Ribonuclease A Family Member 2 Promotes the Malignant Progression of Glioma Through the PI3K/Akt Signaling Pathway

Abstract

The treatment of patients with glioma still faces many difficulties. To further optimize treatment, it is necessary to identify more accurate markers as treatment targets and predict prognostic indicators. RNASE2 was identified as a differentially expressed gene (DEG) in glioma tissues using bioinformatics analysis. In glioma microarrays, 31.21% (54/173) and 68.79% (119/173) patients showed low and high RNASE2 protein expression levels, respectively. RNASE2 protein levels were considerably correlated with age, WHO grade, relapse, and death. Both mRNA and protein levels were associated with the overall survival of patients with glioma. To investigate the role of RNASE2, it was overexpressed or silenced in glioma cells. RNASE2 overexpression promoted cell proliferation, migration, and invasion. In addition, its overexpression promoted the growth of subcutaneous tumors and lung metastasis of glioma cells. Key protein levels in the PI3K/Akt signaling pathway were upregulated by RNASE2 overexpression. In contrast, RNASE2 knockdown had the opposite effects. Furthermore, LY294002 blocked the effects of RNASE2 on the cell function of glioma cells. In conclusion, RNASE2 is a novel marker associated with the diagnosis and prognosis of patients with glioma, and it promotes the malignant progression of gliomas through the PI3K/Akt signaling pathway.

Keywords: PI3K/AKT; RNASE2; glioma; knockdown; overexpression.

Figure 1

Correlation analysis between RNASE2 protein levels and patient prognosis. Based on the RNASE2 protein levels on the glioma tissue microarray, 173 patients with glioma were grouped into low (score <2) and high (score ≥2) expression groups. A Log-rank (Mantel-Cox) test was used to assess the correlation between RNASE2 levels and overall survival, or RNASE2 levels and disease-free survival.

Figure 2

RNASE2 overexpression promotes the proliferation of U87 MG and U251 MG cells, while RNASE2 knockdown suppresses it. RNASE2-overexpressing (ovRNASE2) and RNASE2-silenced (shRNASE2) cells were constructed. The ovNC and shNC groups were used as control cells. (A) Western blot showing successful RNASE2 protein overexpression and knock down. (B) Cell proliferation was promoted by RNASE2 overexpression and suppressed by RNASE2 knockdown after culture for 1, 2, 3, and 4 days. (C, D) The growth of subcutaneous tumors was promoted by RNASE2 overexpression and suppressed by RNASE2 knockdown. Panel (C) shows a photograph of the subcutaneous tumors. Panel (D) shows the statistical results of tumor volume after cell injection for 6, 12, 17, 23, and 26 days. # indicates p < 0.05; ovRNASE2 versus ovNC. & indicates p < 0.05; shRNASE2 versus shNC.

Figure 3

RNASE2 overexpression suppresses the apoptosis of U87 MG and U251 MG cells, while RNASE2 knockdown promotes it. RNASE2-overexpressing (ovRNASE2) and RNASE2-silenced (shRNASE2) cells were constructed. The ovNC and shNC groups were used as control cells. # indicates p < 0.05; ovRNASE2 versus ovNC. & indicates p < 0.05; shRNASE2 versus shNC.

Figure 4

RNASE2 overexpression promotes the migration and invasion of U87 MG and U251 MG cells, while RNASE2 knockdown suppresses it. RNASE2-overexpressing (ovRNASE2) and RNASE2-silenced (shRNASE2) cells were constructed. The ovNC and shNC groups were used as control cells. (A) Transwell assay showing that RNASE2 overexpression increased the number of migrating cells, while RNASE2 knockdown reduced it. (B) Transwell-Matrigel assay showing that RNASE2 overexpression increased the number of invasive cells, while RNASE2 knockdown reduced it. (C) Lung metastasis model in nude mice showing that RNASE2 overexpression increased the number of tumor loci in lung tissue, and that RNASE2 knockdown reduced it. # indicates p < 0.05; ovRNASE2 versus ovNC. & indicates p < 0.05; shRNASE2 versus shNC.

Figure 5

RNASE2 overexpression activates the PI3K/Akt signaling pathway, while RNASE2 knockdown inactivates it. RNASE2-overexpressing (ovRNASE2) and RNASE2-silenced (shRNASE2) cells were constructed. The ovNC or shNC groups were used as control cells. RNASE2 overexpression upregulated, while RNASE2 knockdown downregulated, the protein levels of p-AKT1 and PI3K p85. RNASE2 had no effect on total AKT1 levels.

Figure 6

LY294002, a PI3K inhibitor, blocks the effect of RNASE2 overexpression on the function of U87 MG and U251 MG cells. LY294002 was used to treat cells overexpressing RNASE2 (ovRNASE2+LY294002). DMSO-treated cells were used as a control (ovNC+DMSO, ovRNASE2+DMSO). LY294002 alleviated the effect of RNASE2 overexpression on proliferation (A), apoptosis (B), migration (C), and invasion (D). * indicates p < 0.05; ovRNASE2+LY294002 versus ovRNASE2+DMSO.