Genomic alterations like IDH1/2 mutation, EGFR amplification and chromosomal 1p/19q deletions have been characterized in glioblastoma pathogenesis, molecular subtyping as well as treatment response . In the past decades, signaling pathway deregulation was shown to be finely tuned by genomic and epigenetic changes, in both transcriptional and post-transcriptional ways [46, 47]. Post-translational modifications, including protein ubiquitination, are well-known for its role in protein metabolism, especially in protein degradation . As a hub link in protein ubiquitination and signaling transduction, the alteration of E3 ubiquitin ligases are emerging as oncogenes and tumor suppressors in cancer, including glioblastoma [14, 49]. Meanwhile, technological advances proposed novel strategies for the un-druggable targets in cancer, such as ubiquitin modifiers and transcription factors [50, 51]. Therefore, revealing the function and molecular mechanism of novel E3 modifiers would deepen our understanding of glioblastoma pathogenesis and provide novel options for its treatment.
CRISPR-Cas9 was first proposed as gene knock-out strategy, and then used for gene activation, inhibition and other applications . Based on sgRNAs library, CRISPR screening usually include genome wide and targeted screening, such as kinase libraries . Positive and negative selection strategies were also used for uncovering therapeutic targets, drug sensitive and resistant genes . Here, using the hypothesis that cells with certain genes loss of function may gain growth advantage, we acquired 299 differential genes. Considering that the in-vitro experiment may not really reflect the virtual state in clinical glioblastoma samples, we then studied the expression and prognostic significance of differential genes with both TCGA and CGGA datasets. At last, three genes were left: BIRC3, RNF135 and RNF185. Consistent with previous studies, BIRC3 [33–35] and RNF135  were all previously reported as oncogenes in glioma, while we first revealed that RNF185 may play a tumor suppressor role in glioblastoma.
Ring Finger Protein 185 (RNF185) has been shown to regulate cell autophagy , innate immune responses , osteogenic differentiation  and cystic fibrosis . Moreover, RNF185 was proved to promote gastric cancer metastasis , while its role in glioma remains to be explored. Interestingly, our screening results in U87 cells showed RNF185 as a tumor suppressor. Further validation with other cell lines U251 and DBTRG also supported the CRISPR screening results. The seemingly contradictory function of RNF185 has also been observed in other E3 ubiquitin ligases in different cancer types. For example, E3 ubiquitin-protein ligase (HUWE1) has been shown to promote cancer progression in lung cancer , gastric cancer  and multiple myeloma , while tumor suppressive functions was also reported in prostate cancer  and other cancer types . The converse function of RNF185 may be attributed to its substrates in distinct cancer types.
After validating the RNF185 as a tumor suppressor in glioblastoma, we interrogated the molecular basis for its decreased expression. As a result, we analyzed microRNAs that may induce RNF185 degradation, by bioinformatics prediction, functional studies and molecular validation assays. MiR-587 was finally proved to repress RNF185 expression, and play an oncogenic role in enhancing cell proliferation, repressing cell apoptosis and induce cell migration. This is the first time we prove miR-587 as a tumor promoting miRNA in glioblastoma by targeting RNF185. Moreover, we also studied the transcriptional regulators of RNf185 in glioblastoma samples. Histone transactivation marks peaks in fetal, adult brain and glioma cells shows that RNF185 may also under the transcriptional repression, with development and glioblastoma pathogenesis. At last, the DNA methylation levels in RNF185 promoters also supported the above phenomena. Taken together, we conclude that combined transcriptional repression and elevated miR-587 expression may reduce the RNF185 mRNA expression in glioma.
We also recognize that there are still some problems remains to be solved. First, further in-vivo evidence would make the conclusion that RNF185 play a tumor surpassing role in glioblastoma more solid. Secondly, the substrate and exact functional way of RNF185 in glioblastoma still remain to be revealed. Finally, the strategy of delivering RNF185 for glioblastoma treatment would provide more information for the future clinical application.