Emre Özgür

Long non-coding RNAs with low expression levels in cells are enriched in secreted exosomes.

Long non‐coding RNAs (lncRNAs) are involved in regulating chromatin modifications, gene transcription, mRNA translation, and protein function. We recently reported a high variation in the basal expression levels of a panel of lncRNAs in HeLa and MCF‐7 cells and their differential response to DNA damage induction. Here, we hypothesized that lncRNA molecules with different cellular expression may have a differential abundance in secreted exosomes, and their exosome levels would reflect cellular response to DNA damage. MALAT1, HOTAIR, lincRNA‐p21, GAS5, TUG1, CCND1‐ncRNA in exosomes secreted from cultured cells were characterized. A different expression pattern of lncRNAs in exosomes was seen compared to cells. RNA molecules with relative low expression levels (lincRNA‐p21, HOTAIR, ncRNA‐CCND1) were highly enriched in exosomes. TUG1 and GAS5 levels were moderately elevated in exosomes, whereas MALAT1—which was the most abundant molecule in cells—was present at levels comparable to its cellular levels. lincRNA‐p21 and ncRNA‐CCND1 were the main molecules; exosome levels of them best reflect the change of their cellular levels upon exposure of the cells to bleomycin‐induced DNA damage. In conclusion, we provide evidence that lncRNAs have a differential abundance in exosomes, indicating a selective loading.

Investigation of circulating lncRNAs in B-cell neoplasms.

Long non-coding RNAs (lncRNA) which are longer than 200 base pairs in length, play an important role in cellular machinery. Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are neoplasms of B-cells. In our study we aimed to investigate circulating lncRNA levels of CLL and MM patients. For this purpose we selected 5 candidate lncRNAs (TUG1, LincRNA-p21, MALAT1, HOTAIR, and GAS5) where the first two are regulated by p53. Analyses were performed by real-time PCR using cDNA synthesized from plasma RNAs. In both disease groups differential levels of plasma lncRNAs were observed. LincRNA-p21 was the only molecule displaying significant changes in the CLL group while all remaining lncRNAs showed significant differences in the MM group. In the MM group only TUG1 showed higher levels than the healthy volunteers. In conclusion, the expression levels of the candidate lncRNA molecules display a general trend for tissue- and disease-specific expression which can provide important potential biomarkers specific to the particular disease type. However, further studies are necessary to elucidate their involvement in disease development and progression.

Exosomal lncRNA-p21 levels may help to distinguish prostate cancer from benign disease

Exosomes are membranous vesicles containing various biomolecules including lncRNAs which are involved in cellular communication and are secreted from many cells including cancer cells. In our study, investigated the exosomal GAS5 and lincRNA-p21 lncRNA levels in urine samples from 30 patients with prostate cancer (PCa) and 49 patients with benign prostatic hyperplasia. Quantification of lncRNA molecules was performed by real-time PCR. We observed a significant difference in the exosomal lincRNA-p21 levels between PCa and BPH patients whereas the GAS5 levels did not reveal a difference. Our data suggest that the discriminative potential of exosomal lincRNA-p21 levels may help to improve the diagnostic prediction of the malignant state for patients with PCa.

Induction of p53-inducible microRNA miR-34 by gamma radiation and bleomycin are different

microRNAs (miRNAs) are small molecules in their mature form and master regulators of gene expression. Recent work has shown that miRNAs are involved in the p53 network. Of the various miRNAs, miR-34 is regulated by the p53 protein. miR-34 can be induced by ionizing radiation (IR) in vitro and in vivo. However, there is no data in the literature for induction of miR-34 by a chemical agent inducing DNA damage. Here we studied the expression of miR-34 in HeLa and MCF-7 cells exposed to genotoxic stress-induced by bleomycin (BLM) or γ-radiation. We first analyzed p53 accumulation upon DNA damage induction. The basal level of p53 in MCF-7 cells was higher (approx. 6-fold) than in HeLa cells, and its accumulation was similar for both DNA-damaging agents in both cell lines. We have shown that miR-34 is significantly induced by γ-radiation in HeLa cells, but not in MCF-7 cells. BLM did not significantly affect miR-34 expression in both cell types. In conclusion, our findings reveal that miR-34 induction by genotoxic stress may be cell-type specific.

Accumulation of GAS5 in exosomes is a marker of apoptosis induction

Long non-coding RNAs (lncRNAs) are key regulatory molecules in many fundamental cellular processes and their deregulation is assumed to contribute to carcinogenesis. Exosomal lncRNAs are thought to be involved in the dissemination of cell signals to control local cellular microenvironments. In the current study, exosomal expression of growth arrest specific 5 (GAS5), an inhibitor of cell proliferation and promoter of apoptosis, was evaluated in apoptotic processes initiated by different mechanisms. Therefore, MCF-7 and MDA-MB-231 breast cancer cells were treated with Taxol (2 and 10 nM) and bleomycin (2 and 10 ng/ml) for 24 h. Following cell viability determination and measurement of apoptosis, cellular and exosomal expression levels of GAS5 were investigated using a quantitative polymerase chain reaction assay. The findings indicate that Taxol is more toxic than bleomycin at the indicated doses and the effect was more evident in the MCF-7 cells. Despite varying toxicity rates, comparable levels of apoptotic nucleosomes were measured between Taxol- and bleomycin-treated cells. Upon drug treatment, cellular expression levels of GAS rose (≤1.5-fold) in the two cell lines. It appears that even a small increase in cellular expression leads to exosomal enrichment, as the accumulation of GAS5 in exosomes was marked in the MCF-7 cells (≤5.8-fold). Compared with the MCF-7 cells, the extent of GAS5 enrichment in the exosomes secreted from MDA-MB-231 cells was moderate (≤1.9-fold), potentially as a result of reduced cell death. The present study indicates that GAS5 accumulation in exosomes is a prevalent event in apoptotic processes that are initiated by different mechanisms.

Global quantification of heterochromatin-associated histone methylations in cell lines with differential sensitivity to ionizing radiation

Histone modifications are involved in the DNA damage response (DDR). Here, by utilizing an ELISA immunoassay we assessed the methylation at H3K9 (H3K9me2 and H3K9me3) in two cell lines with differential sensitivity to radiation-induced apoptosis, HeLa (sensitive) and MCF-7 (resistant). We found that DNA damage induction by γ-irradiation leads to considerable accumulation (up to 5-fold) of H3K9me2 and H3K9me3, but not of H4K20me3 (control modification) in MCF-7 cells (p<0.05). Interestingly, a lower dose (2 Gy) was more effective than 5 Gy. In HeLa cells a smaller effect (approx. 1.5-1.8-fold) was evident only at 5 Gy. In conclusion, our findings reveal that DNA damage leads to specific accumulation of H3K9me2 and H3K9me3 in a cell-type specific manner.