Regulation of Receptors Siglec-11 and Siglec-15 by MicroRNAs and their Overall Effect on Glioblastoma

Abstract

Introduction: Glioblastoma is a fast growing and aggressive type of brain tumour. It is the most common type of brain tumour in adults. Although there are developments in current therapies, patient outcomes remain poor. Therefore, new therapeutic strategies are being developed to improve patient prognosis and treatment outcomes. This study aimed to identify novel therapeutic targets for glioblastomas by understanding how Siglec-11 and Siglec-15 are regulated by microRNAs (miRNAs). Siglecs are cell surface receptor molecules that act as a lectin and bind to nine carbon atom sugar sialic acid. Siglec-11 is an inhibitory siglec that is expressed on microglia in the brain. Siglec-15 is expressed in a variety of cancers, however its presence in brain cancer cells has been elusive. Both Siglec-11 and Siglec-15 upon binding with their ligand generate immune inhibitory signals. Thus, they are assumed to be target for cancer immunotherapy. Although there has been active research on their therapeutic targeting, their gene expression regulation has not been extensively researched. Therefore, the aim of this project is to decipher the gene expression regulation of Siglec-11 and Siglec-15 by miRNAs. Methods: Bioinformatics analysis was performed using different online tools; Targetscan (https://www.targetscan.org/vert_72/), miRSystem (http://mirsystem.cgm.ntu.edu.tw), DIANA (https://diana.e-ce.uth.gr/tools) and miRwalk (http://mirwalk.umm.uni-heidelberg.de) were used to identify which miRNAs are involved in the regulation of both Siglecs. Following this, the expression of specific miRNAs was further validated in A172 glioblastoma cell lines using quantitative real time polymerase chain reaction (RT-qPCR). To do so, RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed with the mirVANA miRNA isolation kit. RT-qPCR was performed to validate the expression profiles of the following miRNAs: hsa-miR-138-5p, hsa-miR-153 and hsa-miR-107. Another bioinformatics tool GEPIA2 was used to identify the expression and survival of Siglec-11 and Siglec-15 between cancer patients and healthy individuals. Results: The results from the bioinformatic analysis highlighted that the miRNAs that are highly downregulated in cancer; hsa-miR-138-5p, hsa-miR-153 and hsa-miR-107 were heavily downregulated in cancer. The downregulation of hsa-miR-138-5p, hsa-miR-153 and hsa-miR-107 was confirmed by qRT-PCR in glioblastoma cell line A172 The results from GEPIA2 showed that Siglec-11 and Siglec-15 are highly upregulated in cancer patients as compared to healthy individuals. However, there was no statistical difference in the survival of glioblastoma patients between the high and low expressors of Siglec-11 and Siglec-15. Western blot was carried out for 3 different markers: Siglec-15, E2F3 and CDK6. Conclusion: The expression of the following miRNAs was found to be heavily downregulated in cancer; hsa-miR-138-5p, hsa-miR-153 and hsa-miR-107 indicating that they might manifest tumor suppressor properties. Siglec-11 and Siglec-15 can be used as diagnostic markers and therapeutic targets in glioblastomas as they are regulated by these tumour suppressor miRNAs. However, further functional assays are required to prove the association between these miRNAs and the Siglecs under investigation. Altering miRNA expression levels is regarded to be a possible technique for developing effective cancer therapeutics. A good expression of Siglec-15, CKD6 and E2F3 were shown on protein level using western blot in A172 cell line.