Tumor cells-derived extracellular vesicles carry circ_0064516 competitively inhibit microRNA-6805-3p and promote cervical cancer angiogenesis and tumor growth

The current study tried to elucidate the regulatory role of tumor cell-derived exosomes (Exos)-circ_0064516 in angiogenesis and growth of cervical cancer. Related cirRNAs and downstream target genes were identified through bioinformatics analysis. Exos were isolated from cervical cancer cell line CaSki, followed by co-cultured with human umbilical vein endothelial cells (HUVECs). Then, the roles of circ_0064516, miR-6805-3p, and MAPK1 in migration and angiogenesis of HUVECs were assayed. Furthermore, xenografted tumors were transplanted into nude mice for in vivo validation. In vitro assay validated highly expressed circ_0064516 in cervical cancer cells. Tumor cell-derived Exos carried circ_0064516 to HUVECs. circ_0064516 increased MAPK1 expression by binding to miR-6805-3p, thus enhancing migration and angiogenesis. Exos containing circ_0064516 also promoted tumorigenesis of cervical cancer cells in nude mice. We confirmed the oncogenic role of tumor cell-derived Exos carrying circ_0064516 in cervical cancer progression through miR-6805-3p/MAPK1.</p

Expression of <i>HYH</i> transcripts and proteins in the shoots and roots of WT and <i>hy5-2</i> seedlings.

<p>(<b>A</b>,<b>B</b>) Expression of the <i>HYH</i>::<i>GUS</i> fusion in shoots of 7-d-old light-grown WT (A) and <i>hy5-2</i> (B) seedlings. Scale bar in (A,B): 1 mm. (<b>C</b>,<b>D</b>) Expression of the <i>HYH</i>::<i>HYH-GFP</i> fusion in the cotyledons of WT (C) and <i>hy5-2</i> (D) seedlings. (<b>E</b>,<b>F</b>) Expression of the <i>HYH</i>::<i>HYH-GFP</i> fusion in the hypocotyls of WT (E) and <i>hy5-2</i> (F) seedlings. (<b>G</b>,<b>H</b>) Expression of the <i>HYH</i>::<i>ERGFP</i> fusion in the roots of WT (G) and <i>hy5-2</i> (H) seedlings. (<b>I</b>,<b>J</b>) Expression of the <i>HYH</i>::<i>HYH-GFP</i> fusion in the roots of WT (I) and <i>hy5-2</i> (J) seedlings. Scale bars in (C–J): 50 μm.</p

HY5 binds to the <i>HYH</i> promoter.

<p>(<b>A</b>) Schematic model of <i>HYH</i> genomic sequences. Four fragments of the <i>HYH</i> promoter region (frag 1–4; indicated by lines) were selected for use in a yeast one-hybrid assay (Y1H). Asterisks represent the putative HY5 binding sites. The “ACGT” sequences targeted by HY5 are highlighted for frag 3. (<b>B</b>) Y1H demonstrating that HY5 binds to the <i>HYH</i> promoter at frag 3. <i>COP1</i> serves as a positive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180449#pone.0180449.ref027" target="_blank">27</a>] and the empty vector expressing the AD domain alone is the negative control. (<b>C</b>) An EMSA confirmed the binding of HY5 to the promoter of <i>HYH</i>. Scheme showing the structure of the frag 3 region. The squares indicate ACGT-containing elements (ACE) in frag 3. The corresponding electrophoretic mobility shift assay (EMSA) probes are illustrated as frags 3–1, 3–2, and 3–3. HY5 was produced in an <i>in vitro</i> expression system. The labeled probes were 3' FAM oligonucleotides, while non-labeled probes served as competitors. Arrows indicate the shifted bands.</p

Expression patterns of <i>HY5</i> and <i>HYH</i> and phenotypic analysis in response to light.

<p>(<b>A</b>) Expression patterns of <i>HY5</i>::<i>GUS</i> and <i>HYH</i>::<i>GUS</i> reporter lines in the shoots of plants grown under light, dark, and dark-to-light conditions for 7 d. Scale bar: 1 cm. (<b>B</b>) The increase in hypocotyl length of four-day-old etiolated wild-type (WT), <i>hy5-2</i>, <i>hyh</i>, and <i>hy5-2 hyh</i> seedlings exposed to light for an additional day (n>12). Error bars represent SD (standard deviation). (<b>C</b>) Expression patterns of <i>HY5</i>::<i>GUS</i> and <i>HYH</i>::<i>GUS</i> reporter lines in the roots of plants grown in the light for 7 d. The middle and right panels are cross sections of the roots shown in the left panels, at positions denoted by the upper and lower dashed lines, respectively. Scale bar: 50 μm. (<b>D and E</b>) Primary root length (D) and root apical meristem (RAM) size (E) of four-day-old etiolated WT, <i>hy5-2</i>, <i>hyh</i>, and <i>hy5-2 hyh</i> seedlings transferred to constant light (n = 20). Error bars represent SD. In C, D, and E, error bars with different letters indicate a significant difference at p<0.05 (<i>t</i> test).</p

Dissection of <i>HY5/HYH</i> expression in <i>Arabidopsis</i> reveals a root-autonomous HY5-mediated photomorphogenic pathway

<div><p>ELONGATED HYPOCOTYL 5 (HY5), a member of the bZIP gene family, is a positive regulator of the light signaling pathway in <i>Arabidopsis thaliana</i>. Whereas the <i>hy5</i> mutant exhibits an elongated hypocotyl when grown in the light, the <i>hy5 homolog</i> (<i>hyh</i>) mutant does not. Although the functions of HY5 and HYH in light-mediated seedling development have been revealed, the tissue-specific expression patterns of <i>HY5</i> and <i>HYH</i> and their interconnected regulation are largely unknown. Here, we report that HY5 regulates <i>HYH</i> expression in roots and contributes to root growth under different light conditions. We generated <i>HY5</i> and <i>HYH</i> transcriptional and translational fusion reporter lines to investigate their expression patterns. <i>HY5</i> was constitutively expressed in all root tissues, while <i>HYH</i> was predominantly expressed in root xylem cells. Root growth after a dark-to-light transition was perturbed in the <i>hy5</i> and <i>hy5hyh</i> mutant lines, but not in the <i>hyh</i> mutant line, indicating that HY5 plays a major role in light-regulated root growth. Light-induced <i>HY5</i>/<i>HYH</i> expression occurred autonomously in roots. <i>HYH</i> expression in roots was decreased in the <i>hy5</i> mutant, suggesting that HY5 regulates <i>HYH</i> expression. Collectively, these results indicate that an organ-specific HY5-mediated pathway controls root photomorphogenic development independently of light signaling in the shoot.</p></div

Root-autonomous regulation of <i>HY5</i> and <i>HYH</i> expression in response to light.

<p>(<b>A</b>) Diagram of the decapitation procedure. Dashed lines mark the cutting positions. Letters in brackets represent the corresponding panels. (<b>B</b>,<b>C</b>,<b>F</b>,<b>G</b>) Expression of HY5 protein fusion lines in roots of decapitated or intact plants under dark-to-light and light-to-dark transitions. (<b>D</b>,<b>E</b>,<b>H</b>,<b>I</b>) Expression of HYH protein fusion lines in roots of decapitated or intact plants under dark-to-light and light-to-dark transitions. (<b>J</b>,<b>K</b>) Expression of HY5 protein fusion lines in the roots of intact seedlings under dark and light growth conditions. (<b>L</b>,<b>M</b>) Expression of HYH protein fusion lines in the roots of intact seedlings under dark and light growth conditions. Images were acquired using a fluorescence stereomicroscope. The dotted line indicates the root outline. Scale bar in (B): 50 μm.</p

<i>HY5</i> and <i>HYH</i> transcripts and proteins were induced in the root in response to light.

<p>(<b>A–H</b>) Expression of <i>HY5</i> promoter fusion (<b>A–D</b>) and protein fusion (<b>E–H</b>) lines in roots under light, dark, light-to-dark and dark-to-light transition conditions. (<b>I–P</b>) Expression of the <i>HYH</i> promoter fusion (<b>I–L</b>) and protein fusion (<b>M–P</b>) lines in roots under light, dark, light-to-dark, and dark-to-light transition conditions. Scale bar represents 50 μm.</p