GOLPH3 Regulates Exosome miRNA Secretion in Glioma Cells

Abstract

We aimed to examine whether golgi protein GOLPH3 could affect the secretion of glioma cell-derived exosomes. The exosomes were extracted by ultra-centrifugation from the supernatant of U251 and U87 cell cultures and identified by transmission electron microscopy (TEM), Malvern analyzer, and western blot. The quantity of exosomes was examined by measuring the total protein levels and the number of multiple vesicle bodies (MVBs), the source of exosomes. The exosome miRNAs were analyzed by high-throughput sequencing followed by GO and KEGG analysis, and validated by qRT-PCR. GOLPH3 could not affect the total protein levels of exosomes and the number of MVBs. However, we found 149 differentially expressed miRNAs in exosomes between vector and GOLPH3 over-expression group, and 14 miRNAs were only examined in GOLPH3 over-expression cells. The predicted target genes of these miRNAs had functions in binding and catalytic activity, which were enriched in the pathways of endocytosis, RNA transportation, thyroid hormone signaling and miRNAs in cancer. GOLPH3 could not affect the quantity of exosomes, but rather contribute to miRNA expression in exosomes, which may play some functions in the promotion effect of GOLPH3 on glioma development.

Discussion

Over several decades, exosomes have been found in almost all body fluids, including blood, urine, saliva, cerebrospinal fluid and ascites (Street et al. 2017). These exosomes with specific profiles of miRNAs, proteins, and lipids can act as a “fingerprint” or a “signature” of their parental cell, reflecting their cellular origins and the corresponding physiological conditions. Therefore, exosomes and their cell- or condition-specific cargos may give insight into the cellular processes and be used as biomarkers for many diseases (Zhang et al. 2019).

Because GOLPH3 plays a significant role in the exit of vesicles from the Golgi for trafficking to the plasma membrane (Kuna and Field 2019), we reasoned that GOLPH3 may play a role in exosome secretion. Unfortunately, we found that GOLPH3 could not affect the quantity of the exosome. In addition, Gross et al. showed that, in Drosophila and human cells, wnts were secreted in exosomes and the latter carried wnts on their surface to induce wnt signaling activity in target cells (Gross et al. 2012). However, although we found that wnt2b could be secreted into exosomes, the level of wnt2b, as well as endo- and exo-GOLPH3, showed no change after GOLPH3 over-expression, indicating that the promotion effect of GOLPH3 on wnt2b secretion (Lu et al. 2018) was not performed as exosomes.

As important small non-coding RNA, miRNAs take part in post-transcriptional regulation of biological processes. The expression profiles of miRNAs vary significantly in normal and disease tissues and show physiology-specific characteristics (Heinzelmann et al. 2011). Cumulative evidence indicated that miRNAs could be used as valuable pathological and therapeutic biomarkers in both cells and exosomes (Bekris and Leverenz 2015; Lei et al. 2017). High-throughput small RNA-sequencing technology offers convenience to explore miRNAs in exosomes and has generated a lot of data for exosomes under different circumstance. A comparative study of these data could contribute to explore miRNA biomarkers in exosomes and get to understand their regulatory roles mediated by exosomes in recipient cells. Interestingly, by conducting exosomal high-throughput small RNA-sequencing, we found 149 differentially expressed mature miRNAs between the GOLPH3 over-expression group and its vector control, and many of which, such as miR-21 and miR-182, were dysregulated in GBM (Huang et al. 2018b; Wang et al. 2012; Wang et al. 2019; Zang et al. 2018).

Among the above miRNAs, one of the most studied up-regulations in GBM is miR-21 (Masoudi et al. 2018). It is reported that down-regulation of miR-21 reduces the oncogenic potential of GBM cell lines (Krichevsky and Gabriely 2009) and decreases tumor growth in nude mice (Gaur et al. 2011; Zhou et al. 2010). The well-known targets of miR-21 are tumor suppressors, such as PTEN, RECK and PDCD4, through which miR-21 promotes GBM proliferation (Gaur et al. 2011; Shi et al. 2015; Zhou et al. 2010). Transcriptional profiling of cells whose miR-21 was knocked down revealed changes in the expression of genes correlated with DNA damage response, regulators of cell cycle arrest, and positive regulators of apoptosis (Gabriely et al. 2008). Taken together, miR-21 may be a particularly fascinating subject for exosomal therapeutic exploitation in GBM. In addition, miR-376c-3p was reported to promote or inhibit the progression of many human cancers such as gastric cancer, hepatocellular carcinoma, and neuroblastoma (Bhavsar et al. 2018; Liu et al. 2019; Wang et al. 2018). Because miR-376c-3p was the highest increased miRNA in exosome after GOLPH3 over-expression, it will be interesting to examine the role of miR-376c-3p in glioma progression.

KEGG pathway enrichment analysis showed that those miRNAs target genes were enriched mainly in T cell differentiation, chemokine signaling pathways, cell adhesion molecules, apelin signaling pathways, endocytosis, and RNA transportation, etc. More interestingly, we found that some miRNAs, such as miR-1288, miR-299, and miR-1292, only existed in GOLPH3 over-expression cells, indicating that they may play roles in glioma cell biological functions. A number of papers reported that exosome miRNAs play important roles in tumor progression (Huang et al. 2018b; Zeng et al. 2017) and exosomes may promote cancer cell survival in the metastatic place by forming a pre-metastatic niche (Liang et al. 2016; Liu and Cao 2016); thus, it will be interesting to test the function of these miRNAs in glioma cell growth and migration/invasion.

In summary, GOLPH3 did not promote the secretion quantity of exosomes but affected the expression spectrum of miRNAs in GBM cell-derived exosomes. Since the target genes of the differentially expressed miRNAs in exosomes were involved in T cell differentiation, chemokine signaling pathways, cell adhesion molecules, apelin signaling pathways, endocytosis, and RNA transportation, etc., we deduce that they will play some roles in glioma progression by regulating the above-mentioned cell processes or other biological functions. Therefore, a complete understanding of the roles of GBM cell-derived exosomes will contribute to identify new candidates for treatment of GBM.

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https://link.springer.com/article/10.1007%2Fs12031-020-01535-6