Glioblastoma multiform is a grade IV brain tumor characterized by a heterogenous population of cells that are highly infiltratively, angiogenic and resistant to chemotherapy. As the most widely used chemotherapy drugs, TMZ has been proved to prolong the median survival of GBM patients obviously. However, long-term survival for GBM patients remains uncommon as cells with intrinsic or adapted resistance to TMZ repopulate the tumor. Many mechanisms, such as DNA damage response pathways, cancer stem cells, microenvironment-mediated chemotherapy resistance, tumor-derived endothelial cells, autophagy, were proved to play an important role in TMZ chemoresistance. However, the role of ceRNA networks in TMZ chemoresistance of glioblastomas was still not revealed completely.
The ceRNA hypothesis proposed in 2011 described an intricate post-transcriptional regulatory network that mainly includes lncRNAs, miRNAs, mRNAs, circular RNAs (circRNAs), and other types of RNAs. A number of ceRNAs have been identified to play an important role in the initiation and progression of glioma. The MIR155HG/has-miR-129-5p/C1S axis promoted the proliferation, migration and invasion ability of GBM cells and was a potential marker and therapeutic target for GBM patients. The circ-0043278/miRNA-638/ Homeobox A9 (HOXA9) axis had a vital function in promoting GBM progression. In this study, TMZ chemoresistance related lncRNAs, miRNAs and mRNAs were screened from public databases and the crosstalk networks were predicted and constructed, in finally, the survival analysis was performed to identify the TMZ chemotherapy associated biomarkers in glioblastomas.
In general, 4 mRNAs (LAMA1, COL5A1, ITGA2 and STK11) and 3 related KEGG pathways (hsa04510: Focal adhesion, hsa04512: ECM-receptor interaction, hsa04151: PI3K-Akt signaling pathway) were confirmed to play an important role in TMZ chemoresistance. LAMA1 and COL5A1 were upregulated, while ITGA2 and STK11 were downregulated in TMZ-resistant glioma cells. The gene expression of laminin subunit alpha 1 (LAMA1) was significantly higher in glioblastomas than in non-neoplastic white matter, verified by immunohistochemistry and real time quantitative PCR. By analyzing the expression profiles of recurrent low grade glioma (LGG) and primary LGG, collagen type V alpha 1 chain (COL5A1) was identified as one of the hub DEGs and might participate in the pathological process of recurrence. Furthermore, COL5A1 was a metastasis-associated gene in clear cell renal cell carcinoma and immune cell infiltration related gene in head and neck squamous cell carcinoma or gastric cancer[25-27]. Integrin subunit alpha 2 (ITGA2) was identified as a novel molecular target of GBM and its antibody blockage significantly impeded GBM cell migration. Serine/threonine kinase 11 (STK11) is one member of the serine/threonine kinase family, which is involved in regulating cell polarity, apoptosis, and DNA damage repair.
In addition to mRNAs, we screened 34 lncRNAs and 21 miRNAs through the ceRNA network. As shown in figure 5, miR-9-5p is one of the hub miRNAs in the network and may sensitize GBM cells to TMZ by downregulation of LAMA1 and COL5A1. Consistent with our prediction, miR-9-5p was reported to inhibit GBM cells proliferation by downregulating forkhead box P2 (FOXP2). Of the 34 lncRNAs, LUCAT1, TCL6, MIR4458HG and LINC00114 may play a vital role in TMZ chemotherapy resistance of GBM cells. Yansheng gao et al. revealed that LUCAT1 could act as a oncogenic lncRNA and promote glioma tumorigenesis via regulating miR-375.
Although we screened 4 genes as important biomarkers of TMZ chemoresistance in GBM and established the regulatory ceRNA network. There are a few limitations to this study. First, verification of ceRNA axis based only on public database is insufficient, further cell experiments are needed to confirm our results. Second, we evaluated genes’ effects on TMZ chemoresistance only by survival prognosis analysis based on TCGA database, prospective researches are needed to validate our results. Third, 6 KEGG pathways were overlapped between function enrichment analysis of prognosis associated DE-mRNAs and CTD database. The TMZ resistant cell lines’ expression levels of genes involved in 3 pathways (hsa04540: Gap junction, hsa04912: GnRH signaling pathway, hsa05202: Transcriptional misregulation in cancer) contradicted their prognosis in GBM patients. So we selected 3 KEGG pathways for further analysis.