LncRNA HOXA-AS3 promotes the malignancy of glioblastoma through regulating miR-455-5p/USP3 axis

Abstract

Our objective was to determine the molecular mechanisms by which lncRNA HOXA-AS3 regulates the biological behaviour of glioblastoma multiforme (GBM). We used an lncRNA microarray assay to identify GBM-related lncRNA expression profiles. Qrt-PCR was used to survey the levels of expression of long non-coding RNA (lncRNA) HOXA-AS3 and the target gene. Dual-luciferase reporter assays were used to investigate the interaction of lncRNA HOXA-AS3, the target gene and miRNA. Western blot analysis was used to examine the expression of USP3 and epithelial-mesenchymal transition (EMT) genes. The MTT assay, transwell assay and wound healing assay were used to analyse the effects of lncRNA HOXA-AS3 on GBM cell viability, mobility and invasiveness, respectively. Our results showed that lncRNA HOXA-AS3 was significantly up-regulated in GBM cells and could promote GBM cell proliferation, invasion and migration in vitro and in vivo. HOXA-AS was found to be associated with poor survival prognosis in glioma patients. The dual-luciferase reporter assay also revealed that lncRNA HOXA-AS3 acts as a mir-455-5p sponge by up-regulating USP3 expression to promote GBM progression. Western blot analysis showed that lncRNA HOXA-AS3 could up-regulate EMT-related gene expression in GBM. Experiments showed mir-455-5p could rescue the effect of lncRNA HOXA-AS3 on cell proliferation and invasion. The newly identified HOXA-AS3/mir-455-5p/USP3 pathway offers important clues to understanding the key mechanisms underlying the action of lncRNA HOXA-AS3 in glioblastoma.

Keywords: USP3; competing endogenous RNA; glioblastoma; lncRNA HOXA-AS3; miR-455-5p.

Figure 1

LncRNA HOXA‐AS3 was up‐regulated in glioblastoma patients and glioblastoma cells. A, Volcano plots show differentially expression levels of lncRNA in glioblastoma multiforme (GBM) tissues compared with normal tissues analysis. B, A heatmap is shown the expression of lncRNA for GBM samples (n = 502) compared to normal tissue samples (n = 5). Red represents lncRNAs that were up‐regulated in GBM. C, QRT‐PCR analysis of expression levels of lncRNA HOXA‐AS3 in GBM tissue and normal brain tissues (**P < .01). D, The survival rate between higher expression of lncRNA HOXA‐AS3 (n = 14) and lower expression of lncRNA HOXA‐AS3 (n = 11) in GBM patient was calculated by Kaplan‐Meier curve. E, QRT‐PCR analysis revealed lncRNA HOXA‐AS3 obviously up‐regulated in glioma cell lines compared with NHAs (*P < .05, **P < .01). F, lncRNA HOXA‐AS3 was mainly located in the cytoplasm of the GBM cells

Figure 2

Knock‐down of lncRNA HOXA‐AS3 reduces glioblastoma multiforme cell tumorigenicity and EMT process in vitro. A, The efficiency of lncRNA HOXA‐AS3 knock‐down was detected by qRT‐PCR (**P < .01). B, MTT experiments suggested that lncRNA HOXA‐AS3 knock‐down attenuated the proliferative capacity of LN229 cells (**P < .01, ***P < .001). C, MTT experiments suggested that lncRNA HOXA‐AS3 knock‐down attenuated the proliferative capacity of U251 cells (**P < .01, ***P < .001). D, Colony formation assay manifested lncRNA HOXA‐AS3 knock‐down reduces LN229 and U251 cells tumorigenicity (**P < .01). E, Transwell experiments revealed knock‐down of lncRNA HOXA‐AS3 inhibited LN229 and U251 cell invasion (**P < .01). F, The wound healing assay showed a significant decrease of LN229 and U251 cells migration after transfected lncRNA HOXA‐AS3 inhibitor (**P < .01). G, Western blot detected the different expression levels of EMT‐related gene (E‐cadherin N‐cadherin and vimentin) in si‐HOXA‐AS3 group and blank control group glioma cells (**P < .01, ***P < .001)

Figure 3

LncRNA HOXA‐AS3 depression inhibits glioblastoma tumorigenesis in vivo. A, The volume tumours formed by the LN229/sh‐HOXA‐AS3 cells were significantly smaller relative to those formed by the LN229/sh‐NC cells at the end of the experiment (*P < .05, **P < .01 and ***P < .001). B, Images of dissected tumours after the sh‐NC cells and sh‐HOXA‐AS3 cells were subcutaneous injection into the two groups of nude mice. C, The average weight significantly differed between the two groups (**P < .01). D, lncRNA HOXA‐AS3 expression of LN229/sh‐HOXA‐AS3 group was significantly lower relative to LN229/sh‐NC group in tumour tissues (**P < .01)

Figure 4

LncRNA HOXA‐AS3 acts as a miRNA sponge and negatively regulates miR‐455‐5p expression. A, Network diagram illustrates the regulatory correlation between lncRNA and miRNA based on the data from the bioinformatics software (Starbase v2.0). B, Schematic representation of the wild‐type or mutant miR‐455‐5p binding sequence predicted in lncRNA HOXA‐AS3. C, Luciferase activity analysis showed that lncRNA HOXA‐AS3 expression in miR‐455‐5p‐overexpression group or blank control group (*P < .05). D, qRT‐PCR revealed that knock‐down of lncRNA HOXA‐AS3 significantly up‐regulates miR‐455‐5p expression in LN229 and U251 cells (**P < .01). E, qRT‐PCR revealed that ectopic expression of miR‐455‐5p decrease lncRNA HOXA‐AS3 level in LN229 and U251 cells (*P < .05). F, The negative correlation between lncRNA HOXA‐AS3 and miR‐455‐5p in glioblastoma multiforme (GBM) tissues. G, The expression level of lncRNA HOXA‐AS3 was detected by qRT‐PCR after pull‐down (**P < .01). H, The expression level of miR‐455‐5p was assayed by qRT‐PCR after pull‐down (**P < .01). I, Co‐transfection of lncRNA HOXA‐AS3 3'‐UTR wild‐type or mutant seed region constructed with Over‐miR‐455‐5p in GBM cells (**P < .01)

Figure 5

USP3 is the target gene of miR‐455‐5p in glioblastoma multiforme (GBM). A, The Venn diagram shows the miR‐455‐5p target gene predicted by miRDB (red), Targetscan (green) and miRanda (blue). B, According to the binding site information provided by Targetscan, design USP3‐MUT or USP3‐WT to explore the interaction between USP3 and miR‐455‐5p. C, The luciferase activities of USP3‐WT and USP3‐MUT were measured in GBM cells transfected miR‐455‐5p mimics or miR‐NC with dual‐luciferase reporter assay (**P < .01). D, Pearson correlation showed the correlation between lncRNA HOXA‐AS3 and USP3 in GBM (**P < .01). E, Pearson correlation showed the correlation between miR‐455‐5p and USP3 in GBM. F, Effect of lncRNA HOXA‐AS3 and miR‐455‐5p interactions on USP3 expression was quantified by Western blot

Figure 6

MiR‐455‐5p rescued effect on the proliferation and invasion of lncRNA HOXA‐AS3 overexpression glioblastoma multiforme (GBM) cells. A, MTT experiments suggested that MiR‐455‐5p rescued the effect of lncRNA HOXA‐AS3 on migration of LN229 cell and U251 cell (**P < .01, ***P < .001). B, Transwell assay was conducted to evaluate the effect of miR‐455‐5p on the proliferation of lncRNA HOXA‐AS3 overexpressed LN229 cells and U251 cells (**P < .01, ***P < .001). C, Colony formation assay showed that miR‐455‐5p inhibited the cell proliferative capacity rescued by lncRNA HOXA‐AS3 (**P < .01). D, Representative images of tumour viewed by IVIS after control group, shRNA lncRNA HOXA‐AS3 group and miR‐455‐5p inhibitor group inoculation. E, Western blot analysis showed protein expression of E‐cadherin, N‐cadherin, vimentin and USP3 in orthotopic xenograft tumour lncRNA HOXA‐AS3 knock‐down group and NC control group (*P < .05,**P < .01 and ***P < .001)