Genome-wide CRISPR knockout screening identified G protein pathway suppressor 2 as a novel tumor suppressor for uveal melanoma metastasis.

PURPOSE: Uveal melanoma (UM) is the most common intraocular malignant tumor in adults. Due to the lack of effective treatments for metastatic UM, the survival of UM has not changed over the past 3 decades. Therefore, it is important to identify essential genes regulating the metastasis of UM. METHODS: In this study, a genome-wide CRISPR knockout screen in an orthotopic mouse model of UM was performed to identify the regulatory genes conferring the metastatic phenotype. Loss-of-function analyses were performed to explore the function of G protein pathway suppressor 2 (GPS2) in UM metastasis in vitro and in vivo. RNA sequencing was performed to investigate the molecular mechanism underlying the function of GPS2 as a tumor suppressor in UM. RESULTS: Among the highest-ranking genes, we found several validated tumor suppressors, such as SHPRH, GPS2, PRPH2, and hsa-mir-1229; GPS2 was chosen as the candidate gene for further studies. GPS2 was lower expressed in the tumor tissues of UM patients. Furthermore, knocking-down GPS2 promoted the proliferation and metastatic abilities of UM cells both in vivo and in vitro. Finally, analysis of the transcriptome data revealed that silencing GPS2 upregulates oncogenic signaling pathways MAPK and PI3K-Akt, and in the meantime downregulates tumor suppressor signaling pathway Slit/Robo in UM cells. CONCLUSION: Altogether, our study proved that the GPS2 gene functions as a tumor suppressor and might be a novel potential therapeutic target for UM treatment.

P38 Mitogen-Activated Protein Kinase Protects Against Retinoblastoma Through Regulating USP22/SIRT1/SOST Axis.

Retinoblastoma (RB) is the most common intraocular malignancy in children. It has been previously reported that p38 MAPK is related to the pathogenesis of RB. Here we aim at investigating how p38 MAPK affected RB progression through mediating USP22/SIRT1/SOST axis. In this study, Thirty-two cases of RB and normal retinal tissues were collected. The expression of p38 MAPK, phosphorylation of p38 MAPK (P-p38 MAPK), USP22, SIRT1 and SOST in clinical tissues and cells was measured using RT-qPCR, IHC assay or western blot analysis. Cell proliferation was detected by CCK-8. Apoptosis rate of cells was examined by flow cytometry. Cell migration was evaluated using scratch test. Cell invasion ability was examined by Transwell assay. Co-immunoprecipitation (CO-IP) was utilized to measure the deubiquitination of USP22 on SIRT1. In vivo, mice were respectively injected with plasmids and the tumor growth as well as the tumor weight were detected. Results showed that p38 MAPK, P-p38 MAPK and SOST were poorly expressed in RB tissues and cells whereas USP22 and SIRT1 were overly expressed. P-p38 MAPK inhibited the expression of USP22, and overexpression of USP22 eliminated the inhibitory roles of P-p38 MAPK on tumor growth, as well as cell proliferation, migration and invasion. USP22 stabilized and promoted the expression of SIRT1 through its deubiquitination function. Silencing the expression of SIRT1 contributed to boosted expression of SOST, thus suppressing the growth of tumor cells. Collectively, the phosphorylation of p38 MAPK regulates the SIRT1/SOST axis to protect against RB via silencing USP22. The findings present some cues for a further approach to RB.

miR-133a-5p suppresses gastric cancer through TCF4 down-regulation.

BACKGROUND: The effect of microRNAs (miRNA) on cancer regulations has received a considerable amount of attention recently. MiR-133a-5p has been identified as an anti-tumor miRNA in several types of cancers. However, the effect of miR-133a-5p on gastric cancer (GC) have not been uncovered. In this study, we sought to evaluate the regulation of TCF4 expression by miR-133-5p and the role of the miR-25-3p/TCF4 axis in the progression of GC, with the aim of identifying a potential therapeutic target for GC. METHODS: TCGA (The Cancer Genome Atlas), GTEx (The Genotype-Tissue Expression) and GEO (Gene Expression Omnibus) database were used to analyze the expression and prognosis. We performed MTT and EdU assays to elucidate the effect on cell replication. Apoptotic cells were stained with annexin V-fluorescein isothiocyanate and propidium iodide to stain, and then analyzed by flow cytometry. The effect on cell metastasis was investigated in wound healing and transwell assays. A dual-luciferase reporter assay was used to check for the direct targeting of TCF4 by miR-133a-5p. Bioinformatic analysis of the relationship of TCF4 with tumor microenvironment and the signaling cascade of TCF4 was finally performed. RESULTS: We found that the level of miR-133a-5p was decreased in both tumor tissues and GC cell lines. MiR-133a-5p inhibited cell growth and metastasis, but promoted cell apoptosis. MiR-133a-5p directly targeted TCF4 and downregulated its expression. TCF4 was highly expressed in tumor and higher level of TCF4 indicated poorer prognosis. Moreover, TCF4 overexpression reversed the aforementioned anti-tumor activity of miR-133a-5p. The expression level of TCF4 was significantly correlated with tumor-infiltrating immune cells. CONCLUSIONS: Our findings altogether reveal that miR-133a-5p can serve as a tumor suppressor in gastric cancer via the miR-133a-5p/TCF4 pathway.