Long noncoding RNA AC003092.1 promotes temozolomide chemosensitivity through miR-195/TFPI-2 signaling modulation in glioblastoma

Abstract

Temozolomide (TMZ) and radiation therapy combination for glioblastoma (GB) patients has been considered as the most effective therapy after surgical procedure. However, the overall clinical prognosis remains unsatisfactory due to intrinsic or developing resistance to TMZ. Recently, increasing evidence suggested that long noncoding RNAs (lncRNAs) play a critical role in various biological processes of tumors, and have been implicated in resistance to various drugs. However, the role of lncRNAs in TMZ resistance is poorly understood. Here, we found that the expression of lncRNA AC003092.1 was markedly decreased in TMZ resistance (TR) of GB cells (U87TR and U251TR) compared with their parental cells (U87 and U251). In patients with glioma, low levels of lncRNA AC003092.1 were correlated with increased TMZ resistance, higher risk of relapse, and poor prognosis. Overexpression of lncRNA AC003092.1 enhances TMZ sensitivity, facilitates cell apoptosis, and inhibits cell proliferation in TMZ-resistant GB cells. In addition, we identified that lncRNA AC003092.1 regulates TMZ chemosensitivity through TFPI-2-mediated cell apoptosis in vitro and in vivo. Mechanistically, further investigation revealed that lncRNA AC003092.1 regulates TFPI-2 expression through miR-195 in GB. Taken together, these data suggest that lncRNA AC003092.1 could inhibit the function of miR-195 by acting as an endogenous CeRNA, leading to increased expression of TFPI-2; this promotes TMZ-induced apoptosis, thereby making GB cells more sensitive to TMZ. Our findings indicate that overexpression of lncRNA AC003092.1 may be a potential therapy to overcome TMZ resistance in GB patients.

Fig. 1. Expression of lncRNA AC003092.1 in GB cell lines and glioma tissues and its clinical significance.

a Top five significantly up- and downregulated lncRNAs and mRNA expression of their nearby genes (distance < 300 kb) in U87TR cells. b lncRNA AC003092.1 expression level in TMZ-resistant and its parental GB cells, ^*P < 0.05 compared with U87 or U251 cells. c lncRNA AC003092.1 expression in primary and relapsed GB tissues was examined by qRT-PCR and normalized against U6 expression, ^*P < 0.05 compared with primary GB tissues. d Kaplan–Meier overall survival curves according to high and low lncRNA AC003092.1 expression, P < 0.0001. lncRNA AC003092.1 expression level was assessed by qRT-PCR, and the median value for all 75 GB cases was chosen as the cutoff point with which the cases were separated into high (n = 37) and low (n = 38) lncRNA AC003092.1 expression group. The patients were classified into two groups according to lncRNA AC003092.1 expression; Kaplan–Meier overall survival curves according to lncRNA AC003092.1 expression level, P < 0.0001. Data are presented as the mean ± SD of three independent experiments

Fig. 2. Effects of lncRNA AC003092.1 on TMZ sensitivity, cell apoptosis, and proliferation in GB cells.

a The TMZ sensitivity of U87TR and U251TR cells after V-NC or V-AC transfection measured by CCK-8 assay. ^*P < 0.05 compared with V-NC group cells. b Cell viability of U87TR and U251TR V-NC or V-AC cells treated with 50 μg/ml TMZ for 24, 48, 72, 96, and 120 h. ^*P < 0.05 compared with V-NC group cells. c The TMZ sensitivity of U87 and U251 cells after treatment of si-NC or si-AC. ^*P < 0.05 compared with si-NC group cells. d Cell viability of U87 and U251 si-NC or si-AC cells treated with 50 μg/ml TMZ for 24, 48, 72, 96, and 120 h. ^*P < 0.05 compared with si-NC group cells. The rate of apoptosis of U87TR and U251TR V-NC or V-AC cells after treatment with or without 50 μg/ml TMZ for 48 h determined by FACS-based Annexin-V/FITC double staining (e–g) and TUNEL (h–j). ^*P < 0.05 compared with V-NC cells without TMZ treatment, ^&P < 0.05 compared with V-NC group cells with TMZ treatment. Scale bar = 50 μm. Data are presented as mean ± SD of three independent experiments

Fig. 3. TFPI-2 is the potential target of lncRNA AC003092.1.

a Co-expression analysis between protein-coding RNAs and lncRNA AC003092.1(ENST00000415536). Expression of TFPI-2 was highly positively correlated to lncRNA AC003092.1 expression. b mRNA level of TFPI-2 in U87, U87TR, U251, and U251TR cells measured by qRT-PCR. ^*P < 0.05 compared with U87 or U251 cells. TFPI-2 mRNA (c) and protein expression (d, e) in U87TR and U251TR after V-NC or V-AC transfection. ^*P < 0.05 compared with V-NC group cells. Expression of TFPI-2 mRNA (f) and protein (g, h) in U87 and U251 treated with si-NC or si-AC determined by qRT-PCR. ^*P < 0.05 compared with si-NC group cells. Data are presented as mean ± SD of three independent experiments

Fig. 4. lncRNA AC003092.1 enhances TMZ sensitivity through TFPI-2- mediated cell apoptosis.

a Cell viability of U87TR-V-AC and U251TR-V-AC cells after si-NC or si-TFPI-2 treatment measured by CCK-8 assay. ^*P < 0.05 compared with U87TR-V-AC + si-NC cells or U251TR-V-AC + si-NC cells. b The TMZ sensitivity of U87TR-V-AC and U251TR-V-AC cells after intervention of si-NC or si-TFPI-2 determined by CCK-8 assay. ^*P < 0.05 compared with si-NC-treated cells. The mRNA (c) and protein (d, g) level of TFPI-2 in U87TR and U251TR after transfection of V-NC or V-TFPI-2. ^*P < 0.05 compared with V-NC-treated cells. e Cell viability and (f) the TMZ sensitivity of U87TR and U251TR cells after V-NC or V-TFPI-2 transfection. ^*P < 0.05 compared with V-NC-treated cells. h The protein level of proapoptotic proteins (cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, and cleaved PARP) after V-NC or V-TFPI-2 treatment in U87TR and U251TR cells. ^*P < 0.05 compared with V-NC-treated cells. The ratio of apoptosis of U87TR-V-AC and U251TR-V-AC cells after si-NC or si-TFPI-2 transfection with or without 50 μg/ml TMZ treatment for 48 h determined by flow cytometry (i) and TUNEL (j). ^*P < 0.05 compared with si-NC-treated cells. ^&P < 0.05 compared with si-NC +TMZ-treated cells. Scale bar = 50 μm. Data are presented as mean ± SD of three independent experiments

Fig. 5. lncRNA AC003092.1 functions as ceRNA and negatively modulates miR-195 expression.

a Cellular localization of lncRNA AC003092.1 in U87, U87TR, U87TR-V-AC, U251, U251TR, and U251TR-V-AC cells by RNA-FISH assay. Scale bar = 50 μm. b, c Expression of miRNAs in U87TR and U251TR after V-NC or V-AC transfection measured by qRT-PCR. ^*P < 0.05 compared with U87TR-V-NC or U251TR-V-NC cells. d The luciferase activity of pMIR-lncRNA AC003092.1-Wt and pMIR-lncRNA AC003092.1-Mut after miR-195 or anti-miR-195 transfection in U87TR cells by dual-luciferase reporter assay. ^*P < 0.05. e The expression of lncRNA AC003092.1 and miR-195 in the complexes using anti-Ago2 antibody by RNA-binding protein immunoprecipitation assay. ^*P < 0.05 compared with anti-IgG group. Data are presented as mean ± SD of three independent experiments

Fig. 6. lncRNA AC003092.1 regulates GB TMZ chemosensitivity through miR-195/TFPI-2.

a Expression of miR-195 in U87, U87TR, U251, and U251TR cells measured by qRT-PCR. ^*P < 0.05 compared with U87 or U251 cells. b Expression of miR-195 in primary and relapsed GB tissues by qRT-PCR. ^*P < 0.05 compared with primary GB tissues. c The relationship of lncRNA AC003092.1 and miR-195 expression in GB tissues through qRT-PCR analysis. d Dual-luciferase assay was performed in U87TR cells transfected with luciferase construct alone or co-transfected with miR-195 and V-AC. Firefly luciferase containing a wild or mutant target site of TFPI-2 was constructed and transfected as indicated. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample. ^*P < 0.05. e Expression of TFPI-2 protein level after miR-control or miR-195 transfection in U87TR-V-AC or U251TR-V-AC cells. ^*P < 0.05 compared with miR-control treated cells. f Expression of TFPI-2 in non-tumor and GB tissues by immunohistochemistry (IHC) assay. Scale bar = 100 μm. g IHC scores of TFPI-2 in primary and relapsed GB tissues. ^*P < 0.05 compared with primary GB tissues. h, i The correlation analysis of TFPI-2 IHC scores and lncRNA AC003092.1 (h) or miR-195 (i) expression and in GB tissues. Data are presented as mean ± SD of three independent experiments

Fig. 7. lncRNA AC003092.1 overexpression enhances TMZ sensitivity in vivo.

a, b Photographs of tumors that developed in xenograft-transplanted nude mice tumor model after injection of U87TR-V-NC (1) or U87TR-V-AC (2) cells at 5 weeks treated with 5 μg/g TMZ or PBS. c Weights of tumor xenografts originated from U87TR-V-NC or U87TR-V-AC cells after treatment of TMZ or PBS at 5 weeks. ^*P < 0.05 compared with U87TR-V-NC with TMZ or PBS treatment. d Growth curve of U87TR-V-NC or U87TR-V-AC cells derived subcutaneous tumor xenografts after TMZ or PBS treatment. ^*P < 0.05 compared with U87TR-V-NC treated with TMZ or PBS. e Relative expression of lncRNA AC003092.1 and miR-195 in U87TR-V-NC or U87TR-V-AC cells derived tumor xenograft. f Immunohistochemistry analysis of TFPI-2 and C-PARP expression in U87TR-V-NC or U87TR-V-AC cells derived tumor xenograft. Scale bar = 100 μm. Data are presented as mean ± SD of three independent experiments