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

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.

INTRODUCTION Glioblastoma (GBM) is a primary central nervous system tumor with the highest incidence, high malignancy, and poor prognosis in adults [1][2][3]. Currently, standard treatment of GBM is neurosurgical resection, combined with radiotherapy and chemotherapy [4][5][6][7][8]. Due to the diffuse invasive growth characteristics of GBM, the tumors cannot be completely removed by surgical means [9]. GBM stem cells (GSC) in GBM give tumors the characteristics of genetic heterogeneity and abnormal microvascular proliferation, making them resistant to radiotherapy and chemotherapy [10][11][12]. Temozolomide (TMZ) is the first-line for GBM [13,14], which can cross the blood-brain barrier and induce GBM cell apoptosis. Unfortunately, the clinical application of TMZ has been hampered by resistance of GBM cells [15,16]. Therefore, it is important to elucidate mechanisms of TMZ resistance in GBM.
MicroRNAs (miRNAs) are a class of short-stranded, evolutionarily conserved, single-stranded, non-coding RNA molecules. The mature miRNA is incorporated into the RNA-induced silencing complex (RISC), and then negatively regulate gene expression by binding to target mRNA to prevent protein translation of mRNA [17]. Researchers identified in the blood of glioma patients and found that hsa-miR-27a-3p and its target genes were related to the progression of glioma [18].
The Fanconi anemia complementation group D2 (FANCD2) gene is located on the human chromosome 3p25.3. FANCD2 is the core link of the Fanconi anemia (FA) signaling pathway, and the FA signaling pathway is the main signaling pathway for DNA crosslinking damage repair, which participates in DNA damage repair processes such as nucleotide excision repair, homologous recombination repair, and cross-damage synthesis repair [19]. Studies have shown that FANCD2 re-expression was associated with glioma grade and chemical inhibition of the Fanconi Anaemia pathway sensitises gliomas to chemotherapeutic agents [20]. This research provides a strong basis for developing novel and effective FA signaling pathway inhibitors to improve the treatment of GBM. Although FANCD2 was not up-regulated in TMZ-insensitive U251 cells according to the GSE100736 dataset, but FANCD2 was highly expressed in GBM tumor tissues, it was a ferroptosis-related gene [21], and FANCD2 overexpression improved the viability of TMZ-treated U87 cells [22]. Song et al. [23] and Fathima et al. [24] found that FANCD2 played an active role in the negative regulation of ferroptosis.
The CD44 gene is located on the human chromosome 11p13, and the CD44 protein is related to cell-cell interaction, cell adhesion and migration [25]. High expression of CD44 is associated with poor survival of GBM patients [26], and CD44 GBM is closely related to histopathological grade and cell migration in human [27]. These genes that were differentially expressed during the occurrence of GBM and related to TMZ resistance have attracted our attention. In particular, the FANCD2 gene and CD44 gene (Supplementary materials) [21,28] related to ferroptosis are particularly worthy of our focus on research.
TMZ resistance in GBM is related to different cellular pathways. For example, Wick et al. [29] found that high expression of O-6methylguanine-DNA methyltransferase (MGMT) is related to TMZ resistance. The results of Sun et al. [30] showed that the sensitivity of GBM cells to TMZ increased after prolyl 4-hydroxylase, β polypeptide (P4HB) was inhibited. Schäfer et al. [31] found that GBM resistance to TMZ was mediated by ALDH1A1, which was expected to become a potential target to improve treatment of GBM.
In this study, we analyzed the endogenous expression of lncRNA TMEM161B-AS1, hsa-miR-27a-3p, FANCD2, CD44 and the influence on the malignant biological behavior of GBM cells. Further, we also studied whether lncRNA TMEM161B-AS1 regulated the expression of FANCD2 and CD44 by regulating the expression of hsa-miR-27a-3p. This study aimed to study the possible function of lncRNA TMEM161B-AS1-hsa-miR-27a-3p-FANCD2/CD44 crosstalk in the malignant biological behavior of GBM and TMZ resistance.
Down-regulation of FANCD2 and CD44 expression is associated with ferroptosis in U87 and U251 cells In order to evaluate the effects of FANCD2 and CD44 gene on the proliferation, migration and apoptosis of U87 cells and U251 cells, we successfully constructed two kinds of short hairpin RNA (shRNA) for each gene, namely sh-FANCD2-1, sh-FANCD2-2, sh-CD44-1, sh-CD44-2 ( Figure S1), and the shRNAs with high knockout efficiency (sh-FANCD2-2, sh-CD44-2) were selected for subsequent experiments. According to the CGGA database, the expression levels of FANCD2 and CD44 were positively correlated with the grade of the tumor (Fig. 3a, b) [32,33]. The results showed that the relative expression levels of FANCD2 and CD44 genes in U87 and U251 cells were significantly higher than that in human astrocytes (HA) (Fig. 3c-e). Compared with the sh-NC group, the viability of U87 cells and U251 cells transfected with sh-FANCD2-2 and sh-CD44-2 were significantly decreased (Fig. 3f, g). The migration and invasion capabilities of U87 cells and U251 cells transfected with sh-FANCD2-2 and sh-CD44-2 were significantly decreased (Fig. 3h, i). The apoptosis rate, the concentration of iron and lipid ROS of U87 cells and U251 cells transfected with sh-FANCD2-2 and sh-CD44-2 increased significantly ( Fig. 3j-l).
The above research results indicated that FANCD2 and CD44 genes acted as the oncogenes in U87 cells and U251 cells, and silencing of FANCD2 and CD44 related to ferroptosis in U87 cells and U251 cells.
Silencing of FANCD2 and CD44 were related to the TMZ sensitivity in U87 cells and U251 cells Next, we studied the correlation between the silencing of FANCD2 and CD44 and the TMZ sensitivity in U87 cells and U251 cells. The results of qRT-PCR and Western blot proved that we had successfully silenced FANCD2 and CD44 gene in U87 cells and U251 cells (Fig. 4a, b, c, d). The caspase 3/7 activities and LDH release were significantly increased after FANCD2 and CD44 were silenced, and TMZ treated significantly promoted the caspase 3/7 activities and LDH release (Fig. 4e, f). The above results indicated that silencing of FANCD2 and CD44 were related to the TMZ sensitivity in U87 cells and U251 cells.
Knockdown of lncRNA TMEM161B-AS1 inhibited cell proliferation, migration and invasion, while promoted glioma cell apoptosis Combined with The Encyclopedia of RNA Interactomes (ENCORI) tool [34], we have a keen interest in lncRNA TMEM161B-AS1. According to the results of the GSE100736 dataset, lncRNA TMEM161B-AS1 was significantly up-regulated in TMZ-resistant U251 cells (Supplementary materials). The expression level of lncRNA TMEM161B-AS1 in U87 cells and U251 cells were significantly higher than that of human astrocytes (HA) (Fig. 5a). The results of qRT-PCR proved that we had successfully knocked down lncRNA TMEM161B-AS1 in U87 cells and U251 cells (Fig. 5b). Compared with the si-NC group, the proliferation ability of U87 cells and U251 cells transfected with si-TMEM161B-AS1 were significantly decreased (Fig. 5c, d). The migration ability (Fig. 5e) and invasion ability ( Fig. 5f) of U87 cells and U251 cells transfected with si-TMEM161B-AS1 were inhibited, but the apoptosis rate increased (Fig. 5g).
The above results indicated that knockdown of lncRNA TMEM161B-AS1 inhibited cell proliferation, migration and invasion, while promoted glioma cell apoptosis.
The results showed that the inhibitory effect of lncRNA TMEM161B-AS1 silencing on GBM cells was mediated by hsa-miR-27a-3p.

DISCUSSION
In this study, we analyzed the differential expression genes and ncRNA in GBM cancer tissue and normal tissue from TCGA database and TMZ resistance-related genes and ncRNA in  TMZ-sensitive and TMZ-insensitive U251 cells from GSE100736 dataset. In vitro studies have shown that lncRNA TMEM161B-AS1 regulated the expression of FANCD2 and CD44 by sponging hsa-miR-27a-3p, and changed the malignant biological behavior and TMZ resistance of U87 cells and U251 cells.
In recent years, the results of randomized clinical trials (RCT) and prospective studies have shown that postoperative adjuvant chemotherapy for glioma patients can prolong the overall survival time [35][36][37][38]. However, the emergence of drug resistance casts a shadow over the treatment of glioma. Therefore, further studies will be needed to better understand the pathogenesis and specific resistance mechanisms of glioma. It's of great significance to find more effective therapeutic targets for glioma and to develop a more refined classification system. Ferroptosis is induced by a variety of small molecular substances, leading to disorders of lipid oxide metabolism in cells, resulting in cell death caused by the production of excessive ROS [39]. Studies have shown that ferroptosis played an important role in the occurrence and development of tumors [40,41]. After analyzing the data from CGGA, GSE16011 dataset and the Cancer Genome Atlas dataset, Liu et al. [21] found that several ferroptosis-related genes played an important role in glioma progression.
Long non-coding RNA (lncRNA) is a type of non-coding RNA with a length of more than 200 nucleotides [42]. Recent studies have shown that lncRNA not only plays an important role in many biological processes [43][44][45], but also acts as oncogene or tumor suppressor gene [46]. For example, lncRNA TP73-AS1 was related to the invasiveness of GBM and the sensitivity of GCS cells to TMZ [47]. Zhang et al. [48] found that lncRNA LINC01446 promoted GBM cell tumor progression through the miR-489-3p/ TPT1 axis. TMEM161B-AS1 gene is located on chromosome 5q14.3, one recent study found that lncRNA TMEM161B-AS1 was specifically associated with the light polysomal fraction [45].
However, the role of lncRNA TMEM161B-AS1 in GBM has not been reported until now.
FANCD2 played an important role in bone marrow stromal cells (BMSCs) in resisting ferroptosis-related damage [23]. Wu et al. [49] found that FANCD2 was associated with the risk of clear cell renal cell carcinoma (ccRCC), and high FANCD2 expression corresponded to a high risk of ccRCC. CD44 was closely related to the invasion and migration of glioma cells due to its key role in the adhesion between glioma cells. CD44 promoted the resistance of glioma cells to radiotherapy and chemotherapy, and promoted tumor cell formation and other biological functions [50][51][52]. In this study, we found that FANCD2 gene and CD44 genes act as oncogenes, and knockdown of FANCD2 or CD44 genes in U87 and U251 cells were related to ferroptosis. We found that the expression levels of FANCD2 and CD44 genes were related to the survival of Chinese glioma patients according to the data from the mRNAseq_325 dataset in the CGGA database. Interestingly, we found that knockdown of FANCD2 and CD44 were associated with TMZ sensitive in U87 cells and U251 cells. We suggest that FANCD2 and CD44 may be one of the potential molecular targets to solve the TMZ resistance of U87 cells and U251 cells.
Bioinformation predictions suggested that lncRNA TMEM161B-AS1, FANCD2 and CD44 are potential targets for hsa-miR-27a-3p, which we have also confirmed in the in vitro dual-luciferase reporter assay and cell transfection experiments. Based on the results, we speculate that there may be a lncRNA-miRNA-gene network among lncRNA TMEM161B-AS1, hsa-miR-27a-3p, FANCD2 or CD44, which involves in the occurrence of gliomas.
Excessive consumption of ferritin or transporter caused the accumulation of iron in cells and subsequent peroxidation, leading to lipid peroxides and ferroptosis [54]. In this study, we found that when FANCD2 and CD44 were highly expressed in U87 and U251 cells, the concentration of iron and lipid ROS decreased significantly. When the expression of FANCD2 and CD44 was inhibited, the concentration of iron and lipid ROS were increased significantly, cell proliferation, migration and invasion were inhibited, and the apoptosis rate was significantly increased. Studies in nude mouse models found that knockdown 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.
of lncRNA TMEM161B-AS1 and/or overexpression of hsa-miR-27a-3p inhibited tumor growth. The above findings suggested that FANCD2 and CD44 may be potential target molecules of glioma.
However, there are some limiting factors that need to be further studied and resolved. First of all, the specific molecular mechanisms of the effects of FANCD2 and CD44 on the proliferation, migration and invasion of glioma cells need to be elucidated. Secondly, this study failed to detect cyclin and cell cycle. Third, the failure to study the survival period of nude mice is also one of the shortcomings that need to be remedied. Fourth, the signal pathways involved in the production of malignant biological behaviors of tumor cells are intricate and it is worth studying whether there are other signal molecules involved. Therefore, only studying the regulatory effects of lncRNA and/or miRNA may be due to their effects on different genes. Regulatory effects may adversely affect the regulation of these targets. Fifth, in order to study the role of the genes of study in the regulation of ferroptosis, analysis of the expression of genes related to this process in the different experimental conditions need to be performed.

CONCLUSIONS
In summary, this study demonstrated that the lncRNA TMEM161B-AS1-hsa-miR-27a-3p-FANCD2/CD44 network regulated the malignant biological behavior of GBM cells and TMZ sensitivity. The results of this study not only contributed to the intensive study on the explicit mechanism of occurrence of glioma, but also provided promising therapeutic targets for the treatment of glioma.

MATERIALS AND METHODS Dataset
All the datasets used in this study were obtained from public databases, including RNA-seq data from glioma patients in the TCGA dataset (https:// portal.gdc.cancer.gov/), and the GSE100736 dataset, the Chinese Glioma Genome Atlas (CGGA) dataset (http://www.cgga.org.cn/).

Real-time fluorescent quantitative PCR (qRT-PCR)
Total RNA was isolated from the cells with Trizol reagent (Life Technologies Corporation, Carlsbad, CA, USA). The concentration of the isolated RNA was measured by NanoDrop-2000 Spectrophotometer (Thermo Scientific, Wilmington, USA). According to the supplier's instructions, the PrimeScript RT kit (Takara, Shiga, Japan) was used to reverse-transcribe total RNA into cDNA, and then the SYBR Premix Ex Taq kit (Takara, Shiga, Japan) was used for PCR analysis. The primer sequences used for qRT-PCR analysis in this study were listed in Supplemental Table S1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were used as endogenous controls, the expression level was normalized to the endogenous control, and the fold change was calculated using the 2 -ΔΔCt method.

Transwell assay
Transfected U87 or U251 cells were harvested after 24 h transfection. The cell migration and invasion abilities were determined by Transwell assay. For migration ability detection, 2× 10 5 U87 and U251 cells were plated in the upper chamber of Transwell assay inserts (Millipore, Billerica, MA, USA) containing 200 μl of serum-free DMEM with a membrane (8-mm pores). For the invasion assay, the transfected cells were plated in the top chamber with a Matrigel-coated membrane (BD Biosciences, Franklin Lakes, NJ, USA) in 500 μl serum-free DMEM with 750 μl 10% FBS-DMEM in the bottom chamber. Then, the inserts were placed into the wells of the bottom chamber of a 24-well plate filled with conditioned medium. The lower surface of the membrane was fixed with methanol and glacial acetic acid (v/v: 3:1) and stained with crystal violet after 24 h of incubation. Cell numbers were calculated in 5 random fields after photographed with a digital microscope (×200).

Apoptosis detection assay
U87 cells and U251 cells apoptosis were evaluated by Annexin V-FITC/PI staining (BD Biosciences, San Jose, CA, USA) followed by flow cytometry (FACScan, BD Biosciences). After being washed three times with PBS, U87 cells and U251 cells were harvested in binding buffer at a concentration of 1×10 6 cells/ml, and 5 μl of PI and 5 μl FITC were added to the cell suspension and incubated for 15 min at room temperature in the dark condition.

Western blot
The collected cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) for 0.5 h on ice, and centrifuged at 15 were then washed three times with TBST and incubated with horseradish  peroxidase-labeled goat anti-rabbit IgG (1:2000, Abcam, Cambridge, MA, USA) for 2 h at room temperature. After adding developer for 1 min, the immunoblots were analyzed by the ImageJ2X software (National Institutes of Health, Bethesda, MD, USA), and the relative integrated density values (IDVs) were calculated using FluorChem FC2 system (Alpha Innotech, San Leandro, CA, USA) based on β-actin as an internal control.

RNA immunoprecipitation
The Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) was used to performed the RNA immunoprecipitation (RIP) assay according to the manufacturer's protocol. Cells were lysed by RIP lysis buffer and 0.1 ml U87 cells or U251 cells extract were incubated with RIPA buffer containing magnetic beads conjugated with human anti-Argonaute2 (Ago2) antibody (Abcam, Cambridge, MA, USA) at 4°C for 8 h. Then washing buffer was used to wash the samples and isolated the RNA-protein complexes from beads by proteinase K for 0.5 h at 55°C. Then the RNA was extracted and analyzed by qRT-PCR.

Caspase 3/7 activity measurements
U87 cells and U251 cells were collected and incubated for 1 h in the dark with 100 μL Caspase-Glo 3/7 reagent. The absorbance at 485/530 nm was detected by a microplate reader.

Lipid ROS measurement
The details of the procedures had been previously described [39,55,56]. U87 cells and U251 cells were collected and resuspended in DMEM with 10% FBS, then 10 μM C11 BODIPY (Thermo Fisher, Bonn, Germany) was added and incubated for 0.5 h at 37°C, 5% CO 2 in the dark. Then washed the cells with PBS for three times to remove the excess C11 BODIPY. The level of lipid ROS was proportional to the change in fluorescence emission peak from 590 nm to 510 nm caused by oxidation of the polyunsaturated butadienyl moiety of the dye. This analysis was performed by a flow cytometry.

Iron measurement
The total iron level in U87 cells and U251 cells were detected by Iron Assay Kit (Sigma, Missouri, USA) according to the manufacturer's protocol. The details of the procedures had been previously described [39,55,56].

Tumor xenograft assay
In this study, 4-week-old specific pathogen free male nude mice were selected and assigned into 4 groups, each of which was respectively injected with U251 cells and U87 cells transfected with NC, hsa-miR-27a-3p angomir, si-TMEM161B-AS1, si-TMEM161B-AS1 + hsa-miR-27a-3p angomir. Each nude mouse was subcutaneously injected with 4×10 5 cells in the right flank area for subcutaneous implantation. Tumors volume were measured 45days after injection, according to the formula: volume (mm 3 ) = length × width 2 /2. Then the nude mice were sacrificed and tumors were isolated. All mice were randomly assigned to each experimental group.

Statistical analysis
Data were presented as means ± standard deviation (mean ± SD). All statistical analyses were performed by the Prism 8.0 GraphPad software (GraphPad Software, La Jolla, CA, US). All experiments were repeated at least three times and the differences were analyzed by one-way ANOVA or the Student's t-test (two tailed). Differences were considered as statically significant when p < 0.05.

DATA AVAILABILITY
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.