The lncRNA CASC2/miR-18b/RORA Axis Suppresses Invasion and the Epithelial-Mesenchymal Transition in Multifocal Glioblastomas via the TGF-β1 /Smad Signaling Pathway

Background: Multifocal glioblastoma is an uncommon and refractory subtype of high grade glioma with poor prognosis. The epithelial-mesenchymal transition (EMT) is an important cause of enhanced glioblastoma invasiveness and tumor recurrence. Our previous study found that retinoic acid receptor-related orphan receptor A (RORA) is a nuclear receptor, and plays an important role in inhibiting proliferation and tumorigenesis of glioma. We further confirmed that RORA was downregulated in multifocal glioblastoma (GBM), therefore, we determined whether RORA was involved in the migration, invasion, and EMT of multifocal GBM. Methods: Human glioma cell lines, U87 and T98G, and patient-derived glioma stem cells (GSCs), GSC2C and GSC4D, were used for in vitro and in vivo experiments. The expressions of RORA, miR-18b, and lncRNA CASC2 in glioma cells were detected by RT-qPCR, western blotting. The biological effects of RORA, miR-18b, and CASC2 on glioma migration, invasion, and EMT were evaluated using the migration assay, Transwell, immunofluorescence staining, and xenograft experiments. Results: RORA was expressed at lower levels in multifocal glioblastoma and negatively regulated the migration, invasion, and EMT of multifocal GBM via the TGF-β1/Smad signaling pathway. MiR-18b functioned as a promotor in multifocal glioblastoma, while CASC2 played the role of a tumor suppressor. MiR-18b inhibited RORA expression via binding to its mRNA 3'-UTR in multifocal glioblastoma, while CASC2 upregulated RORA expression via miR-18b-mediated competitive endogenous RNA mechanism. Conclusions: The lncRNA CASC2/miR-18b/RORA/TGF- β1/Smad signaling pathway axis, which lead to the EMT and enhanced invasiveness of multifocal GBM, may be a promising therapeutic strategy for multifocal glioblastoma. Funding Statement: This work was supported by the Major Disease Prevention and Control Technology Action Plan of China (2018ZX-07S-006), the Liaoning BaiQianWan Talents Program (No. 2019-B45), the Social Development Program from Shenyang Science and Technology Bureau, China (20-205-4-075), the Project of JiangSu XianSheng Pharmaceutical Co. (JSXSZD-SA2020-06-004), and the Shanghai Sailing Program (No. 19YF1439000). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: Patients and controls were acquired with informed consent, under the protocol approved by the First Hospital of China Medical University research ethics committee.