Li et al. Molecular Medicine (2020) 26:43 Molecular Medicine https://doi.org/10.1186/s10020-020-00171-5 RESEARCH ARTICLE Open Access Overexpression of RPN2 suppresses radiosensitivity of glioma cells by activating STAT3 signal transduction Changyu Li1†, Haonan Ran2†, Shaojun Song1, Weisong Liu3, Wenhui Zou1, Bei Jiang4, Hongmei Zhao5 and Bin Shao6* Abstract Background: Radiation therapy is the primary method of treatment for glioblastoma (GBM). Therefore, the suppression of radioresistance in GBM cells is of enormous significance. Ribophorin II (RPN2), a protein component of an N-oligosaccharyl transferase complex, has been associated with chemotherapy drug resistance in multiple cancers, including GBM. However, it remains unclear whether this also plays a role in radiation therapy resistance in GBM. Methods: We conducted a bioinformatic analysis of RPN2 expression using the UCSC Cancer Genomics Browser and GEPIA database and performed an immunohistochemical assessment of RPN2 expression in biopsy specimens from 34 GBM patients who had received radiation-based therapy. We also studied the expression and function of RPN2 in radiation-resistant GBM cells. Results: We found that RPN2 expression was upregulated in GBM tumors and correlated with poor survival. The expression of RPN2 was also higher in GBM patients with tumor recurrence, who were classified to be resistant to radiation therapy. In the radiation-resistant GBM cells, the expression of RPN2 was also higher than in the parental cells. Depletion of RPN2 in resistant cells can sensitize these cells to radiation-induced apoptosis, and overexpression of RPN2 had the reverse effect. Myeloid cell leukemia 1 (MCL1) was found to be the downstream target of RPN2, and contributed to radiation resistance in GBM cells. Furthermore, STAT3 was found to be the regulator of MCL1, which can be activated by RPN2 dysregulation. Conclusion: Our study has revealed a novel function of RPN2 in radiation-resistant GBM, and has shown that MCL1 depletionorsuppressioncouldbeapromisingmethodoftherapytoovercometheresistancepromotedbyRPN2 dysregulation. Keywords: GBM, RPN2, STAT3, Signal transduction Background radiation, and temozolomide chemotherapy (Stupp et al., Glioblastomas (GBM) are tumors of the central nervous 2005). Despite these aggressive treatments, survival rates system and result in 1.2 ~ 1.4 × 104 deaths in the U.S. after treatment remain low, at an average of around 12– alone every year (Hess et al., 2004). The existing treat- 15 months (Preusser et al., 2011). It is thought that GBM ment methods are usually limited to surgical excision, tumors can obtain chemo and radiation therapy resist- ance by stimulating DNA repair systems or by initiating * Correspondence: [email protected] changes to the cell cycle and apoptosis (Mirimanoff †Changyu Li and Haonan Ran both authors contributed equally to this work et al., 2006; Mrugala & Chamberlain, 2008). Resistance and should be considered as equal first coauthors. 6 to chemotherapy has been widely investigated and has Neurosurgery, The First Affiliated Hospital of Harbin Medical University, MGMT – No.199 Dazhi Street, Nangang District, Harbin 150001, Heilongjiang, China mostly focused on the expression of (0 6 Full list of author information is available at the end of the article methylguanine-DNA Methyltransferase) (Perazzoli et al., © The Author(s). 2020 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://creativecommons.org/licenses/by/4.0/. Li et al. Molecular Medicine (2020) 26:43 Page 2 of 9 2015). However, the fundamental mechanisms under- Radiotherapy lying radiotherapy resistance and its generation are still Radiotherapy was conducted in the Radiotherapy Oncol- unclear. Radiation therapy remains a primary method of ogy department of the First Affiliated Hospital of Harbin treatment for GBM (Ghotme et al., 2017), and therefore Medical University, using a Varian 2100C linear acceler- the reduction of radioresistance in GBM cells and thera- ator (dose, 5 Gy; dose rate, 5 Gy/min). The cells were peutic targets is of huge significance. seeded in a 12-well plate and preserved under adjustable Ribophorin II (RPN2) is a protein component of an N- conditions for 1 day, and subsequently treated with radi- oligosaccharyl transferase complex, the downregulation ation, and cultivated again under identical conditions for of which can trigger apoptosis in human breast cancer 1 day more. cells resistant to docetaxel., and its silencing confers sen- sitivity of the tumor to cisplatin treatment (Honma Clonal radioresistant cell generation et al., 2008). In addition, gastric cancers with high RPN2 A172 and U87 cells were seeded in culture plates (100 expression have exhibited dramatically higher recurrence mm) and treated with a 1 Gy radiation dose until an ac- rates and lower 5-year survival rates relative to those cumulated dose of 5 Gy was reached. All dissociated with low expression (Fujimoto et al., 2017). These obser- cells were recovered using cloning cylinders (Sigma Al- vations suggest that RPN2 expression could serve as a drich) and seeded in a 96-well plate. Once proliferating, predictive biomarker for chemotherapy resistance. In a these cells were transferred to a 12-well plate, and subse- recent study, RPN2 was reported to be modulated by quently a 6-well plate. Colonel radioresistant cells were circNFIX, and promoted GBM tumor growth in vivo then obtained. and in vitro (Ding et al., 2019). However, the correlation of RPN2 expression and radiotherapy resistance in GBM Cell transfection remains unknown. 5 This study explored the function of RPN2 in radiore- U87 cells were inoculated in 12-well plates (1 × 10 cells/ RPN2 MCL1 STAT3 sistant GBM, and found that its high expression contrib- well). , , and siRNA or control siRNA utes to the tolerance of GBM to radiotherapy. The (GenePharma, Co., Ltd., Shanghai, China) were trans- ™ dysregulation of RPN2 led to abnormal myeloid cell fected into cells through Lipofectamine 2000 (Invitro- – leukemia 1 (MCL1) expression through the promotion gen, Shanghai, China) at 50 60% confluency. Two days of STAT3 transcription activity. Our study, therefore, later, the resulting cells were harvested and preserved provides a new target to overcome radioresistance in for future use. The siRNA sequences involved were: RPN2 ′ ′ MCL1 GBM therapy. ,5-GGCCACUGUUAAACUAGAACA-3 ; , 5′-CGCCGAAUUCAUUAAUUUAUU-3′; STAT3,5′- CUUCAGACCCGUCAACAAA-3′. The negative (mock) Methods siRNA sequence involved was 5′-ACGCCUCCCGAACG Bioinformatics analysis UTTUUCUUGUCGUC-3′.ThepcDNA-V5-RPN2 was The abnormal expression of RPN2 and MCL1 was constructed by inserting the RPN2 cDNA into pcDNA™3.1/ investigated through the UCSC Cancer Genomics V5-His TOPO vector (Thermofisher, Waltham, MA. USA) Browser (https://xena.ucsc.edu/welcome-to-ucsc-xena/) according to the manufacturer instructions. and GEPIA online database (http://gepia.cancer-pku.cn/). qPCR Patient samples and cell culture RNA was isolated using TRIzol (Thermo Fisher Scien- GBM samples were taken from 34 patients admitted to tific) and quantified using a NanoDrop 2000 (Thermo the First Affiliated Hospital of Harbin Medical Univer- Fisher). RNA was treated with RNase R (Geneseed, sity. These GBM patients had all received radiation ther- Guangzhou, China) to achieve circRNA purification. The apy, with 12 patients experiencing GBM recurrence. The cDNA was prepared from 0.5 μg RNA using a Prime- corresponding brain samples were harvested and pre- Script RT reagent kit (TaKaRa, Dalian, China) or Taq- served at − 80 °C. Informed consent was obtained from Man miRNA Reverse Transcription Kit (Applied Biosys- all participants, and the study was approved by the Eth- tems, FosterCity, CA, USA). qPCR was conducted using ics Committee of the First Affiliated Hospital of Harbin a PCR System (Bio-Rad). The following were primer se- Medical University. quences involved: RPN2 (Forward, 5′- AGGAAGTGGT The normal glioma cell lines (U87, T98, U251, U- GTTTGTTGCC-3′; Reverse, 5′-CAGTCGAGGGAGCT 118MG and A172) and astrocyte cell line (HA) were TCTTC-3′); MCL1 (5′-AAAGCCTGTCTGCCAAAT-3′ provided by BeNa Culture Collection (Beijing, China). and reverse primer 5′-CCTATAAACCCACCACTC-3′); These cells were cultivated in DMEM (Sigma, St. Louis, and GAPDH (Forward, 5′-GAATGGGCAGCCGTTAG- MO, USA) with 10% FBS at 37 °C under 5% CO2. GAA-3′; Reverse, 5′-AAAAGCATCACCCGGAGGAG-3′). Li et al. Molecular Medicine (2020) 26:43 Page 3 of 9 Western blot (WB) Results Western blot analysis was conducted to isolate the target Abnormal expression of RPN2 is correlated to poor proteins using RIPA buffer (Beyotime) containing prote- survival of GBM patients ase inhibitors. The protein concentrations were deter- To study the role of RPN2 in GBM radiotherapy, we first mined using