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RBM5-AS1 promotes radioresistance in medulloblastoma through stabilization of SIRT6 protein

RBM5-AS1 promotes radioresistance in medulloblastoma through stabilization of SIRT6 protein Cancer stem cells (CSCs) contribute to radioresistance in medulloblastoma. Thus, identification of key regulators of medulloblastoma stemness is critical for improving radiotherapy for medulloblastoma. In the present study, we pro- filed CSC-related long non-coding RNAs (lncRNAs) between radioresistant and parental medulloblastoma cells. The roles of the lncRNA RBM5-AS1 in the stemness and radiosensitivity of medulloblastoma cells were investigated. We found that RBM5-AS1, a novel inducer of medulloblastoma stemness, was significantly upregulated in radioresistant medulloblastoma cells compared to parental cells. Knockdown of RBM5-AS1 diminished the viability and clonogenic survival of both radioresistant and parental medulloblastoma cells after radiation. Silencing of RBM5-AS1 significantly enhanced radiation-induced apoptosis and DNA damage. In vivo studies confirmed that depletion of RBM5-AS1 inhibited tumor growth and increased radiosensitivity in a medulloblastoma xenograft model. In contrast, overexpres- sion of RBM5-AS1 reduced radiation-induced apoptosis and DNA damage in medulloblastoma cells. Mechanistically, RBM5-AS1 interacted with and stabilized sirtuin 6 (SIRT6) protein. Silencing of SIRT6 reduced the stemness and rein- forced radiation-induced DNA damage in medulloblastoma cells. Overexpression of SIRT6 rescued medulloblastoma cells from RBM5-AS1 depletion-induced radiosensitization and DNA damage. Overall, we identify RBM5-AS1 as an inducer of stemness and radioresistance in medulloblastoma. Targeting RBM5-AS1 may represent a potential strategy to overcome the resistance to radiotherapy in this malignancy. Keywords: Medulloblastoma, RBM5-AS1, Protein stability, Radioresistance, Stemness Introduction are recommended as a standard treatment for medul- Medulloblastoma is one of the most common pediat- loblastoma [3]. Craniospinal irradiation after surgery ric malignancies, accounting for 15–20% of all tumors has been suggested to improve long-term outcome of the central nervous system in children [1]. Activa- in patients with medulloblastoma [1, 4, 5]. However, tion of oncogenic networks including HIPPO-YAP/ the development of radioresistance hampers thera- TAZ and AURORA-A/MYCN pathways has been peutic efficacy. Accumulating evidence suggests that shown to promote medulloblastoma tumorigenesis cancer stem cells (CSCs) contribute to tumor radiore- and relapse [2]. Multimodal regimens including maxi- sistance [6]. For instance, Yan et  al. reported that AhR mal surgical resection, radiotherapy, and chemotherapy activation enhances cancer stem-like properties and radioresistance [7]. Shi et  al.reported that pharmaco- logical inhibition of bone marrow and X-linked (BMX) disrupts glioma stem cells and reduces radioresist- *Correspondence: jiangmawei@xinhuamed.com.cn; siyu.chen@shsmu.edu.cn Department of Oncology, Xin Hua Hospital Affiliated to Shanghai ance [8]. CD133 is a widely used CSC marker [9, 10]. Jiaotong University School of Medicine, Shanghai 200092, China Garg et  al.reported that CD133 CSCs contribute to © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhu et al. acta neuropathol commun (2021) 9:123 Page 2 of 13 medulloblastoma recurrence through the signal trans- parental medulloblastoma cells. The function of RBM5- ducer and activator of transcription 3 (STAT3) signal- AS1 in the growth, stemness, and radiosensitivity of ing axis [10]. Identification of novel cancer stemness medulloblastoma cells was clarified. The RBM5-AS1- regulators is of significance in improving radiotherapy interacting partner was also investigated. for medulloblastoma. Sirtuin 6 (SIRT6) belongs to the sirtuin family of Results protein deacetylases and participates in various bio- RBM5‑AS1 depletion sensitizes medulloblastoma cells logical processes, including growth, differentiation, and to radiation treatment inflammation [11– 14]. SIRT6 is involved in base exci- To identify lncRNAs involved in radioresistance of sion repair and thus contributes to genomic stability medulloblastoma cells, we profiled 84 CSC-related lncR - [12]. In hepatocellular carcinoma (HCC), SIRT6 can NAs in radioresistant and parental DAOY cells using potentiate apoptosis resistance by repressing the tran- quantitative real-time PCR arrays. CSC-related lncRNAs scription of the pro-apoptotic gene Bax [15]. Similarly, were chosen given the causal relationship between cancer SIRT6 confers resistance to DNA damage in multiple stemness and radioresistance [7, 8]. Among the lncRNAs myeloma cells through downregulation of mitogen- tested (Additional file  1: Table  S1), 4 lncRNAs showed activated protein kinase (MAPK) pathway genes [16]. significant expression changes: i.e., XIST with 2.6-fold Depletion of SIRT6 blocks DNA repair responses and downregulation and RBM5-AS1, DANCR, and MALAT1 enhances the sensitivity of acute myeloid leukemia cells with 3.5-, 6.9-, and 2.2-fold upregulation, respectively to DNA-damaging agents [17]. Therefore, SIRT6 plays (Fig.  1A, B). To ascertain the role of these deregulated an important role in tumor progression. lncRNAs in the radiosensitivity of medulloblastoma cells, Long non-coding RNAs (lncRNAs) are a family of we knocked down RBM5-AS1, DANCR, and MALAT1 regulatory RNA molecules of > 200 nucleotides in and overexpressed XIST in DAOY cells. Silencing of length [18]. Although they lack protein-coding poten- RBM5-AS1 increased the radiosensitivity of radiore- tial, lncRNAs can interact with proteins or other RNA sistant and parental DAOY cells (Fig.  1C, D). However, molecules to modulate gene expression and activ- depletion of DANCR or MALAT1 or overexpression of ity [19, 20]. For instance, the lncRNA ARHGAP5- XIST did not affect the radiosensitivity of radioresistant AS1 enhances chemoresistance in gastric cancer cells DAOY cells (Additional file  1: Figure S1). Clonogenic through stabilization of ARHGAP5 mRNA [19]. Chen survival assay further demonstrated that knockdown of et  al. reported that the lncRNA IHS activates the ERK RBM5-AS1 significantly diminished surviving fraction and AKT signaling pathways to stimulate the prolifera- after radiation in radioresistant and parental DAOY cells tion and metastasis in HCC [20]. LncRNA RBM5-AS1 (Fig.  1E). These results suggest that RBM5-AS1 plays a has been suggested as a critical modulator of colon critical role in radiation resistance of medulloblastoma cancer stemness [21]. Biochemically, RBM5-AS1 is cells. localized in the nucleus of colon cancer cells and can Next, we validated the role of RBM5-AS1 in the main- directly interact with β-catenin to enhance the tran- tenance of medulloblastoma stemness. The stem cell scription of specific β-catenin targets [21]. The upregu - marker CD133 has been used to identify medulloblas- lation of β-catenin by RBM5-AS1 has also been noted toma stem cells [9, 10]. Analysis of CD133 expression in bone cells [22]. Li et al. reported that RBM5-AS1 can showed that radioresistant DAOY cells expressed a promote the proliferation and invasion of oral squa- higher level of CD133 than parental cells (Fig. 2A). More- mous cell carcinoma cells via the miR-1285-3p/YAP1 over, knockdown of RBM5-AS1 led to a reduction in the axis [23]. However, the biological role of RBM5-AS1 in level of CD133 in radioresistant DAOY cells. We also medulloblastoma remains unclear. investigated the effect of RBM5-AS1 on the expression In the present study, we profiled CSC-related lncR - of other stemness markers CD44 and SOX2. As shown in NAs including RBM5-AS1 between radioresistant and Fig.  2B, RBM5-AS1 depletion decreased the expression (See figure on next page.) Fig. 1 RBM5-AS1 depletion sensitizes medulloblastoma cells to radiation treatment. A Quantitative real-time PCR arrays were performed to identify dysregulated lncRNAs between radioresistant and parental DAOY cells. The heatmap shows the lncRNAs that significantly differ between the 2 groups. B Bar graphs show the level of RBM5-AS1, DANCR, MALAT1, and XIST between radioresistant and parental DAOY cells. C Measurement of RBM5-AS1 levels in radioresistant and parental DAOY cells transfected with control shRNA (shCtrl) or shRBM5-AS1. D Radioresistant and parental DAOY cells in serum-free medium were exposed to different doses of X-rays, and cell viability was measured after 3 days. E Clonal formation assays showed that RBM5-AS1 knockdown increased the sensitivity of radioresistant and parental DAOY cells to radiation. Left panels: representative photographs of dishes with colonies. *P < 0.05 Zhu  et al. acta neuropathol commun (2021) 9:123 Page 3 of 13 Fig. 1 (See legend on previous page.) Zhu et al. acta neuropathol commun (2021) 9:123 Page 4 of 13 Fig. 2 RBM5-AS1 contributes to medulloblastoma stemness. A Western blot analysis of the stem cell marker CD133 in radioresistant and parental DAOY cells transfected with control shRNA (shCtrl) or shRBM5-AS1. B Quantitative real-time PCR analysis of CD44 and SOX2 in radioresistant and parental DAOY cells transfected with indicated constructs. C Radioresistant and parental DAOY cells transfected with shCtrl or shRBM5-AS1 were cultured in the suspension condition to allow the formation of tumorspheres. Top panels: representative photographs of tumorspheres. Scale bar = 40 μm. *P < 0.05 of both CD44 and SOX2 in parental and radioresist- ant DAOY cells. Sphere-forming assay was then used to determine stem cell self-renewal. We found that knock- down of RBM5-AS1 impaired the formation of spheres by radioresistant DAOY cells (Fig.  2C). These findings confirm the regulation of medulloblastoma stemness by RBM5-AS1. RBM5‑AS1 knockdown induces apoptosis and DNA damage response Next, we investigated the effects of knockdown of RBM5- AS1 on apoptosis and DNA damage response in medul- loblastoma cells. We found that radiation exposure led to a significant apoptosis in parental DAOY cells, which was enhanced by knockdown of RBM5-AS1 (Fig.  3A). Similarly, silencing of RBM5-AS1 significantly enhanced radiation-induced apoptosis in radioresistant DAOY cells (Fig. 3B). DNA damage response was evaluated by exam- ining the expression of γ-H2AX, a biomarker of double strand breaks [24]. Of note, RBM5-AS1 silencing rein- forced DNA damage response after radiation (Fig. 3C, D). Taken together, these findings suggest that knockdown of RBM5-AS1 augments radiation-induced apoptosis and DNA damage responses. Depletion of RBM5‑AS1 suppresses tumor growth and increases radiosensitivity in vivo Next, we validated the effects of depletion of RBM5- AS1 on tumor growth and radiosensitivity in athymic nude mice. As shown in Fig. 4A, depletion of RBM5-AS1 reduced the growth of DAOY xenograft tumors. Fur- thermore, RBM5-AS1-depleted DAOY xenograft tumors showed more sensitive to radiation exposure than con- trol tumors (Fig.  4A, B). Immunohistochemical staining indicated a reduction of Ki-67-positive proliferative cells (Fig. 4C) and increase of terminal dUTP nick-end labeling (TUNEL)-positive apoptotic cells (Fig. 4D) in the RBM5- AS1-depleted tumors after radiation treatment. Taken together, RBM5-AS1 knockdown restrains tumor growth and enhances radiosensitivity in medulloblastoma. ▸ Zhu  et al. acta neuropathol commun (2021) 9:123 Page 5 of 13 Fig. 3 RBM5-AS1 knockdown induces apoptosis and DNA damage response. A, B Cells transfected with shCtrl or shRBM5-AS1 were radiated with X-rays at 8 Gy. Apoptosis was measured using Annexin-V/PI staining. C, D DNA damage response was evaluated by immunostaining for γ-H2AX. Scale bar = 20 μm. *P < 0.05 Ectopic expression of RBM5‑AS1 confers radioresistance vector-transfected medulloblastoma cells. Similar find - to medulloblastoma cells ings were noted in D283Med medulloblastoma cells Next, we asked whether overexpression of RBM5- (Fig.  5A, B). Moreover, overexpression of RBM5-AS1 AS1 could induce radioresistance in medulloblas- protected medulloblastoma cells from radiation-induced toma cells. We observed that enforced expression of DNA damage (Fig.  5C). In addition, clonogenic survival RBM5-AS1 (Fig.  5A) attenuated radiation-induced assay demonstrated that RBM5-AS1-overexpressing apoptosis in DAOY cells (Fig.  5B), compared to empty cells were more resistant to radiation than control cells Zhu et al. acta neuropathol commun (2021) 9:123 Page 6 of 13 Fig. 4 Depletion of RBM5-AS1 suppresses tumor growth and increases radiosensitivity in vivo. A, B DAOY xenograft tumors were exposed to irradiation (IR), and tumor growth was determined. A Macroscopic view of xenograft tumors. C Immunohistochemical staining for Ki-67 in xenograft tumor sections. Scale bar = 100 μm. (D) TUNEL staining in xenograft tumor sections. Scale bar = 100 μm. *P < 0.05 (Fig. 5D). These data collectively indicate that RBM5-AS1 RBM5-AS1 regulates the radioresistant phenotype of overexpression induces radioresistance in medulloblas- medulloblastoma cells, we performed RBM5-AS1 pull- toma cells. down assays. RBM5-AS1 interacting proteins were iden- tified by mass spectrometry. We validated the presence of RBM5‑AS1 associates with SIRT6 protein SIRT6 in the RBM5-AS1 pulldown complex by Western in medulloblastoma cells blot analysis (Fig.  6A). In this study, SIRT6 was selected A previous study has shown that RBM5-AS1 directly for further validation because of its importance in DNA interacts with β-catenin and promotes β-catenin acti- damage response [15–17]. We performed RNA immu- vation in colon cancer cells [21]. However, we did not noprecipitation (RIP) assay using anti-SIRT6 antibody. observe the enhanced activation of β-catenin signal- Compared to control IgG immunoprecipitated sample, ing by RBM5-AS1 in medulloblastoma cells (Additional the SIRT6 antibody-bound complex contained a signifi - file  1: Figure S2). To determine the mechanism by which cantly greater level of RBM5-AS1 (Fig. 6B). Zhu  et al. acta neuropathol commun (2021) 9:123 Page 7 of 13 Fig. 5 Ectopic expression of RBM5-AS1 confers radioresistance to medulloblastoma cells. A Quantitative real-time PCR analysis of RBM5-AS1 levels in both DAOY and D283Med cells transfected with empty vector or RBM5-AS1-expressing plasmid. B Detection of apoptosis in DAOY and D283Med cells transfected with empty vector or RBM5-AS1-expressing plasmid after irradation. C Assessment of DNA damage response in the cells treated as in B. D Clonogenic survival assay showing that RBM5-AS1-overexpressing cells were more resistant to radiation than control cells. *P < 0.05 Given the interaction between RBM5-AS1 and SIRT6, to block protein synthesis. As expected, overexpression we asked whether RBM5-AS1 could modulate the of RBM5-AS1 increased the stability of SIRT6 protein expression of SIRT6 in medulloblastoma cells. Notably, (Fig.  6E). Taken together, RBM5-AS1 associates with we found that overexpression of RBM5-AS1 led to an and stabilizes SIRT6 protein in medulloblastoma cells. increase in the protein level of SIRT6 in both DAOY and D283Med cells (Fig.  6C). However, RBM5-AS1 SIRT6 mediates the oncogenic activity of RBM5‑AS1 overexpression did not affect the mRNA level of SIRT6 in medulloblastoma (Fig.  6D), suggesting that RBM5-AS1 modulates the To investigate the importance of SIRT6 in RBM5-AS1- expression of SIRT6 at post-transcriptional level. Next mediated aggressive phenotype, we knocked down SIRT6 we asked whether RBM5-AS1-mediated upregulation in medulloblastoma cells (Fig.  7A). Similar to the phe- of SIRT6 expression was a result of increased protein notype of RBM5-AS1-depleted cells, depletion of SIRT6 stability. To address this, we used cycloheximide (CHX) reduced the stem-like properties (Fig.  7B) and enhanced Zhu et al. acta neuropathol commun (2021) 9:123 Page 8 of 13 Fig. 6 RBM5-AS1 associates with SIRT6 protein in medulloblastoma cells. A Western blot analysis of SIRT6 protein in the RNA–protein complex that was pulled down by RBM5-AS1 probes. B RIP assay performed with anti-SIRT6 antibody. The levels of RBM5-AS1 in immunoprecipitated samples were detected. *P < 0.05. C Western blot analysis showed that overexpression of RBM5-AS1 increased the protein level of SIRT6 in both DAOY and D283Med cells transfected with empty vector or RBM5-AS1-expressing plasmid. D Quantitative real-time PCR analysis of SIRT6 mRNA levels in the cells treated as in C. ns no significance. E Cycloheximide (CHX) was used to block protein synthesis, and SIRT6 protein stability was assessed radiation-induced DNA damage (Fig. 7C) in medulloblas- in medulloblastoma cells [27, 28]. In this study, we iden- toma cells. In addition, overexpression of SIRT6 (Fig. 7D) tify a novel medulloblastoma driver lncRNA. We show reversed RBM5-AS1 depletion-induced radiosensitiza- that RBM5-AS1 is upregulated in radioresistant DAOY tion (Fig.  7E) and DNA damage (Fig.  7F). Thus, SIRT6 is cells (Fig.  1A). Depletion of RBM5-AS1 overcomes the functionally relevant to RBM5-AS1 in medulloblastoma. radioresistance of medulloblastoma cells, as evidenced by reduced cell viability and clonogenic survival after Discussion exposure to radiation (Fig.  1C, D). Conversely, ectopic Several lncRNAs have been reported to contribute to expression of RBM5-AS1 enhances clonogenic survival medulloblastoma growth and survival [25, 26]. For of medulloblastoma cells in response to radiation (Fig. 5). instance, silencing of the lncRNA TP73-AS1 induces Our findings provide first evidence for the role of RBM5- apoptosis and inhibits cell proliferation and migration AS1 in inducing radioresistance in medulloblastoma. Zhu  et al. acta neuropathol commun (2021) 9:123 Page 9 of 13 Fig. 7 SIRT6 mediates the oncogenic activity of RBM5-AS1 in medulloblastoma. A Western blot analysis showed depletion of SIRT6 in medulloblastoma cells. B Eec ff t of SIRT6 knockdown on self-renewal capacity of cancer stem cells. C Depletion of SIRT6 enhanced radiation-induced DNA damage. D Western blot analysis confirmed overexpression of SIRT6 in medulloblastoma cells. E Overexpression of SIRT6 rescued RBM5-AS1 depletion-induced radiosensitization. F Overexpression of SIRT6 attenuated RBM5-AS1 depletion-induced DNA damage response. *P < 0.05 It has been suggested that CSCs play a critical role in carcinoma [30]. It has been previously demonstrated that the development of radioresistance [7, 8]. Chen et  al. RBM5-AS1 is involved in the enrichment of colon cancer reported that C D133 CSCs are enriched upon radia- CSCs [21]. Consistently, RBM5-AS1 also promotes the tion and confer radioresistance in non-small cell lung self-renewal of medulloblastom CSCs (Fig.  2B). Moreo- cancer [29]. JARID1B silencing-mediated suppression of ver, knockdown of RBM5-AS1 reduces the expression of stemness results in increased radiation sensitivity in oral the CSC marker CD133 (Fig. 2A). These findings suggest Zhu et al. acta neuropathol commun (2021) 9:123 Page 10 of 13 that RBM5-AS1-mediated radioresistance may be caus- of ubiquitin-mediated SIRT1 degradation [35]. USP10 ally linked to enhanced cancer stemness. It has been has exhibited the ability to prevent SIRT6 protein from documented that the population of medulloblastoma ubiquitination and degradation [36, 37]. These studies CD133 cell is enlarged upon hypoxic stimulation, and suggest the possibility that the association with RBM5- + − CD133 cells exhibit more radioresistant than CD133 AS1 may reinforce USP10-dependent suppression of cells [31]. Therefore, it is interesting to determine the role SIRT6 degradation. However, the detailed mechanism for of RBM5-AS1 in medulloblastoma radioresistance under RBM5-AS1-mediated stabilization of SIRT6 remains to hypoxic conditions. be uncovered in future work. Previous studies have indicated RBM5-AS1 as an onco- SIRT6 has been extensively reported to modulate gene in colon cancer and oral squamous cell carcinoma tumor progression [13, 14, 38]. For example, SIRT6 [21, 23]. Li et al. reported that RBM5-AS1 has the ability contributes to the invasiveness and metastasis in lung to promote oral squamous cell carcinoma cell prolifera- cancer and ovarian cancer [14, 39]. Cagnetta et  al. has tion and invasion [23]. In this study, we show that RBM5- indicated that SIRT6 mediates DNA repair in leukemia AS1 knockdown enhances radiation-induced apoptosis [17]. Similarly, Lee et  al. reported that SIRT6 prevents in medulloblastoma cells (Fig. 3A, B). However, radiation DNA damage and cellular senescence in HCC cells caused comparable apoptosis in radioresistant and paren- [40]. Our data confirm the role of SIRT6 in the regula - tal DAOY cells transfected with control shRNA, which tion of DNA damage response of medulloblastoma cells may be due to short observation time, i.e. 48 h after X-ray (Fig.  7). We show that knockdown of SIRT6 enhances exposure. Indeed, we noted that the parental group had radiation-induced DNA damage, which is similar to significantly profound apoptosis than the radioresistant the finding in RBM5-AS1-depleted cells. Moreover, group 72  h after radiation (data not shown). We further enforced expression of SIRT6 reverses RBM5-AS1 demonstrate that overexpression of RBM5-AS1 protects depletion-induced radiosensitization and DNA dam- medulloblastoma cells from radiation-induced apoptosis age response. However, the key pathways involved in (Fig.  5). Consistently, ectopic expression of RBM5-AS1 SIRT6 oncogenic activity remain to be clarified. There attenuates radiation-induced DNA damage in medul- is evidence that SIRT6 safeguards human mesenchy- loblastoma cells (Fig.  5C). These results confirm the mal stem cells from oxidative stress [41]. Consistently, prosurvival capacity of RBM5-AS1 in medulloblastoma our data indicate that SIRT6 knockdown impairs the cells. In agreement with our data, several lncRNAs such stemness of medulloblastoma cells. However, a previ- as Meg3 and PVT1 have been found to regulate DNA ous study reported that SIRT6 overexpression inhib- damage repair and apoptosis responses [32, 33]. We its cancer stem-like capacity in breast cancer with also validated the role of RBM5-AS1 in the xenograft PI3K activation [42]. Therefore, SIRT6 likely plays mouse model. Of note, depletion of RBM5-AS1 slows the distinct roles in different origins of stem cells. Nev - growth of DAOY xenograft tumors (Fig.  4). Moreover, ertheless, SIRT6 mediates the oncogenic activity of RBM5-AS1 silencing sensitizes DAOY xenograft tumors RBM5-AS1 in medulloblastoma through promotion to radiation, which is associated with increased apopto- of stem-like capacity and attenuation of DNA damage. sis. Therefore, RBM5-AS1 may represent a promising tar - These findings warrant investigation of the therapeutic get to overcome radioresistance in medulloblastoma. potential of targeting both RBM5-AS1 and SIRT6 in lncRNAs exert their biological activities via multi- medulloblastoma. ple mechanisms, such as sponging of microRNAs [23], Although in this study we indicate that RBM5-AS1 epigenetic silencing of gene expression [34], and post- regulates growth and radioresistance of medulloblas- translational modification of proteins [35]. Di Cecilia toma cells, the clinical significance of RBM5-AS1 et  al. revealed the interaction between RBM5-AS1 has not been addressed yet. Future work is needed to and β-catenin protein in colon cancer cells [21]. How- explore the expression, tissue distribution, and clinical ever, such interaction was not detected in medulloblas- relevance of RBM5-AS1 in medulloblastoma. Moreo- toma cells, suggesting another mechanism involved in ver, direct evidence is required to confirm the causal the oncogenic activity of RBM5-AS1 in medulloblas- link between RBM5-AS1-induced stemness and radi- toma. Specially, we discover that RBM5-AS1 interacts oresistance of medulloblastoma cells. In addition, the with SIRT6 protein in medulloblastoma cells (Fig.  6). findings in radioresistant DAOY cells will be validated RBM5-AS1 overexpression increases the protein lev- in other radioresistant medulloblastoma cell lines. els, which is at least, in part, a result of increased stabi- In conclusion, RBM5-AS1 is a radiation respon- lization of SIRT6 protein. The lncRNA HULC has been sive lncRNA that contributes to increased stemness shown to stabilize SIRT1 protein through induction of and radioresistance in medulloblastoma. The onco - ubiquitin-specific peptidase (USP) 22 and thus inhibition genic activity of RBM5-AS1 is ascribed to stabilization Zhu  et al. acta neuropathol commun (2021) 9:123 Page 11 of 13 of SIRT6. Thus, targeting RBM5-AS1 may offer a Cell transfection was performed using the Lipo- potential strategy for improving radiotherapy in fectamine LTX Plus (Invitrogen) as per the manufac- medulloblastoma. turer’s protocol. Twenty-four hours after transfection, transfected cells were subjected to gene expression analy- sis. For selection of stable cell lines, transfected cells were Materials and methods selected in the medium containing 1  μg/mL puromycin Cell culture (Sigma-Aldrich). Medulloblastoma cell lines DAOY and D283Med were obtained from the American Type Culture Collection In vitro sphere‑forming assay (Manassas, VA, USA) and cultured in Dulbecco’s modi- Cells were seeded onto ultra-low attachment 24-well fied Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, plates (Corning, Lowell, MA, USA) at a density of 1000 MO, USA) containing 10% fetal bovine serum (FBS; cells per well. They were cultured in serum-free DMEM/ HyClone, Logan, UT, USA) at 37 °C in 5% CO . No myco- F12 medium supplemented with basic fibroblast growth plasma contamination was found. factor and epidermal growth factor (20 ng/mL each; Inv- itrogen) for 2  weeks. Culture media were replenished Establishment of radioresistant cell lines every 3  days. The number of spheres in each well was Radioresistant DAOY-IR cells were developed as determined. described previously [43]. Briefly, DAOY cells were repeatedly exposed to 4  Gy of X-rays. After treatment Cell viability assay with a cumulative dose of 80 Gy, the surviving cell clones, Cells in serum-free medium were exposed to dif- namely DAOY-IR, were recovered and expanded. ferent doses of X-rays and cultured for 3  days. Cell viability was measured using the 3-(4,5-dimethythiazol- Quantitative real‑time PCR analysis 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Total RNA was extracted from cells using TRIzol reagent Cells were incubated with 0.5  mg/mL MTT (Sigma- (Invitrogen, Grand Island, NY, USA) following the manu- Aldrich) at 37  °C for 4  h. After addition of dimethyl facturer’s instructions. Reverse transcription was per- sulfoxide, absorbance was measured at 570  nm with a formed using the SuperScript III Reverse Transcriptase microplate spectrophotometer. kit (Invitrogen). Quantitative PCR was then done using the following PCR primers: RBM5-AS1 forward: 5′-GCT Clonogenic assay TCA ACA CTG CGT GAC AA-3′, reverse: 5′-CGT GGA Cells were plated onto 6-well plates (4000 cells/well) ATC AAA TGG AGT GG-3′; SIRT6 forward: 5′-CGT GGA overnight and then radiated with 2 or 8  Gy. Media were TGA GGT GAT GTG -3′, reverse: 5′-GGC TTA TAG GAA changed every 3 days until colonies were formed. Fifteen CCA TTG AGA-3′; CD44 forward: 5′-GCC CAA TGC CTT days later, cells were fixed with 4% of buffered forma - TGA TGG ACC-3′, reverse: 5′-GCA GGG ATT CTG TCT lin for 15  min and stained with 0.25% crystal violet for GTG CTG-3′; SOX2 forward: 5′-GCC TGG GCG CCG 20 min. The colonies were counted for each well. The sur - AGT GGA -3′, reverse: 5′-GGG CGA GCC GTT CAT GTA viving fraction was calculated as a ratio of the number of GGT CTG -3′; B-Actin forward: 5′-GGT GGC TTT TAG colonies divided by the total number of cells seeded. GAT GGC AAG-3′, reverse: 5′-ACT GGA ACG GTG AAG GTG ACAG-3′. We also performed quantitative real-time Flow cytometry analysis of apoptosis PCR arrays to profile 84 CSC-related lncRNAs in radiore - Apoptotic response was evaluated 48  h after expo- sistant and parental DAOY cells. The candidate lncRNAs sure to 8 Gy of X-rays. In brief, cells were washed and are listed in Additional file  1: Table S1. The relative gene resuspended in the Annexin Binding Buffer. The cell −ΔΔCt expression was calculated by the 2 method [44]. suspension was added with Annexin V conjugated with fluorescein isothiocyanate (FITC; Sigma-Aldrich) Plasmids and cell transfection and incubated for 15  min at 4  °C. Afterwards, the RBM5-AS1- and SIRT6-targeting short hairpin RNAs cells were stained with propidium iodide (PI; Sigma- (shRNAs) were synthesized by Beijing Hanyu Biomed (Bei- Aldrich). Apoptotic cells were immediately analyzed jing, China) and cloned to pLKO.1 vector (Sigma-Aldrich). using the FACScantoII cytometer with the FlowJo 10.2 The targeting sequence for RBM5-AS1 and SIRT6 was software (BD Bioscience, San Jose, CA, USA). 5′-GAG UCA CAU UCC UUA GCC AUG-3′ and 5′-GAC AAA CUG GCA GAG CUC CAC-3′, respectively. The Immunofluorescent staining RBM5-AS1- and SIRT6-expressing plasmids were con- DNA damage was assessed by analyzing γ-H2AX foci structed by Beijing Hanyu Biomed. All plasmids were veri- formation [45]. Briefly, cells were radiated with a dose fied by DNA sequencing. Zhu et al. acta neuropathol commun (2021) 9:123 Page 12 of 13 of 8  Gy, fixed with 4% paraformaldehyde, and permea - proteinase K at 55  °C for 30  min. The precipitated RNA bilized with 0.2% TritonX-100. After blocking, the cells samples were tested for RBM5-AS1 by quantitative PCR were incubated with anti-phospho-γ-H2AX (ab26350, analysis. Abcam, Cambridge, MA, USA; 1:50 dilution). An Alexa- Fluor 488 conjugated goat anti-rabbit IgG was used as the Western blot analysis secondary antibody. Nuclei were counter-stained with Cells were lysed using Radioimmunoprecipitation Assay 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Buffer (RIPA) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were Animal studies determined using the Bio-Rad Protein Assay Kit (Bio- Five-week-old male BALB/c nude mice (nu/nu) were Rad, Hercules, CA, USA). Protein samples were sepa- maintained in a pathogen-free environment and allowed rated by 12% sodium dodecyl sulphate‐polyacrylamide free access to water and food. These mice were randomly gel electrophoresis and transferred onto nitrocellulose assigned to 4 groups (5 mice each group) and received membranes. The membranes were incubated with pri - subcutaneous inoculation of RBM5-AS1-depleted or mary antibodies recognizing CD133 (ab19898, Abcam; control DAOY cells (2 × 10 cells/mouse) with or without 1:1000 dilution), SIRT6 (1:500 dilution) or GAPDH radiation exposure. For radiation treatment, mice were (ab181602, Abcam; 1:5000 dilution) overnight at 4 °C, fol- given a single dose of 10 Gy on day 3 after cell injection. lowed by a horseradish peroxidase-conjugated secondary Tumor volume was calculated weekly for 4  weeks. After antibody (Sigma-Aldrich). The protein bands were visu - the last measurement, mice were sacrificed and tumors alized using the ECL Plus Chemiluminescence Detection were removed. Tumor sections were deparaffinized with Kit (Thermo Fisher Scientific, Rockford, IL, USA). xylene and rehydrated by alcohol gradient. Endogenous peroxidase activity was blocked using a 0.3% hydrogen Statistics peroxide solution. The sections were subjected to immu - Results are expressed as mean ± standard deviation and nostaining with anti-Ki-67 antibody (ab15580, Abcam; were analyzed by the Student’s t test or one-way ANOVA 1:100 dilution). For apoptosis analysis, TUNEL stain- followed by the Bonferroni’s test. P < 0.05 was considered ing was performed on tumor sections with the TUNEL statistically significant. Staining kit (Beyotime, Haimen, China). The percentage of Ki-67- and TUNEL-positive cells was calculated in 5 Supplementary Information random microscopic fields. All studies involving animals The online version contains supplementary material available at https:// doi. were approved by the Animal Care and Use Commit- org/ 10. 1186/ s40478- 021- 01218-2. tee of Shanghai Jiaotong University School of Medicine (Shanghai, China; approval number: 2018-0269). Additional file 1. Supplementary materials. RNA pulldown assay Acknowledgements Biotin-labeled sense or antisense RBM5-AS1 RNAs were This work was supported by the Hospital Funded Clinical Research, Xin Hua generated by in  vitro transcription using Biotin RNA Hospital Affiliated to Shanghai Jiao Tong University School of Medicine of China (17CSY04). Labeling Mix (Sigma-Aldrich) and T7 RNA polymerase (Promega, Madison, WI, USA). The labeled RNA was Competing interests purified using the RNeasy Mini Kit (Qiagen, Valencia, The authors declare that they have no competing interests. CA, USA) and then incubated with precleared DAOY cell Received: 18 May 2021 Accepted: 16 June 2021 lysates at 4 °C overnight. The RNA–protein binding com - plexes were captured by streptavidin agarose beads (Inv- itrogen). The released proteins were detected by Western blot analysis or mass spectrometry. References 1. Grill J, Dufour C, Guerrini-Rousseau L, Ayrault O (2021) New research directions in medulloblastoma. Neurochirurgie 67:87–89 RIP assay 2. 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RBM5-AS1 promotes radioresistance in medulloblastoma through stabilization of SIRT6 protein

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Abstract

Cancer stem cells (CSCs) contribute to radioresistance in medulloblastoma. Thus, identification of key regulators of medulloblastoma stemness is critical for improving radiotherapy for medulloblastoma. In the present study, we pro- filed CSC-related long non-coding RNAs (lncRNAs) between radioresistant and parental medulloblastoma cells. The roles of the lncRNA RBM5-AS1 in the stemness and radiosensitivity of medulloblastoma cells were investigated. We found that RBM5-AS1, a novel inducer of medulloblastoma stemness, was significantly upregulated in radioresistant medulloblastoma cells compared to parental cells. Knockdown of RBM5-AS1 diminished the viability and clonogenic survival of both radioresistant and parental medulloblastoma cells after radiation. Silencing of RBM5-AS1 significantly enhanced radiation-induced apoptosis and DNA damage. In vivo studies confirmed that depletion of RBM5-AS1 inhibited tumor growth and increased radiosensitivity in a medulloblastoma xenograft model. In contrast, overexpres- sion of RBM5-AS1 reduced radiation-induced apoptosis and DNA damage in medulloblastoma cells. Mechanistically, RBM5-AS1 interacted with and stabilized sirtuin 6 (SIRT6) protein. Silencing of SIRT6 reduced the stemness and rein- forced radiation-induced DNA damage in medulloblastoma cells. Overexpression of SIRT6 rescued medulloblastoma cells from RBM5-AS1 depletion-induced radiosensitization and DNA damage. Overall, we identify RBM5-AS1 as an inducer of stemness and radioresistance in medulloblastoma. Targeting RBM5-AS1 may represent a potential strategy to overcome the resistance to radiotherapy in this malignancy. Keywords: Medulloblastoma, RBM5-AS1, Protein stability, Radioresistance, Stemness Introduction are recommended as a standard treatment for medul- Medulloblastoma is one of the most common pediat- loblastoma [3]. Craniospinal irradiation after surgery ric malignancies, accounting for 15–20% of all tumors has been suggested to improve long-term outcome of the central nervous system in children [1]. Activa- in patients with medulloblastoma [1, 4, 5]. However, tion of oncogenic networks including HIPPO-YAP/ the development of radioresistance hampers thera- TAZ and AURORA-A/MYCN pathways has been peutic efficacy. Accumulating evidence suggests that shown to promote medulloblastoma tumorigenesis cancer stem cells (CSCs) contribute to tumor radiore- and relapse [2]. Multimodal regimens including maxi- sistance [6]. For instance, Yan et  al. reported that AhR mal surgical resection, radiotherapy, and chemotherapy activation enhances cancer stem-like properties and radioresistance [7]. Shi et  al.reported that pharmaco- logical inhibition of bone marrow and X-linked (BMX) disrupts glioma stem cells and reduces radioresist- *Correspondence: jiangmawei@xinhuamed.com.cn; siyu.chen@shsmu.edu.cn Department of Oncology, Xin Hua Hospital Affiliated to Shanghai ance [8]. CD133 is a widely used CSC marker [9, 10]. Jiaotong University School of Medicine, Shanghai 200092, China Garg et  al.reported that CD133 CSCs contribute to © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhu et al. acta neuropathol commun (2021) 9:123 Page 2 of 13 medulloblastoma recurrence through the signal trans- parental medulloblastoma cells. The function of RBM5- ducer and activator of transcription 3 (STAT3) signal- AS1 in the growth, stemness, and radiosensitivity of ing axis [10]. Identification of novel cancer stemness medulloblastoma cells was clarified. The RBM5-AS1- regulators is of significance in improving radiotherapy interacting partner was also investigated. for medulloblastoma. Sirtuin 6 (SIRT6) belongs to the sirtuin family of Results protein deacetylases and participates in various bio- RBM5‑AS1 depletion sensitizes medulloblastoma cells logical processes, including growth, differentiation, and to radiation treatment inflammation [11– 14]. SIRT6 is involved in base exci- To identify lncRNAs involved in radioresistance of sion repair and thus contributes to genomic stability medulloblastoma cells, we profiled 84 CSC-related lncR - [12]. In hepatocellular carcinoma (HCC), SIRT6 can NAs in radioresistant and parental DAOY cells using potentiate apoptosis resistance by repressing the tran- quantitative real-time PCR arrays. CSC-related lncRNAs scription of the pro-apoptotic gene Bax [15]. Similarly, were chosen given the causal relationship between cancer SIRT6 confers resistance to DNA damage in multiple stemness and radioresistance [7, 8]. Among the lncRNAs myeloma cells through downregulation of mitogen- tested (Additional file  1: Table  S1), 4 lncRNAs showed activated protein kinase (MAPK) pathway genes [16]. significant expression changes: i.e., XIST with 2.6-fold Depletion of SIRT6 blocks DNA repair responses and downregulation and RBM5-AS1, DANCR, and MALAT1 enhances the sensitivity of acute myeloid leukemia cells with 3.5-, 6.9-, and 2.2-fold upregulation, respectively to DNA-damaging agents [17]. Therefore, SIRT6 plays (Fig.  1A, B). To ascertain the role of these deregulated an important role in tumor progression. lncRNAs in the radiosensitivity of medulloblastoma cells, Long non-coding RNAs (lncRNAs) are a family of we knocked down RBM5-AS1, DANCR, and MALAT1 regulatory RNA molecules of > 200 nucleotides in and overexpressed XIST in DAOY cells. Silencing of length [18]. Although they lack protein-coding poten- RBM5-AS1 increased the radiosensitivity of radiore- tial, lncRNAs can interact with proteins or other RNA sistant and parental DAOY cells (Fig.  1C, D). However, molecules to modulate gene expression and activ- depletion of DANCR or MALAT1 or overexpression of ity [19, 20]. For instance, the lncRNA ARHGAP5- XIST did not affect the radiosensitivity of radioresistant AS1 enhances chemoresistance in gastric cancer cells DAOY cells (Additional file  1: Figure S1). Clonogenic through stabilization of ARHGAP5 mRNA [19]. Chen survival assay further demonstrated that knockdown of et  al. reported that the lncRNA IHS activates the ERK RBM5-AS1 significantly diminished surviving fraction and AKT signaling pathways to stimulate the prolifera- after radiation in radioresistant and parental DAOY cells tion and metastasis in HCC [20]. LncRNA RBM5-AS1 (Fig.  1E). These results suggest that RBM5-AS1 plays a has been suggested as a critical modulator of colon critical role in radiation resistance of medulloblastoma cancer stemness [21]. Biochemically, RBM5-AS1 is cells. localized in the nucleus of colon cancer cells and can Next, we validated the role of RBM5-AS1 in the main- directly interact with β-catenin to enhance the tran- tenance of medulloblastoma stemness. The stem cell scription of specific β-catenin targets [21]. The upregu - marker CD133 has been used to identify medulloblas- lation of β-catenin by RBM5-AS1 has also been noted toma stem cells [9, 10]. Analysis of CD133 expression in bone cells [22]. Li et al. reported that RBM5-AS1 can showed that radioresistant DAOY cells expressed a promote the proliferation and invasion of oral squa- higher level of CD133 than parental cells (Fig. 2A). More- mous cell carcinoma cells via the miR-1285-3p/YAP1 over, knockdown of RBM5-AS1 led to a reduction in the axis [23]. However, the biological role of RBM5-AS1 in level of CD133 in radioresistant DAOY cells. We also medulloblastoma remains unclear. investigated the effect of RBM5-AS1 on the expression In the present study, we profiled CSC-related lncR - of other stemness markers CD44 and SOX2. As shown in NAs including RBM5-AS1 between radioresistant and Fig.  2B, RBM5-AS1 depletion decreased the expression (See figure on next page.) Fig. 1 RBM5-AS1 depletion sensitizes medulloblastoma cells to radiation treatment. A Quantitative real-time PCR arrays were performed to identify dysregulated lncRNAs between radioresistant and parental DAOY cells. The heatmap shows the lncRNAs that significantly differ between the 2 groups. B Bar graphs show the level of RBM5-AS1, DANCR, MALAT1, and XIST between radioresistant and parental DAOY cells. C Measurement of RBM5-AS1 levels in radioresistant and parental DAOY cells transfected with control shRNA (shCtrl) or shRBM5-AS1. D Radioresistant and parental DAOY cells in serum-free medium were exposed to different doses of X-rays, and cell viability was measured after 3 days. E Clonal formation assays showed that RBM5-AS1 knockdown increased the sensitivity of radioresistant and parental DAOY cells to radiation. Left panels: representative photographs of dishes with colonies. *P < 0.05 Zhu  et al. acta neuropathol commun (2021) 9:123 Page 3 of 13 Fig. 1 (See legend on previous page.) Zhu et al. acta neuropathol commun (2021) 9:123 Page 4 of 13 Fig. 2 RBM5-AS1 contributes to medulloblastoma stemness. A Western blot analysis of the stem cell marker CD133 in radioresistant and parental DAOY cells transfected with control shRNA (shCtrl) or shRBM5-AS1. B Quantitative real-time PCR analysis of CD44 and SOX2 in radioresistant and parental DAOY cells transfected with indicated constructs. C Radioresistant and parental DAOY cells transfected with shCtrl or shRBM5-AS1 were cultured in the suspension condition to allow the formation of tumorspheres. Top panels: representative photographs of tumorspheres. Scale bar = 40 μm. *P < 0.05 of both CD44 and SOX2 in parental and radioresist- ant DAOY cells. Sphere-forming assay was then used to determine stem cell self-renewal. We found that knock- down of RBM5-AS1 impaired the formation of spheres by radioresistant DAOY cells (Fig.  2C). These findings confirm the regulation of medulloblastoma stemness by RBM5-AS1. RBM5‑AS1 knockdown induces apoptosis and DNA damage response Next, we investigated the effects of knockdown of RBM5- AS1 on apoptosis and DNA damage response in medul- loblastoma cells. We found that radiation exposure led to a significant apoptosis in parental DAOY cells, which was enhanced by knockdown of RBM5-AS1 (Fig.  3A). Similarly, silencing of RBM5-AS1 significantly enhanced radiation-induced apoptosis in radioresistant DAOY cells (Fig. 3B). DNA damage response was evaluated by exam- ining the expression of γ-H2AX, a biomarker of double strand breaks [24]. Of note, RBM5-AS1 silencing rein- forced DNA damage response after radiation (Fig. 3C, D). Taken together, these findings suggest that knockdown of RBM5-AS1 augments radiation-induced apoptosis and DNA damage responses. Depletion of RBM5‑AS1 suppresses tumor growth and increases radiosensitivity in vivo Next, we validated the effects of depletion of RBM5- AS1 on tumor growth and radiosensitivity in athymic nude mice. As shown in Fig. 4A, depletion of RBM5-AS1 reduced the growth of DAOY xenograft tumors. Fur- thermore, RBM5-AS1-depleted DAOY xenograft tumors showed more sensitive to radiation exposure than con- trol tumors (Fig.  4A, B). Immunohistochemical staining indicated a reduction of Ki-67-positive proliferative cells (Fig. 4C) and increase of terminal dUTP nick-end labeling (TUNEL)-positive apoptotic cells (Fig. 4D) in the RBM5- AS1-depleted tumors after radiation treatment. Taken together, RBM5-AS1 knockdown restrains tumor growth and enhances radiosensitivity in medulloblastoma. ▸ Zhu  et al. acta neuropathol commun (2021) 9:123 Page 5 of 13 Fig. 3 RBM5-AS1 knockdown induces apoptosis and DNA damage response. A, B Cells transfected with shCtrl or shRBM5-AS1 were radiated with X-rays at 8 Gy. Apoptosis was measured using Annexin-V/PI staining. C, D DNA damage response was evaluated by immunostaining for γ-H2AX. Scale bar = 20 μm. *P < 0.05 Ectopic expression of RBM5‑AS1 confers radioresistance vector-transfected medulloblastoma cells. Similar find - to medulloblastoma cells ings were noted in D283Med medulloblastoma cells Next, we asked whether overexpression of RBM5- (Fig.  5A, B). Moreover, overexpression of RBM5-AS1 AS1 could induce radioresistance in medulloblas- protected medulloblastoma cells from radiation-induced toma cells. We observed that enforced expression of DNA damage (Fig.  5C). In addition, clonogenic survival RBM5-AS1 (Fig.  5A) attenuated radiation-induced assay demonstrated that RBM5-AS1-overexpressing apoptosis in DAOY cells (Fig.  5B), compared to empty cells were more resistant to radiation than control cells Zhu et al. acta neuropathol commun (2021) 9:123 Page 6 of 13 Fig. 4 Depletion of RBM5-AS1 suppresses tumor growth and increases radiosensitivity in vivo. A, B DAOY xenograft tumors were exposed to irradiation (IR), and tumor growth was determined. A Macroscopic view of xenograft tumors. C Immunohistochemical staining for Ki-67 in xenograft tumor sections. Scale bar = 100 μm. (D) TUNEL staining in xenograft tumor sections. Scale bar = 100 μm. *P < 0.05 (Fig. 5D). These data collectively indicate that RBM5-AS1 RBM5-AS1 regulates the radioresistant phenotype of overexpression induces radioresistance in medulloblas- medulloblastoma cells, we performed RBM5-AS1 pull- toma cells. down assays. RBM5-AS1 interacting proteins were iden- tified by mass spectrometry. We validated the presence of RBM5‑AS1 associates with SIRT6 protein SIRT6 in the RBM5-AS1 pulldown complex by Western in medulloblastoma cells blot analysis (Fig.  6A). In this study, SIRT6 was selected A previous study has shown that RBM5-AS1 directly for further validation because of its importance in DNA interacts with β-catenin and promotes β-catenin acti- damage response [15–17]. We performed RNA immu- vation in colon cancer cells [21]. However, we did not noprecipitation (RIP) assay using anti-SIRT6 antibody. observe the enhanced activation of β-catenin signal- Compared to control IgG immunoprecipitated sample, ing by RBM5-AS1 in medulloblastoma cells (Additional the SIRT6 antibody-bound complex contained a signifi - file  1: Figure S2). To determine the mechanism by which cantly greater level of RBM5-AS1 (Fig. 6B). Zhu  et al. acta neuropathol commun (2021) 9:123 Page 7 of 13 Fig. 5 Ectopic expression of RBM5-AS1 confers radioresistance to medulloblastoma cells. A Quantitative real-time PCR analysis of RBM5-AS1 levels in both DAOY and D283Med cells transfected with empty vector or RBM5-AS1-expressing plasmid. B Detection of apoptosis in DAOY and D283Med cells transfected with empty vector or RBM5-AS1-expressing plasmid after irradation. C Assessment of DNA damage response in the cells treated as in B. D Clonogenic survival assay showing that RBM5-AS1-overexpressing cells were more resistant to radiation than control cells. *P < 0.05 Given the interaction between RBM5-AS1 and SIRT6, to block protein synthesis. As expected, overexpression we asked whether RBM5-AS1 could modulate the of RBM5-AS1 increased the stability of SIRT6 protein expression of SIRT6 in medulloblastoma cells. Notably, (Fig.  6E). Taken together, RBM5-AS1 associates with we found that overexpression of RBM5-AS1 led to an and stabilizes SIRT6 protein in medulloblastoma cells. increase in the protein level of SIRT6 in both DAOY and D283Med cells (Fig.  6C). However, RBM5-AS1 SIRT6 mediates the oncogenic activity of RBM5‑AS1 overexpression did not affect the mRNA level of SIRT6 in medulloblastoma (Fig.  6D), suggesting that RBM5-AS1 modulates the To investigate the importance of SIRT6 in RBM5-AS1- expression of SIRT6 at post-transcriptional level. Next mediated aggressive phenotype, we knocked down SIRT6 we asked whether RBM5-AS1-mediated upregulation in medulloblastoma cells (Fig.  7A). Similar to the phe- of SIRT6 expression was a result of increased protein notype of RBM5-AS1-depleted cells, depletion of SIRT6 stability. To address this, we used cycloheximide (CHX) reduced the stem-like properties (Fig.  7B) and enhanced Zhu et al. acta neuropathol commun (2021) 9:123 Page 8 of 13 Fig. 6 RBM5-AS1 associates with SIRT6 protein in medulloblastoma cells. A Western blot analysis of SIRT6 protein in the RNA–protein complex that was pulled down by RBM5-AS1 probes. B RIP assay performed with anti-SIRT6 antibody. The levels of RBM5-AS1 in immunoprecipitated samples were detected. *P < 0.05. C Western blot analysis showed that overexpression of RBM5-AS1 increased the protein level of SIRT6 in both DAOY and D283Med cells transfected with empty vector or RBM5-AS1-expressing plasmid. D Quantitative real-time PCR analysis of SIRT6 mRNA levels in the cells treated as in C. ns no significance. E Cycloheximide (CHX) was used to block protein synthesis, and SIRT6 protein stability was assessed radiation-induced DNA damage (Fig. 7C) in medulloblas- in medulloblastoma cells [27, 28]. In this study, we iden- toma cells. In addition, overexpression of SIRT6 (Fig. 7D) tify a novel medulloblastoma driver lncRNA. We show reversed RBM5-AS1 depletion-induced radiosensitiza- that RBM5-AS1 is upregulated in radioresistant DAOY tion (Fig.  7E) and DNA damage (Fig.  7F). Thus, SIRT6 is cells (Fig.  1A). Depletion of RBM5-AS1 overcomes the functionally relevant to RBM5-AS1 in medulloblastoma. radioresistance of medulloblastoma cells, as evidenced by reduced cell viability and clonogenic survival after Discussion exposure to radiation (Fig.  1C, D). Conversely, ectopic Several lncRNAs have been reported to contribute to expression of RBM5-AS1 enhances clonogenic survival medulloblastoma growth and survival [25, 26]. For of medulloblastoma cells in response to radiation (Fig. 5). instance, silencing of the lncRNA TP73-AS1 induces Our findings provide first evidence for the role of RBM5- apoptosis and inhibits cell proliferation and migration AS1 in inducing radioresistance in medulloblastoma. Zhu  et al. acta neuropathol commun (2021) 9:123 Page 9 of 13 Fig. 7 SIRT6 mediates the oncogenic activity of RBM5-AS1 in medulloblastoma. A Western blot analysis showed depletion of SIRT6 in medulloblastoma cells. B Eec ff t of SIRT6 knockdown on self-renewal capacity of cancer stem cells. C Depletion of SIRT6 enhanced radiation-induced DNA damage. D Western blot analysis confirmed overexpression of SIRT6 in medulloblastoma cells. E Overexpression of SIRT6 rescued RBM5-AS1 depletion-induced radiosensitization. F Overexpression of SIRT6 attenuated RBM5-AS1 depletion-induced DNA damage response. *P < 0.05 It has been suggested that CSCs play a critical role in carcinoma [30]. It has been previously demonstrated that the development of radioresistance [7, 8]. Chen et  al. RBM5-AS1 is involved in the enrichment of colon cancer reported that C D133 CSCs are enriched upon radia- CSCs [21]. Consistently, RBM5-AS1 also promotes the tion and confer radioresistance in non-small cell lung self-renewal of medulloblastom CSCs (Fig.  2B). Moreo- cancer [29]. JARID1B silencing-mediated suppression of ver, knockdown of RBM5-AS1 reduces the expression of stemness results in increased radiation sensitivity in oral the CSC marker CD133 (Fig. 2A). These findings suggest Zhu et al. acta neuropathol commun (2021) 9:123 Page 10 of 13 that RBM5-AS1-mediated radioresistance may be caus- of ubiquitin-mediated SIRT1 degradation [35]. USP10 ally linked to enhanced cancer stemness. It has been has exhibited the ability to prevent SIRT6 protein from documented that the population of medulloblastoma ubiquitination and degradation [36, 37]. These studies CD133 cell is enlarged upon hypoxic stimulation, and suggest the possibility that the association with RBM5- + − CD133 cells exhibit more radioresistant than CD133 AS1 may reinforce USP10-dependent suppression of cells [31]. Therefore, it is interesting to determine the role SIRT6 degradation. However, the detailed mechanism for of RBM5-AS1 in medulloblastoma radioresistance under RBM5-AS1-mediated stabilization of SIRT6 remains to hypoxic conditions. be uncovered in future work. Previous studies have indicated RBM5-AS1 as an onco- SIRT6 has been extensively reported to modulate gene in colon cancer and oral squamous cell carcinoma tumor progression [13, 14, 38]. For example, SIRT6 [21, 23]. Li et al. reported that RBM5-AS1 has the ability contributes to the invasiveness and metastasis in lung to promote oral squamous cell carcinoma cell prolifera- cancer and ovarian cancer [14, 39]. Cagnetta et  al. has tion and invasion [23]. In this study, we show that RBM5- indicated that SIRT6 mediates DNA repair in leukemia AS1 knockdown enhances radiation-induced apoptosis [17]. Similarly, Lee et  al. reported that SIRT6 prevents in medulloblastoma cells (Fig. 3A, B). However, radiation DNA damage and cellular senescence in HCC cells caused comparable apoptosis in radioresistant and paren- [40]. Our data confirm the role of SIRT6 in the regula - tal DAOY cells transfected with control shRNA, which tion of DNA damage response of medulloblastoma cells may be due to short observation time, i.e. 48 h after X-ray (Fig.  7). We show that knockdown of SIRT6 enhances exposure. Indeed, we noted that the parental group had radiation-induced DNA damage, which is similar to significantly profound apoptosis than the radioresistant the finding in RBM5-AS1-depleted cells. Moreover, group 72  h after radiation (data not shown). We further enforced expression of SIRT6 reverses RBM5-AS1 demonstrate that overexpression of RBM5-AS1 protects depletion-induced radiosensitization and DNA dam- medulloblastoma cells from radiation-induced apoptosis age response. However, the key pathways involved in (Fig.  5). Consistently, ectopic expression of RBM5-AS1 SIRT6 oncogenic activity remain to be clarified. There attenuates radiation-induced DNA damage in medul- is evidence that SIRT6 safeguards human mesenchy- loblastoma cells (Fig.  5C). These results confirm the mal stem cells from oxidative stress [41]. Consistently, prosurvival capacity of RBM5-AS1 in medulloblastoma our data indicate that SIRT6 knockdown impairs the cells. In agreement with our data, several lncRNAs such stemness of medulloblastoma cells. However, a previ- as Meg3 and PVT1 have been found to regulate DNA ous study reported that SIRT6 overexpression inhib- damage repair and apoptosis responses [32, 33]. We its cancer stem-like capacity in breast cancer with also validated the role of RBM5-AS1 in the xenograft PI3K activation [42]. Therefore, SIRT6 likely plays mouse model. Of note, depletion of RBM5-AS1 slows the distinct roles in different origins of stem cells. Nev - growth of DAOY xenograft tumors (Fig.  4). Moreover, ertheless, SIRT6 mediates the oncogenic activity of RBM5-AS1 silencing sensitizes DAOY xenograft tumors RBM5-AS1 in medulloblastoma through promotion to radiation, which is associated with increased apopto- of stem-like capacity and attenuation of DNA damage. sis. Therefore, RBM5-AS1 may represent a promising tar - These findings warrant investigation of the therapeutic get to overcome radioresistance in medulloblastoma. potential of targeting both RBM5-AS1 and SIRT6 in lncRNAs exert their biological activities via multi- medulloblastoma. ple mechanisms, such as sponging of microRNAs [23], Although in this study we indicate that RBM5-AS1 epigenetic silencing of gene expression [34], and post- regulates growth and radioresistance of medulloblas- translational modification of proteins [35]. Di Cecilia toma cells, the clinical significance of RBM5-AS1 et  al. revealed the interaction between RBM5-AS1 has not been addressed yet. Future work is needed to and β-catenin protein in colon cancer cells [21]. How- explore the expression, tissue distribution, and clinical ever, such interaction was not detected in medulloblas- relevance of RBM5-AS1 in medulloblastoma. Moreo- toma cells, suggesting another mechanism involved in ver, direct evidence is required to confirm the causal the oncogenic activity of RBM5-AS1 in medulloblas- link between RBM5-AS1-induced stemness and radi- toma. Specially, we discover that RBM5-AS1 interacts oresistance of medulloblastoma cells. In addition, the with SIRT6 protein in medulloblastoma cells (Fig.  6). findings in radioresistant DAOY cells will be validated RBM5-AS1 overexpression increases the protein lev- in other radioresistant medulloblastoma cell lines. els, which is at least, in part, a result of increased stabi- In conclusion, RBM5-AS1 is a radiation respon- lization of SIRT6 protein. The lncRNA HULC has been sive lncRNA that contributes to increased stemness shown to stabilize SIRT1 protein through induction of and radioresistance in medulloblastoma. The onco - ubiquitin-specific peptidase (USP) 22 and thus inhibition genic activity of RBM5-AS1 is ascribed to stabilization Zhu  et al. acta neuropathol commun (2021) 9:123 Page 11 of 13 of SIRT6. Thus, targeting RBM5-AS1 may offer a Cell transfection was performed using the Lipo- potential strategy for improving radiotherapy in fectamine LTX Plus (Invitrogen) as per the manufac- medulloblastoma. turer’s protocol. Twenty-four hours after transfection, transfected cells were subjected to gene expression analy- sis. For selection of stable cell lines, transfected cells were Materials and methods selected in the medium containing 1  μg/mL puromycin Cell culture (Sigma-Aldrich). Medulloblastoma cell lines DAOY and D283Med were obtained from the American Type Culture Collection In vitro sphere‑forming assay (Manassas, VA, USA) and cultured in Dulbecco’s modi- Cells were seeded onto ultra-low attachment 24-well fied Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, plates (Corning, Lowell, MA, USA) at a density of 1000 MO, USA) containing 10% fetal bovine serum (FBS; cells per well. They were cultured in serum-free DMEM/ HyClone, Logan, UT, USA) at 37 °C in 5% CO . No myco- F12 medium supplemented with basic fibroblast growth plasma contamination was found. factor and epidermal growth factor (20 ng/mL each; Inv- itrogen) for 2  weeks. Culture media were replenished Establishment of radioresistant cell lines every 3  days. The number of spheres in each well was Radioresistant DAOY-IR cells were developed as determined. described previously [43]. Briefly, DAOY cells were repeatedly exposed to 4  Gy of X-rays. After treatment Cell viability assay with a cumulative dose of 80 Gy, the surviving cell clones, Cells in serum-free medium were exposed to dif- namely DAOY-IR, were recovered and expanded. ferent doses of X-rays and cultured for 3  days. Cell viability was measured using the 3-(4,5-dimethythiazol- Quantitative real‑time PCR analysis 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Total RNA was extracted from cells using TRIzol reagent Cells were incubated with 0.5  mg/mL MTT (Sigma- (Invitrogen, Grand Island, NY, USA) following the manu- Aldrich) at 37  °C for 4  h. After addition of dimethyl facturer’s instructions. Reverse transcription was per- sulfoxide, absorbance was measured at 570  nm with a formed using the SuperScript III Reverse Transcriptase microplate spectrophotometer. kit (Invitrogen). Quantitative PCR was then done using the following PCR primers: RBM5-AS1 forward: 5′-GCT Clonogenic assay TCA ACA CTG CGT GAC AA-3′, reverse: 5′-CGT GGA Cells were plated onto 6-well plates (4000 cells/well) ATC AAA TGG AGT GG-3′; SIRT6 forward: 5′-CGT GGA overnight and then radiated with 2 or 8  Gy. Media were TGA GGT GAT GTG -3′, reverse: 5′-GGC TTA TAG GAA changed every 3 days until colonies were formed. Fifteen CCA TTG AGA-3′; CD44 forward: 5′-GCC CAA TGC CTT days later, cells were fixed with 4% of buffered forma - TGA TGG ACC-3′, reverse: 5′-GCA GGG ATT CTG TCT lin for 15  min and stained with 0.25% crystal violet for GTG CTG-3′; SOX2 forward: 5′-GCC TGG GCG CCG 20 min. The colonies were counted for each well. The sur - AGT GGA -3′, reverse: 5′-GGG CGA GCC GTT CAT GTA viving fraction was calculated as a ratio of the number of GGT CTG -3′; B-Actin forward: 5′-GGT GGC TTT TAG colonies divided by the total number of cells seeded. GAT GGC AAG-3′, reverse: 5′-ACT GGA ACG GTG AAG GTG ACAG-3′. We also performed quantitative real-time Flow cytometry analysis of apoptosis PCR arrays to profile 84 CSC-related lncRNAs in radiore - Apoptotic response was evaluated 48  h after expo- sistant and parental DAOY cells. The candidate lncRNAs sure to 8 Gy of X-rays. In brief, cells were washed and are listed in Additional file  1: Table S1. The relative gene resuspended in the Annexin Binding Buffer. The cell −ΔΔCt expression was calculated by the 2 method [44]. suspension was added with Annexin V conjugated with fluorescein isothiocyanate (FITC; Sigma-Aldrich) Plasmids and cell transfection and incubated for 15  min at 4  °C. Afterwards, the RBM5-AS1- and SIRT6-targeting short hairpin RNAs cells were stained with propidium iodide (PI; Sigma- (shRNAs) were synthesized by Beijing Hanyu Biomed (Bei- Aldrich). Apoptotic cells were immediately analyzed jing, China) and cloned to pLKO.1 vector (Sigma-Aldrich). using the FACScantoII cytometer with the FlowJo 10.2 The targeting sequence for RBM5-AS1 and SIRT6 was software (BD Bioscience, San Jose, CA, USA). 5′-GAG UCA CAU UCC UUA GCC AUG-3′ and 5′-GAC AAA CUG GCA GAG CUC CAC-3′, respectively. The Immunofluorescent staining RBM5-AS1- and SIRT6-expressing plasmids were con- DNA damage was assessed by analyzing γ-H2AX foci structed by Beijing Hanyu Biomed. All plasmids were veri- formation [45]. Briefly, cells were radiated with a dose fied by DNA sequencing. Zhu et al. acta neuropathol commun (2021) 9:123 Page 12 of 13 of 8  Gy, fixed with 4% paraformaldehyde, and permea - proteinase K at 55  °C for 30  min. The precipitated RNA bilized with 0.2% TritonX-100. After blocking, the cells samples were tested for RBM5-AS1 by quantitative PCR were incubated with anti-phospho-γ-H2AX (ab26350, analysis. Abcam, Cambridge, MA, USA; 1:50 dilution). An Alexa- Fluor 488 conjugated goat anti-rabbit IgG was used as the Western blot analysis secondary antibody. Nuclei were counter-stained with Cells were lysed using Radioimmunoprecipitation Assay 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Buffer (RIPA) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were Animal studies determined using the Bio-Rad Protein Assay Kit (Bio- Five-week-old male BALB/c nude mice (nu/nu) were Rad, Hercules, CA, USA). Protein samples were sepa- maintained in a pathogen-free environment and allowed rated by 12% sodium dodecyl sulphate‐polyacrylamide free access to water and food. These mice were randomly gel electrophoresis and transferred onto nitrocellulose assigned to 4 groups (5 mice each group) and received membranes. The membranes were incubated with pri - subcutaneous inoculation of RBM5-AS1-depleted or mary antibodies recognizing CD133 (ab19898, Abcam; control DAOY cells (2 × 10 cells/mouse) with or without 1:1000 dilution), SIRT6 (1:500 dilution) or GAPDH radiation exposure. For radiation treatment, mice were (ab181602, Abcam; 1:5000 dilution) overnight at 4 °C, fol- given a single dose of 10 Gy on day 3 after cell injection. lowed by a horseradish peroxidase-conjugated secondary Tumor volume was calculated weekly for 4  weeks. After antibody (Sigma-Aldrich). The protein bands were visu - the last measurement, mice were sacrificed and tumors alized using the ECL Plus Chemiluminescence Detection were removed. Tumor sections were deparaffinized with Kit (Thermo Fisher Scientific, Rockford, IL, USA). xylene and rehydrated by alcohol gradient. Endogenous peroxidase activity was blocked using a 0.3% hydrogen Statistics peroxide solution. The sections were subjected to immu - Results are expressed as mean ± standard deviation and nostaining with anti-Ki-67 antibody (ab15580, Abcam; were analyzed by the Student’s t test or one-way ANOVA 1:100 dilution). For apoptosis analysis, TUNEL stain- followed by the Bonferroni’s test. P < 0.05 was considered ing was performed on tumor sections with the TUNEL statistically significant. Staining kit (Beyotime, Haimen, China). The percentage of Ki-67- and TUNEL-positive cells was calculated in 5 Supplementary Information random microscopic fields. All studies involving animals The online version contains supplementary material available at https:// doi. were approved by the Animal Care and Use Commit- org/ 10. 1186/ s40478- 021- 01218-2. tee of Shanghai Jiaotong University School of Medicine (Shanghai, China; approval number: 2018-0269). Additional file 1. Supplementary materials. RNA pulldown assay Acknowledgements Biotin-labeled sense or antisense RBM5-AS1 RNAs were This work was supported by the Hospital Funded Clinical Research, Xin Hua generated by in  vitro transcription using Biotin RNA Hospital Affiliated to Shanghai Jiao Tong University School of Medicine of China (17CSY04). Labeling Mix (Sigma-Aldrich) and T7 RNA polymerase (Promega, Madison, WI, USA). The labeled RNA was Competing interests purified using the RNeasy Mini Kit (Qiagen, Valencia, The authors declare that they have no competing interests. CA, USA) and then incubated with precleared DAOY cell Received: 18 May 2021 Accepted: 16 June 2021 lysates at 4 °C overnight. The RNA–protein binding com - plexes were captured by streptavidin agarose beads (Inv- itrogen). The released proteins were detected by Western blot analysis or mass spectrometry. References 1. Grill J, Dufour C, Guerrini-Rousseau L, Ayrault O (2021) New research directions in medulloblastoma. Neurochirurgie 67:87–89 RIP assay 2. 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Journal

Acta Neuropathologica CommunicationsSpringer Journals

Published: Jul 5, 2021

Keywords: Medulloblastoma; RBM5-AS1; Protein stability; Radioresistance; Stemness

References