Hsa_circ_0072309 enhances autophagy and TMZ sensitivity in glioblastoma

Abstract

Aims: Circular RNAs have been reported to play key roles in the progression of various cancers, including gliomas. The present study was designed to investigate the role of hsa_circ_0072309 in autophagy and temozolomide (TMZ) sensitivity in glioblastoma (GBM).

Methods: The effect of hsa_circ_0072309 on autophagy and TMZ sensitivity were examined by GFP-RFP-LC3, transmission electron microscopy(TEM), flow cytometry, Western blot, and immunofluorescence. The mechanism of hsa_circ_0072309 regulating p53 signaling pathway was analyzed using Western blot, IP, and rescue experiments.

Results: Low hsa_circ_0072309 expression predicts poor prognosis for glioma patients. The regulation of hsa_circ_0072309 on autophagy and TMZ sensitivity depends on the status of p53. Hsa_circ_0072309 promoted autophagy by p53 signaling pathway and enhanced sensitivity of glioblastoma to temozolomide (TMZ) in p53 wild-type GBM, but not in p53 mutant GBM. Hsa_circ_0072309 inhibits p53 ubiquitination and increases the stability of p53 protein in the context of p53 wild-type. MiR-100 mediates hsa_circ_0072309 regulating p53. P53 inhibitor or autophagy inhibitor could reverse the effect of hsa_circ_0072309 on TMZ sensitivity in p53 wild-type GBM.

Conclusions: This study revealed a function of hsa_circ_0072309 promoting autophagy by p53 signaling pathway and enhancing TMZ sensitivity. These findings demonstrated that hsa_circ_0072309 may be a potential and promising target in designing the treatment strategy for GBM.

Keywords: autophagy; circRNA; glioblastoma; temozolomide.

FIGURE 1

Low hsa_circ_0072309 expression predicts poor prognosis for glioma patients. (A) Kaplan–Meier survival analysis for hsa_circ_0072309 expression in glioma patients (n = 32). Low‐expression group: n = 16. High‐expression group: n = 16. P value was calculated by log rank test. (B) The expression pattern of hsa_circ_0072309 in a variety of human tissues from circAtlas database. (C) The expression pattern of LIFR (the host gene of hsa_circ_0072309) in a variety of human tissues from circAtlas database. (D)The junction ratio of hsa_circ_0072309 in a variety of human tissues from circAtlas database

FIGURE 2

Hsa_circ_0072309 promotes autophagy in p53 wild‐type GBM. (A) Autophagic flux was determined by diploid adenovirus (mRFP‐GFP‐LC3). Representative images of fluorescent LC3 puncta in U87‐PCDH and U87‐72309 cells are shown. Red dots represent autolysosomes and yellow dots autophagosomes, scale bars =10 μm. (B) U87‐PCDH and U87‐72309 cells were analyzed by transmission electron microscopy. Black arrow indicates autophagosome. Scale bars represent 5 μm and 1 μm. (C) Immunofluorescence was used to determine the effect of hsa_circ_0072309 overexpression on p62 in A172 cells. Scale bars =20 μm. (D) Western blot was used to determine the effect of hsa_circ_0072309 overexpression or depletion on the expression of ATG7, ATG16L1, Beclin‐1, P62, and LC3B in A172 cells. (E) Western blot was used to determine the effect of hsa_circ_0072309 overexpression or depletion on the expression of ATG7, ATG16L1, Beclin‐1, P62, and LC3B in U87 cells. A172/U87‐PCDH: the control group. A172/U87‐72309: the hsa_circ_0072309 overexpressed group. A172/U87‐shNC: the negative control group. A172/U87‐sh72309: the hsa_circ_0072309 knockdown group

FIGURE 3

Hsa_circ_0072309 enhances TMZ sensitivity in p53 wild‐type GBM. (A) Annexin V‐PE/7‐AAD staining and flow cytometric analysis were performed to detect the effect of hsa_circ_0072309 overexpression on TMZ‐induced apoptosis in A172 cells. *p < 0.05. (B) Annexin V‐PE/7‐AAD staining and flow cytometric analysis were performed to detect the effect of hsa_circ_0072309 depletion on TMZ‐induced apoptosis in A172 cells. *p < 0.05. (C) Western blot was used to determine the effect of hsa_circ_0072309 overexpression on the expression of Bax, caspase‐9, cleaved‐caspase‐3, total‐caspase‐3, and Bcl‐2 in A172 cells treated with TMZ. (D) Western blot was used to determine the effect of hsa_circ_0072309 overexpression on the expression of Bax, caspase‐9, cleaved‐caspase‐3, total‐caspase‐3, and Bcl‐2 in U87 treated with TMZ cells. The graphs are representative of three independent experiments with similar results. A172/U87‐PCDH: the control group. A172/U87‐72309: the hsa_circ_0072309 overexpressed group. A172‐shNC: the negative control group. A172‐sh72309: the hsa_circ_0072309 knockdown group

FIGURE 4

Hsa_circ_0072309 had no significant effect on autophagy, TMZ sensitivity, and p53 expression in p53 mutant GBM. (A) Western blot was used to determine the effect of hsa_circ_0072309 overexpression or depletion on the expression of ATG7, Beclin‐1, P62, and LC3B in U251 cells. (B) Immunofluorescence was used to determine the effect of hsa_circ_0072309 overexpression on p62 in U251 cells. scale bars =20 μm. (C) Annexin V‐PE/7‐AAD staining and flow cytometric analysis were performed when the hsa_circ_0072309 overexpressed group and the control group were treated with TMZ; ns, not significant. U251‐PCDH: the control group. U251‐72309: the hsa_circ_0072309 overexpressed group. U251‐shNC: the negative control group. U251‐sh72309: the hsa_circ_0072309 knockdown group. (D) Western blot was performed to determine the effect of hsa_circ_0072309 overexpression on the expression of p53 in U251 cells treated with or without TMZ

FIGURE 5

Hsa_circ_0072309 increases the stability of p53 protein by inhibiting p53 ubiquitination via miR‐100 in the context of wild‐type p53. (A) Western blot was performed to determine the effect of hsa_circ_0072309 overexpression on the expression of p53, MDM2, ATG16L1, ATG7, LC3B, and p62 in U87 cells treated with or without TMZ. (B) Immunofluorescence was used to determine the effect of hsa_circ_0072309 depletion on p53 in U87 cells. scale bars =50 μm. (C) A cycloheximide (CHX)‐chase experiment was performed to compare the rate of p53 degradation between the hsa_circ_0072309 overexpressed group and the control group. (D) Western blot was used to detect the level of p53 when the hsa_circ_0072309 depletion group and the control group were treated with MG132 or not. (E) U87‐PCDH and U87‐72309 cells were transfected with His‐ubiquitin, followed by treatment of MG132. The proteins were immunoprecipitated using anti‐p53 antibody, followed by immunoblotting with anti‐ubiquitin antibodies. (F) Immunofluorescence was performed to determine the expression of p53 in U87‐PCDH and U87‐72309 cells transfected with miR‐100 mimic or NC mimic. The graphs are representative of three independent experiments with similar results. U87‐PCDH: the control group. U87‐72309: the hsa_circ_0072309 overexpressed group. U87‐shNC: the negative control group. U87‐sh72309: the hsa_circ_0072309 knockdown group

FIGURE 6

Hsa_circ_0072309 promotes autophagy by p53 signaling pathway. (A) The hsa_circ_0072309 overexpressed cells and the control cells were treated with pifithrin‐α(PFT‐α) or not. Autophagic flux was determined by diploid adenovirus (mRFP‐GFP‐LC3). Representative images of fluorescent LC3 puncta are shown. Red dots represent autolysosomes and yellow dots autophagosomes, scale bars =10 μm. (B) The statistics of LC3 puncta in Figure 6A. (C) Western blot was used to detect the protein level of ATG16L1, ATG7, and p62 when the hsa_circ_0072309 overexpressed cells and the control cells were treated with pifithrin‐αor not. The graphs are representative of three independent experiments with similar results. U87‐PCDH: the control group. U87‐72309: the hsa_circ_0072309 overexpressed group. (D) The quantification of Figure 6C

FIGURE 7

Pifithrin‐α or 3‐MA could reverse the effect of hsa_circ_0072309 on TMZ sensitivity. (A) Annexin V‐PE/7‐AAD staining and flow cytometric analysis were performed when the hsa_circ_0072309 overexpressed group and the control group were treated with TMZ or TMZ+3‐MA or TMZ+Pft‐αor not. *p < 0.05. (B) The statistics of apoptosis rates in Figure 7A. The graphs are representative of three independent experiments with similar results. U87‐PCDH: the control group. U87‐72309: the hsa_circ_0072309 overexpressed group

FIGURE 8

Hsa_circ_0072309 promotes the autophagy of glioblastoma in vivo. (A) HE and IHC analyses of p53, p62, and LC3B in orthotopic tumor sections. (B) The weight of tumors was analyzed. *p < 0.05. (C) Mouse survival is shown by Kaplan–Meier curves. P value was calculated by log rank test. (D) Mechanistic model for hsa_circ_0072309 regulating autophagy and TMZ sensitivity. In the context of p53 wild type, hsa_circ_0072309 may inhibits p53 ubiquitination and prevents p53 degradation via sponging miR‐100. With the amount of p53 increased, autophagic cell death is activated and the sensitivity to TMZ is enhanced