,the prognosis of patients with glioma is still not optimistic.Studies have shown that long non-codingRNA(lncRNA)plays an important role in the occurrence and development of tumors,especially in the process of tumor invasion and infiltration.The extracellular matrix(ECM)acts as a tissue barrier for tumor invasion and invasion,and it also plays an important role in the regulation of glioma invasion and invasion.At present,the role of ECM genes in gliomas and the mechanism by which lncRNA regulates ECM gene expression are still unclear.This study aimed to discover the ECM genes that play a key role in the occurrence and development of gliomas,construct a ceRNA(competing endogenousRNA,ceRNA)network that regulates key ECM genes,and explore the important regulatory mechanisms in the ceRNA network.Methods:1.Screening ECM genes closely related to the prognosis,immune microenvironment and EMT of glioma:(1)Key ECM genes closely related to WHO classification and prognosis of glioma was identified through weighted gene co-expression network analysis(WGCNA),(2)Validated their prognostic value of these ECM genes in the Oncomine,The Cancer Genome Atlas(TCGA)and Chinese Glioma Genome Atlas(CGGA)databases.(3)The correlation between the expression of these ECM genes and the stromal scores,immune scores,immunosuppressive cell recruitment factors,immunosuppressive factors,and the infiltration of various immune cells were further analyzed.(4)The correlation between these ECM genes and glioma epithelialmesenchymal transition(EMT)was also analyzed.2.Constructing a ceRNA network regulating these key ECM genes:We utilized the lncRNA and microRNA(miRNA)data in the TCGA database and the prediction results of the star Base database to construct a ceRNA network that regulates these ECM genes.3.LncRNA FOXD3-AS1 regulates the expression of ECM genes by competitively binding miR-128-3p:(1)RT-q PCR was used to detect the expression of FOXD3-AS1 in human normal astrocyte cell lines(HEB)and human glioma cell lines(HS683,SHG44 and A172).(2)After knocking down the expression of FOXD3-AS1 in glioma cell lines(HS683,SHG44 and A172)by transfection of siRNA,using CCK-8 proliferation experiment,clone formation experiment,would healing experiment,invasion experiment and Western blotting analysis Detect the changes of glioma cell proliferation,migration,invasion and EMT;(3)After overexpressing FOXD3-AS1 in the glioma cell line by plasmid,using CCK-8 proliferation experiment,clone formation experiment,would healing experiment,invasion experiment and Western blotting analysis to detect the proliferation,migration,invasion and EMT of glioma cell line(HS683 and SHG44);(4)FISH(fluorescence in situ hybridization,FISH)experiment was used for subcellular localization of FOXD-AS1;RNA immunoprecipitation(RIP)and luciferase reporter gene experiments were used to detect FOXD3-AS1 and miR-128-3p can be combined;(5)CCK-8 proliferation experiment,clone formation experiment,would healing experiment,invasion experiment and Western blotting analysis were used to detect the effects of miR-128-3p on the proliferation,migration,invasion and EMT of glioma cell lines(HS683 and SHG44).(6)Using CCK-8 proliferation experiment,clone formation experiment,would healing experiment,invasion experiment and Western blotting analysis to detect the proliferation,migration,invasion and EMT of glioma cell lines(HS683 and SHG44)by FOXD3-AS1/miR-128-3p axis;(7)Using fluorescein reporter gene experiment,RT-q PCR,Western blotting analysis to verify whether FN1 and COL3A1 are the target genes of miR-128-3p.(8)Western blotting analysis was used to detect whether the FOXD3-AS1/miR-128-3p axis can regulate the expression of multiple ECM genes.(9)Western blotting analysis and immunofluorescence experiment were used to verify whether the FOXD3-AS1/miR-128-3p axis can regulate the Wnt/β-catenin signaling pathway.(10)Detect the influence of FOXD3-AS1/miR-128-3p axis on the growth of glioma in vivo and the regulation of ECM gene through mouse subcutaneous transplantation tumor model.Results:1.Screening ECM genes closely related to the prognosis,immune microenvironment and EMT of glioma:(1)We screened out 9 key ECM genes(COL1A1,COL1A2,COL3A1,COL4A1,COL4A2,COL5A2,FN1,LAMB1 and LAMC1)closely related to the prognosis and the WHO classification of glioma.(2)It was also found that the expression of these ECM genes was significantly positively correlated with stromal and immune scores,the expression of immunosuppressive cell recruitment and immunosuppressive factors,and a variety of immune infiltrating cells.(3)It was also found that these ECM genes are involved in the EMT process of gliomas.2.Constructing a ceRNA network regulating these key ECM genes:A prognostic related ceRNA network that regulates these 9 ECM genes was constructed.3.LncRNA FOXD3-AS1 regulates the expression of ECM genes by competitively binding miR-128-3p:(1)The results of q RT-PCR showed that FOXD3-AS1 was highly expressed in glioma cell lines(HS683,SHG44 and A172);(2)After using siRNA to interfere with FOXD3-AS1,experiments showed that the proliferation,migration,invasion and EMT of glioma cell lines(HS683,SHG44 and A172)were significantly inhibited;(3)After the expression plasmid overexpressed FOXD3-AS1 in the glioma cell line,experiments showed that the proliferation,migration,invasion and EMT of the glioma cell line(HS683 and SHG44)were significantly enhanced;(4)RIP experiment and luciferase reporter gene experiment confirmed that FOXD3-AS1 and miR-128-3p can directly bind;(5)The results of experiments showed that miR-128-3p can inhibit the proliferation,migration,invasion and EMT of glioma cell lines(HS683and SHG44);(6)The results of experiments showed that the FOXD3-AS1/miR-128-3p axis can promote the proliferation,migration,invasion and EMT of glioma cell lines(HS683,SHG44 and A172);(7)Luciferase reporter gene experiment,RT-q PCR,Western blotting analysis confirmed that FN1 and COL3A1 are the target genes of miR-128-3p;(8)Western blotting analysis results showed that FOXD3-AS1/miR-128-3p axis can regulate the expression of multiple ECM genes(FN1,COL3A1,COL1A1,LAMB1 and LAMC1);(9)Western blotting analysis and immunofluorescence experiments showed that the FOXD3-AS1/miR-128-3p axis can regulate the Wnt/β-catenin signaling pathway;(10)In vivo experiments show that the FOXD3-AS1/miR-128-3p axis can regulate tumor growth and ECM gene expression.Conclusion: 1.The expression of ECM genes(COL1A1,COL1A2,COL3A1,COL4A1,COL4A2,COL5A2,FN1,LAMB1,and LAMC1)is negatively correlated with the prognosis,and can regulate the immunosuppressive microenvironment and participate in the EMT process of glioma.2.A prognostic related ceRNA network that regulates these ECMs was constructed,which could contribute to providing some new potential prognostic biomarkers or therapeutic targets for glioma research.3.FOXD3-AS1 can regulte the expression of multiple ECM genes(FN1,COL3A1,COL1A1,LAMB1 and LAMC1)and promote the progression of glioma by sponge miR-128-3p. |
PDF Full Text Request