Abstract
Accumulating evidences revealed that long noncoding RNAs (lncRNAs) have been participated in cancer malignant progression, including glioblastoma multiforme (GBM). Despite much studies have found the precise biological role in the regulatory mechanisms of GBM, however the molecular mechanisms, particularly upstream mechanisms still need further elucidated. RT-QPCR, cell transfection, western blotting and bioinformatic analysis were executed to detect the expression of EGR1, HNF1A-AS1, miR-22-3p and ENO1 in GBM. Cell proliferation assay, colony formation assay, wound healing, migration and invasion assays were performed to detect the malignant characters of GBM cells. The molecular regulation mechanism was confirmed by luciferase reporter assay, ChIP and RIP. Finally, orthotopic mouse models were established to examine the effect of HNF1A-AS1 in vivo. In the current study, we analyzed clinical samples to show that the HNF1A-AS1 expression is upregulated and associated with poor patient survival in GBM. Functional studies revealed that HNF1A-AS1 knockdown markedly inhibits malignant phenotypes of GBM cells, whereas overexpression of HNF1A-AS1 exerts opposite effect. Mechanistically, the transcription factor EGR1 forced the HNF1A-AS1 expression by directly binding the promoter region of HNF1A-AS1. Furthermore, combined bioinformatics analysis with our mechanistic work, using luciferase reporter assays and RIP, we first demonstrated that HNF1A-AS1 functions as a competing endogenous RNA (ceRNA) with miR-22-3p to regulate ENO1 expression in GBM cells. HNF1A-AS1 directly binds to miR-22-3p and significantly inhibits miR-22-3p expression, while ENO1 expression was increased. miR-22-3p inhibitor offsets the HNF1A-AS1 silencing induced suppression in malignant behaviors of GBM cells. ENO1 was verified as a direct target of miR-22-3p and its expression levels was negatively with the prognosis in GBM patients. Taken together, our study illuminated the definite mechanism of HNF1A-AS1 in promoting GBM malignancy, and provided a novel therapeutic target for further clinical application.
Highlights
glioblastoma multiforme (GBM), the most aggressive subtype of glioma in adults, highly malignant and high risk of recurrence, accounting for 47.1% of all malignant tumors of the nervous system [1]
Colony formation assays found that HNF1A-AS1 was obviously upregulated in GBM, as indicated that the clone numbers and colony size was attenuated compared to low-grade glioma tissues and normal brain tissues, in the HNF1A-AS1 knockdown group, suggesting that depletion of but there was no significant difference between low-grade glioma HNF1A-AS1 slow down the growth of GBM cells (Fig. 2C)
Transwell assay showed that knockdown of indicated that HNF1A-AS1 was highly upregulated in four GBM cell HNF1A-AS1 significantly reduced migratory and invasive capacity lines in comparison with human astrocyte (HA) (Fig. 1B)
Summary
GBM, the most aggressive subtype of glioma in adults, highly malignant and high risk of recurrence, accounting for 47.1% of all malignant tumors of the nervous system [1]. In the light of the World Health Organization (WHO) classification of tumor in the central nervous system (CNS), GBM is classified as a grade IV glioma, with an unfavorable prognosis and a five-year overall survival rate less than 10% [2, 3]. An increasing number of reports have demonstrated that lncRNAs play a vital role in cancer progression, and are dysregulated in various human cancers, including GBM [15,16,17]. LncRNA AC016405.3 endpin cell proliferation and metastasis by regulating TET2, via sponging of miR-19a-5p in GBM cells [19]. LncRNA HOTAIRM1 is highly upregulated in GBM, which is positively correlated with tumor grade in patients with glioma, and aggravates the progression of GMB by regulating HOXA1 gene [20]. Despite several lncRNAs have been well studied, the functional mechanism of most lncRNAs in GBM remain largely unknow
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