CRISPR screening of E3 ubiquitin ligases reveals Ring Finger Protein 185 as a novel tumor suppressor in glioblastoma repressed by promoter hypermethylation and miR-587

SgRNAs of 557 E3 ubiquitin ligases were designed, and gene knockdown efficiency was validated with qRT-PCR examinations. The ubiquitin ligase gene list and validated sgRNA sequences were shown in Additional file 1: Table S1.

Based on the amplicons sequencing, pooled sequencing reads were compared with human genome HG38. The reads of E3 ligase genes in the fourth passage were compared with the passage 0. By using reads fold change ≥ 2 or ≤ 0.5 and p value < 0.05, differential genes were screened.

Class-three gene expression of GDC TCGA Glioblastoma (GBM) dataset was downloaded from UCSC Xena website (https://​xena.​ucsc.​edu/​), and applied for subsequent survival analysis. Log-Rank test of patients with higher and lower gene expression was conducted, and Kaplan–Meier survival plot was shown. For Chinese Glioma Genome Atlas (CGGA) analysis, gene expression and prognostic significance in WHO II–III grade glioma samples, the relationship of gene methylation patterns and parameter of brain cancer etiology (including Histology type, WHO grade, IDH mutation status and 1p/19q deletion) was conducted with the online tool.

Glioblastoma cells lines U87, U251 and DBTRG were purchased from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10% of fetal bovine serum (Gibco) and 1% of penicillin–streptomycin at 37 °C, 5% CO₂ humidified atmosphere. For cell transfection, the cells were cultured in 6 well plates, when the cell reached 75% confluence. The negative control, pcDNA3.1-RNF185, negative control, and miR-587 inhibitors were synthesized by Shanghai Gene Pharma Co., Ltd (Shanghai, China). The negative control, pcDNA3.1-RNF185 were transfected into glioblastoma cells using Lipofectamine 2000 transfection reagent (Invitrogen, USA) according to manufacturer’s instructions. Negative control or miR-587 inhibitors at concentration of 5 nM, 10 nM, 20 nM, 40 nM were transfected into glioblastoma cells using lipofectamine 2000 transfection reagent according to manufacturer’s instructions. The overexpression and knockdown efficiency were validated with qRT-PCR. GAPDH and U6 were used as the house keeping gene. Primers for RNF185, miR-587, GAPDH and U6 are as follows: RNF185: F-5′-GTGTTTACATCAGTGGTTGGAGA-3′; R-5′-GTGCTGCCCCTTCCATAGAG-3′;

GAPDH: F-5′-GGAGCGAGATCCCTCCAAAAT-3′; R-5′-GGCTGTTGTCATACTTCTCATGG-3′;

miR-587: F-5′-CCAGGCAAGAGAGAGTTGCTG-3′; R-5′-AGTCACAGGTGCAGACACATT-3′;

U6: F-5′-CTCGCTTCGGCAGCACA′; R-5′-AACGCTTCACGAATTTGCGT-3′. Anti-miR-587 miScript miRNA inhibitor was purchased from Qiagen Inc. (Valencia, CA, USA).

U251 and DBTRG cells were seeded into 96-well plates (2 × 10⁴ cells/well) and cultured for 12 h. After washing, U251 and DBTRG cells were incubated with 10% CCK-8 (Dojindo Molecular Technologies, Inc., Minato-ku, Tokyo, Japan) and optical density measured using a xMark Microporous Plate Absorption Spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The apoptotic rate of U251 and DBTRG cells was detected using an Annexin V, 633 Apoptosis Detection Kit (Dojindo Molecular Technologies), following the kit instructions. U251 and DBTRG cells were seeded into 6-well plates (5 × 10⁵ cells/well) and cultured for 12 h. Next, U251 and DBTRG cells were incubated with Annexin V, followed by propidium iodide (PI) buffer for 15 min at 25 °C in a dark room. Subsequently, apoptotic cells were quantified using a NovoCyte 1040 flow cytometer (ACEA Biosciences, Inc., Zhejiang, China).

Cells were lysed in a mixed buffer that contained RIPA, NaF, and PMSF. Protein concentrations were analyzed using a BCA protein assay kit (Tiangen Biotech Co., Ltd., Beijing, China). Protein was resolved by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were incubated overnight with the indicated primary antibodies at 4 °C and then incubated with appropriate secondary antibodies for 2 h at room temperature. Protein bands were detected by ImageQuant LAS4000 (General Electric Company, Boston, MA, USA) and quantified by ImageJ software. GAPDH was detected as a loading control. Primary antibodies used in the study were listed as the following: TNFR1 (Proteintech, Cat No. 60192-1-AP), BAD (Proteintech, Cat No. 10435-1-AP), FAS (Proteintech, Cat No. 13098-1-AP), Cleaved Caspase3 (Proteintech, Cat No. 66470-2-Ig), and GAPDH (Proteintech, 60004-1-Ig).

The migratory ability was assessed through wound-healing assay. U251 and DBTRG cells (2 × 10⁵) were seeded into a 6-well plate allowed to reach confluence. Then, uniform wounds were scraped using a 200-µl pipette tip across the cell monolayer. Cells were rinsed with phosphate-buffered saline and cultured in the medium. Then, the wound closures were observed after 24 h. The initial gap length (0 h) and the residual gap length (24 h) after wounding were calculated from photomicrographs using an Olympus fluorescence microscope (Olympus).

PmirGLO Dual-Luciferase miR Target Expression Vector (Promega) was used to assess the direct binding of miR-587 to RNF185 3′UTR. The wild-type reporter construct pmirGLO-RNF185-WT or the mutant reporter construct (pmirGLO-RNF185-MUT) with miR-587 inhibitors were co-transfected in 293T cells. After transfection for 24 h, firefly luciferase levels were measured using a Dual-Luciferase Reporter Assay System (Promega, Wisconsin) and normalized to Renilla luciferase activity. Each experiment was repeated at least three times.

For the trans-well assay, Matrigel were seeded into the upper chamber. The 3 × 10⁵ (without Matrigel) or 5 × 10⁵ cells (with Matrigel) in serum‐free medium were seeded into the upper chamber. The lower chamber was filled with medium supplemented with 10% FBS as a chemo-attractant. After 48 h of incubation, the cells on the upper surface of the filter were removed with a cotton swab, and cells that invaded through the filter or Matrigel sticked to the lower surface of the filter, were fixed and stained with 0.5% crystal violet, and counted under a light microscope.

All statistical analyses were performed using Graphpad v 8.0 software. Each experiment was performed in triplicates, and the data were shown as the mean ± SD, unless otherwise stated. Kaplan–Meier analysis with log-rank test was used for survival analysis. Student’s t-test was used to compare the mean values. Pearson chi-square test was used to analyze the association between RNF185 expression and miRNAs. P value < 0.05 was considered to be statistically significant.

To explore the function of E3 ubiquitin ligases on tumor growth phenotype of glioma, a sgRNAs library of 557 E3 ligase genes were constructed, and followed by lentivirus packaging. Then glioma cancer cell U87 was used as a representative cell line for the subsequent cell growth screening. After the cell passage for 4 generations, cells were collected, and the DNA amplicons were applied for sequencing. The identified gene reads in 4^th generation were compared with the initial gene reads, and cell with enriched and lost reads were statically compared (Fig. 1A). By using reads number cutoff of ≥ 2 or ≤ 0.5 and p-value < 0.05, all 299 (9 enriched and 290 lost) significantly enriched or lost genes (SELGs) were acquired (Fig. 1B). The reads of enriched or lost genes in all 3 replicates were shown in Fig. 1C. At last, the domain of significant enriched proteins was analyzed, and as shown in Fig. 1D, proteins with Ring finger ranked top one, followed by Broad-Complex, BTC and C-termincal Kelch. The sgRNAs sequences of 557 E3 ubiquitin ligases and analyzed results were shown in Additional file 1: Table S1.

Then the clinical significance of all SCGs was conducted for further analysis. First, the overall survival analysis was conducted with TCGA glioma multiforme (GBM) dataset. By using median expression of each SCG as cut-off, Log-Rank analysis was applied to calculate the Hazard ratio and p-value. All 14 genes (BIRC3, IRF2BPL, KBTBD3, KLHL10, RABGEF1, RNF39, RNF135, RNF185, RNF186, SOCS4, TRIM48, ZBTB5, ZBTB6 and ZBTB8A) showed p-value < 0.05, and the Kaplan–Meier survival plot was shown in Fig. 2. Then the 14 prognostic genes were conducted for further analysis, with CGGA datasets. First, the expression of these 14 genes was compared in WHO II, III and IV grade. As shown in Fig. 3, 8 genes showed significant deregulation in GBM II-IV grade groups, such as BIRC3, IRF2BPL and RNF135 showed consistent higher expression, and decreased expression was observed of genes KBTBD3, RNF185, RNF39, ZBTB5 and ZBTB6. Finally, the prognostic significance of these 14 genes was further analyzed in CGGA datasets, and four genes showed significance. BIRC3, KLHL10 and RNF135 showed unfavorable biomarker performance in glioma samples, and only RNF185 may serve as a favorable marker (Fig. 4).

Of the 3 genes (BIRC3, RNF135 and RNF185) with both differential expression and prognostic significance, BIRC3 [33‐35] and RNF135 [36] were all previously reported as oncogenes in glioma. Ring Finger Protein 185 (RNF185) has been shown to regulate cell autophagy [37], innate immune responses [38], osteogenic differentiation [39] and cystic fibrosis [40]. Moreover, RNF185 was proved to promote gastric cancer metastasis [41], while its role in glioma remains to be explored. Interestingly, though the function of RNF185 was shown to promote gastric cancer, our screening results in U87 cells showed RNF185 as a tumor suppressor. So, we further validated the function of RNF185 in other glioma cell lines, U251 and DBTRG, with overexpression strategy. As the CCK8 assay results showed in Fig. 5A, RNF185 overexpressed U251 and DBTRG cells exhibits decreased cell number, suggesting that RNF185 inhibits cell proliferation. Then we examined the cell apoptosis rate, and RNF185 overexpressed U251 and DBTRG cells demonstrated significant higher apoptosis rate, compared with control cell lines (Fig. 5B). Western blotting assay revealed that overexpression of RNF185 significantly increased the protein level of TNFR1, BAD, FAS, and Cleaved Caspase3 (Fig. 5C), indicating the promotion of apoptosis. At last, the migration capability was studied with wound healing assays, and as shown in Fig. 5D, RNF185 overexpression significantly attenuated the migration distance in both cell lines. Transwell assay also found that overexpression of RNF185 inhibited the invasion of U251 and DBTRG cells (Fig. 5E). In summary, the above results proved RNF185 as a tumor suppressor in glioma cell lines.

Next, we intend to explore the reason for the decreased expression of RNF185 in glioma cancer samples. MicroRNAs (miRNAs) have been shown to mediated mRNA degradation and repress protein translation by targeting mRNA 3′UTRs, in post-transcriptional level. In order to explore miRNAs that may regulate RNF185 expression in glioma, we first predicted miRNAs with three online tools: miRWalk, TargetScan and miRDB. By comparing the miRNAs predicted in all three tools, 88 common miRNAs were obtained (Fig. 6A). Then the correlation of these 88 miRNAs with RNF185 expression were analyzed with TCGA glioma multiforme (GBM) dataset, and miR-525 and miR-587 showed significant negative correlation with RNF185 (Fig. 6B). As miR-525 was shown to repress glioma cells proliferation in other studies [42], we chose miR-587 for subsequent analysis, for its oncogenic role in multiple cancer types [43‐45] except glioma. So, we first studied the function of miR-587 in glioma cells with CCK8, apoptosis, wound healing assays and transwell assays. As shown in Fig. 6C–F, miR-587 inhibitors phenol-copied the cell proliferation, apoptosis and migration and invasion function of RNF185 overexpressed U251 and DBTRG cells, implying that miR-587 play an oncogenic role in glioma. Next, to explore whether miR-587 regulate RNF185 expression, we examined the expression of RNF185 in miR-587 inhibitor treated glioma cells. As a result, miR-587 inhibitors significantly increased the RNF185 (Fig. 6G). Finally, a dual luciferase assay was conducted to validate the binding of miR-587 on RNF185 3’UTR sequences, by mutating the predicted sites of RNF185 3’UTR sequences. As shown in Fig. 6H, RNF185 wide type (WT) cells showed increased luciferase activity, while no significant changes were observed in mutant (MUT) cells. RNF185 wide type (WT) cells showed increased luciferase activity with dose dependence of miR-587 inhibitor reagent (Fig. 6I). Taken together, elevated miR-587 expression contributes to the decreased expression of RNF185 and promotes glioma proliferation, migration and reduces cell apoptosis.

At last, the transcription molecular events of RNF185 in glioma were analyzed, and the histone activation marks and DNase-Seq peaks in the promoter region of RNF185 was analyzed in fetal brain, adult brain and glioma cells U87 and U251. As shown in Fig. 7A, strong peak signals were observed in fetal brain, followed by adult brain and glioma cells, suggesting that RNF185 may also under transcriptional repression in glioma. Finally, the DNA methylation level in RNF185 promoter and RNF185 expression was analyzed, with TCGA and CGGA datasets (Fig. 7B, C). The strong negative correlation of methylation with mRNA expression suggests that promoter hypermethylation may play an important role for the decreased expression of RNF185 in glioma samples. What’s more, the gene methylation has significantly relationship with parameter of brain cancer etiology (including Histology type, WHO grade, IDH mutation status and 1p/19q deletion) (Fig. 7D). Especially, gene methylation was remarkably different IDH mutation status (P = 0.032). indicating its clinical significance. Collectively, this study shows that Ring Finger Protein 185 as a novel tumor suppressor in glioma multiforme, which is repressed by promoter hypermethylation and miR-587.