Knockdown of lncRNA HOXD-AS2 Improves the Prognosis of Glioma Patients by Inhibiting the Proliferation and Migration of Glioma Cells

Abstract

Objective: Increasing studies reported that long noncoding RNAs are involved in regulating glioma progression. However, the specific roles and mechanisms of lncRNAs in glioma remain unclear. Here, we sought to explore the functions of HOXD-AS2 in glioma progression.

Methods: Gene expressions of lncRNAs in 5 normal brain tissue specimens and 5 glioblastoma tissue specimens were detected by gene expression profile chip technology. Bioinformatic analysis was performed to see whether differential expression of lncRNAs played any significant role in glioma occurrence and progression. The relationship between HOXD-AS2 level and clinical prognosis of the patients was analyzed. HOXD-AS2 was specifically interfered with by siRNA technology to observe its effects on U251 cell growth, proliferation, apoptosis, and invasion.

Results: The expression level of HOXD-AS2 gene in glioma was significantly higher than that in the normal brain tissue, which was related to the tumor grade. The level of HOXD-AS2 gene in patients with high-grade glioma was higher than that in patients with low-grade glioma. High expression of HOXD-AS2 gene was a risk factor for poor prognosis of glioma patients. Knocking down the expression of HOXD-AS2 in glioma cell line U251 arrested the cell cycle and reduced the cell proliferation. Furthermore, it could significantly reduce the migration ability of the cells but had no significant effect on the invasion.

Conclusion: HOXD-AS2 is an oncogenic lncRNA associated with the poor prognosis of glioma. Knockdown of HOXD-AS2 may reduce the growth of glioma, which may provide a new avenue for treatment.

Figure 1

Results of RT-qPCR showed good consistency with the microarray results.

Figure 2

Increased HOXD-AS2 expression confers poor prognosis in patients with glioma. (a) The relative expression values of HOXD-AS2/GAPDH in normal brain tissue and grade I, II, III, and IV gliomas were 0.0073 ± 0.0046, 0.0519 ± 0.0514, 1.3477 ± 2.2276, 1.9717 ± 2.9306, and 1.8884 ± 3.1063, respectively, showing significant differences between the normal brain tissue and tumor tissues of different grades (P < 0.05). (b) The Kaplan–Meier overall survival curves according to HOXD-AS2 expression level. Glioma patients with high HOXD-AS2 expression had significantly shorter median survival than patients with low HOXD-AS2 expression (P < 0.001). (c, d) Low HOXD-AS2 expression was significantly associated with longer median survival in patients with low-grade glioma (P < 0.05) but not in patients with high-grade glioma. NB, normal brain tissue; I–IV, I–IV glioma. ^∗P < 0.05.

Figure 3

Levels of expression of HOXD-AS2 in U251 cell lines after siRNA transfection. Knockdown of HOXD-AS2 inhibits U251 cell growth. (a) The levels of expression of HOXD-AS2 in three glioma cell lines were analyzed by RT-qPCR. (b) The levels of expression of HOXD-AS2 in the U251 cell line were analyzed by RT-PCR; the relative gene expression level of SiHOXD-AS2-1—48 hr, SiHOXD-AS2-2—48 hr, and SiHOXD-AS2-3—48 hr was 0.20 ± 0.04, 0.34 ± 0.09, and 0.83 ± 0.12. NC, negative control; ^∗∗P < 0.01. (c) Knockdown of HOXD-AS2 reduced the growth of U251 cells compared with the NC group in the CCK-8 assay. (d) U251 cells were transfected with siRNAs, and after 48 h, cells were stained and imaged under a fluorescence microscope. Scale bars represent 50 μm. (e) The ratio of EdU‐positive cells was calculated and plotted on the histogram. (f) Cell cycle profiling was analyzed using flow cytometry. The image shows the flow cytometry distribution of each cycle in the three groups of cells 48 h after transfection. (g) The proportion of cells in G1 phase in interference groups 1 and 2 was higher than that in the NC group, indicating that the number of cells in S phase and G2 phase was reduced significantly. Data were based on at least three independent experiments and shown as mean ± SD. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 4

Detection of apoptosis by flow cytometry. (a) The percentage of dead cells (UL), late apoptotic cells (UR), surviving cells (LL), and early apoptotic cells (LR) in the three cell groups was observed by flow cytometry. (b) The proportion of phase 4 cells in the three cell groups did not change significantly. Data were based on at least three independent experiments and shown as mean ± SD. ^∗P > 0.05.

Figure 5

Silencing of HOXD-AS2 inhibited cell migration in glioma cells. (a) Wound scratch assays were adopted to examine the migratory capacity. (b) The healing area in the SiHOXD-AS2-1 group and SiHOXD-AS2-2 group was significantly smaller than that in the NC group 36 and 72 h after scratching (^∗∗∗P < 0.001). (c) The invasion ability of U251 cells from different groups was detected in Transwell assays. (d) The number of invasive cells in the SiHOXD-AS2-1 group and SiHOXD-AS2-2 group vs. the NC group. Data were based on at least three independent experiments and shown as mean ± SD. ^∗P > 0.05.