Over-expression of lncRNA TMEM161B-AS1 promotes the malignant biological behavior of glioma cells and the resistance to temozolomide via up-regulating the expression of multiple ferroptosis-related genes by sponging hsa-miR-27a-3p

Abstract

A growing body of evidence suggests that long-chain non-coding RNA (lncRNA) plays an important role in the malignant biological behavior and drug resistance of glioblastoma (GBM) cells. In this study, we analyzed the role and potential mechanism of lncRNA TMEM161B-AS1 in the malignant biological behavior of GBM cells and temozolomide (TMZ) resistance. Studies have found that FANCD2 and CD44 are significantly related to the occurrence of GBM, TMZ resistance and the survival of GBM patients. Knockdown of TMEM161B-AS1 down-regulated the expression of FANCD2 and CD44 by sponging hsa-miR-27a-3p, inhibited the proliferation, migration, invasion and promoted apoptosis, ferroptosis of U87 cells and U251 cells. Down-regulation of lncRNA TMEM161B-AS1 and/or over-expression of hsa-miR-27a-3p down-regulated the expression of FANCD2 and CD44, and inhibited the tumor growth in nude mice. These results demonstrated that the lncRNA TMEM161B-AS1-hsa-miR-27a-3p-FANCD2/CD44 signal axis regulated the malignant biological behavior of GBM and TMZ resistance. These findings were expected to provide promising therapeutic targets for the treatment of glioma.

Fig. 1. Differential expression analysis results of genes and non-coding RNAs related to glioma and temozolomide resistance.

a Volcano plot analysis of differentially expressed gene in the TCGA-GBM dataset. It contains data on 5 normal tissue samples and 156 tumor tissue samples. b Volcano plot analysis of differentially expressed genes in TMZ-sensitive and TMZ-resistant U251 glioma cell lines in the GSE100736 dataset. c Venn diagram of up-regulated gene distribution in TCGA-GBM dataset and GSE100736 dataset. d Analysis results of differentially expressed microRNAs in TMZ-resistant GBM cell line and TMZ-sensitive GBM cell line in the GSE100775 dataset. e The analysis results of differentially expressed lncRNAs in TMZ-sensitive and TMZ-resistant U251 glioma cell lines in GSE100736 dataset.

Fig. 2. Kaplan–Meier survival analysis for glioma patients with low and high gene expression in CGGA datasets.

a–l Comparison of Survival of Primary Glioma and Recurrent Glioma patients with high and low ZIC1, TMEM154, PRRX14, ITGA4, APOBEC3F, ADAM12, ANTXR2, PHLDA1, CALCRL, GALNT5, FANCD2, CD44 expression.

Fig. 3. Down-regulation of FANCD2 and CD44 promoted ferroptosis of U87 cells and U251 cells.

a, b Comparison of FANCD2 and CD44 gene expression level in patients with different WHO Grade gliomas. c, d The relative expression level of FANCD2 mRNA and CD44 mRNA in human astrocytes (HA), U87 and U251 cells were detected by qRT-PCR. e The relative expression levels of FANCD2 and CD44 in HA, U87 and U251 cells were detected by western blot. f, g The viability of U87 and U251 cells after sh-NC, sh-FANCD2-2, sh-CD44-2 transfection were detected by Cell Counting Kit-8 assay. h, i The migration and invasion ability of U87 and U251 cells after sh-NC, sh-FANCD2-2, sh-CD44-2 transfection were detected by Transwell assay. j The apoptosis rate of U87 and U251 cells after sh-NC, sh-FANCD2-2, sh-CD44-2 transfection were detected by Flow cytometry. k, l Comparison of the total iron levels and lipid ROS levels in U87 and U251 cells after sh-NC, sh-FANCD2-2, and sh-CD44-2 transfection. n = 3 per group.

Fig. 4. FANCD2 and CD44 silenced U87 cells and U251 cells were sensitive to TMZ.

a The relative expression levels of FANCD2 protein in the non-transfected group (Control), blank control (sh-NC), sh-FANCD2-1, sh-FANCD2-2 transfected U87 and U251 cells were detected by western blot. b The relative expression levels of CD44 protein in Control, sh-NC, sh-CD44-1, sh-CD44-2 transfected U87 and U251 cells were detected by western blot. c Comparison of Caspase 3/7 activities in TMZ treated or without TMZ (Ctrl) treated U87 and U251 cells after transfected with sh-NC, sh-FANCD2, and sh-CD44. d Comparison of LDH release in TMZ treated or Ctrl treated U87 and U251 cells after transfected with sh-NC, sh-FANCD2, and sh-CD44. *p < 0.05, compared with sh-NC group; **p < 0.01, compared with sh-NC group; #p < 0.05, compared with Ctrl group. n = 3 per group.

Fig. 5. Knockdown TMEM161B-AS1 inhibited the proliferation, migration, invasion of glioma cells and promoted apoptosis.

a The TMEM161B-AS1 levels in U87, U251 and human astrocytes (HA) cells were detected by qRT-PCR. b The TMEM161B-AS1 levels in the Control, si-NC, si-TMEM161B-AS1 transfected U87 and U251 cells. c, d The cell viability of U87, U251 cells transfected with Control, si-NC and si-TMEM161B-AS1 were analyzed by Cell Counting Kit-8 assay. e, f The cell migration and invasion ability of Control, si-NC, si-TMEM161B-AS1 transfected U87 and U251 cells were detected by Transwell assay. g The apoptosis rate of Control, si-NC, si-TMEM161B-AS1 transfected U87 and U251 cells were detected by Flow cytometry. n = 3 per group.

Fig. 6. TMEM161B-AS1 acted as a sponge of hsa-miR-27a-3p.

a Prediction of binding sites between TMEM161B-AS1 and hsa-miR-27a-3p by StarBase v2.0. b Compared of TMEM161B-AS1 and hsa-miR-27a-3p levels between RIP assay with antibody Ago2, IgG, or input from U87 cell extracts. *p < 0.05, compared with NC. c Compared of TMEM161B-AS1 and hsa-miR-27a-3p levels between RIP assay with antibody Ago2, IgG, or input from U251 cell extracts. *p < 0.05, compared with NC. d Luciferase reporter assay was performed to detect luciferase activity in U87 and U251 cells co-transfected with the constructed luciferase reporter plasmids (TMEM161B-AS1 wt or TMEM161B-AS1 mut) and hsa-miR-27a-3p angomir or no temple control (NC). *p < 0.05, compared with NC. n = 3 per group.

Fig. 7. The tumor suppressor effect caused by TMEM161B-AS1 knockout was mediated by hsa-miR-27a-3p.

a, b The hsa-miR-27a-3p, TMEM161B-AS1 expression level in U87 cells and U251 cells transfected by no template control (NC), si-TMEM161B-AS1 + hsa-miR-27a-3p antagomir, hsa-miR-27a-3p antagomir, si-TMEM161B-AS1 were detected by qRT-PCR. c, d Comparison of cell viability of U87 cells and U251 cells after transfected with NC, si-TMEM161B-AS1 + hsa-miR-27a-3p antagomir, hsa-miR-27a-3p antagomir, and si-TMEM161B-AS1. e, f Comparison of cell migration and invasion ability of U87 cells and U251 cells after transfected with NC, si-TMEM161B-AS1 + hsa-miR-27a-3p antagomir, hsa-miR-27a-3p antagomir, si-TMEM161B-AS1. g. Comparison of apoptosis rate of U87 cells and U251 cells after transfection of NC, si-TMEM161B-AS1 + hsa-miR-27a-3p antagomir, hsa-miR-27a-3p antagomir, si-TMEM161B-AS1. *p < 0.05, compared with NC, **p < 0.01, compared with NC. n = 3 per group.

Fig. 8. hsa-miR-27a-3p down-regulated the expression of FANCD2 and CD44, inhibited the proliferation, migration, invasion of glioma cells and promoted apoptosis and ferroptosis.

a, c The predicted wild-type or mutated hsa-miR-27a-3p binding sites in FANCD2 or CD44. b, d The regulatory relationship between hsa-miR-27a-3p and FANCD2 or CD44 was validated in luciferase reporter assay. e, f The transfection efficiency of U87 and U251 cells were estimated by qRT-PCR. g, h FANCD2 and CD44 expression level of U87 cells and U251 cells was estimated by Western Blot. i, j Influence of hsa-miR-27a-3p/FANCD2 axis and hsa-miR-27a-3p/CD44 axis on the proliferation of U87 and U251 cells was detected by Cell Counting Kit-8. k, l The migration and invasion ability of U87 and U251 cells were detected by Transwell assay. m Effects of hsa-miR-27a-3p/FANCD2 axis and hsa-miR-27a-3p/CD44 axis on apoptosis of U87 cells and U251 cells. n. Relative Total Iron level in U87 and U251 cells detected by Iron Assay Kit (Sigma Aldrich, Missouri, USA). o Lipid ROS was measured by C11-BODIPY staining coupled to flow cytometry in U87 and U251 cells. *p < 0.05, **p < 0.01, ***p < 0.001, compared with NC. n = 3 per group.