Exosomal circRNA_100284 from arsenite-transformed cells, via microRNA-217 regulation of EZH2, is involved in the malignant transformation of human hepatic cells by accelerating the cell cycle and promoting cell proliferation

Abstract

Intercellular communication between malignant cells and neighboring nonmalignant cells is involved in carcinogenesis. In the progression of carcinogenesis, exosomes are messengers for intercellular communication. Circular RNAs (circRNAs) are noncoding RNAs with functions that include regulation of the cell cycle and proliferation. However, the functions of exosomal circRNAs are not clear. The present research aimed to determine whether circRNAs secreted from arsenite-transformed human hepatic epithelial (L-02) cells are transferred into normal L-02 cells and become functionally active in the normal cells. The results showed that circRNA_100284 is involved in the malignant transformation of L-02 cells induced by arsenite. The medium from transformed L-02 cells induced upregulation of circRNA_100284, accelerated the cell cycle, and promoted proliferation of normal L-02 cells. Transformed cells transferred circRNA_100284 into normal L-02 cells via exosomes and led to the malignant transformation of the non-transformed cells. Knockdown of circRNA_100284, which reduced circRNA_100284 levels in exosomes derived from transformed L-02 cells, blocked the accelerated cell cycle and reduced proliferation and malignancy. In addition, in normal L-02 cells, exosomal circRNA_100284 derived from arsenite-transformed L-02 cells induced acceleration of the cell cycle and promoted proliferation via acting as a sponge of microRNA-217. Further, exosomal circRNA_100284 was upregulated in the sera of people exposed to arsenite. Thus, exosomes derived from transformed L-02 cells transferred circRNA_100284 to surrounding cells, which induced an accelerated cell cycle and promoted proliferation of normal liver cells and led to the malignant transformation of the non-transformed cells. The findings support the concept that exosomal circRNAs are involved in cell-cell communication during carcinogenesis induced by arsenite.

Fig. 1. Effects of circRNA_100284 on the neoplastic capacity of arsenite-transformed L-02 cells.

C-L-02, passage-control L-02 cells; T-L-02, arsenite-transformed L-02 cells. L-02 cells were treated with 0 or 2 μM of arsenite for 0, 3, 6, 12, or 24 h. a The levels of circRNA_100284 were determined by qRT-PCR assays (means ± SD, n = 3). L-02 cells were exposed to 0 or 2 μM of arsenite for 0, 10, 20, or 30 passages. b The levels of circRNA_100284 were determined by qRT-PCR assays (means ± SD, n = 3). *P < 0.05, different from control cells. c Colonies and (d) their numbers (means ± SD, n = 3) of C-L-02 cells and T-L-02 cells in soft agar (bars = 250 μm). e Tumors were examined, and (f) their volumes were measured (means ± SD, n = 6) at 4 weeks after C-L-02 cells and T-L-02 cells were inoculated into nude mice. *P < 0.05, different from C-L-02 cells. T-L-02 cells were transfected with 10 nM control siRNA or 10 nM circRNA_100284 siRNA for 24 h. g The levels of circRNA_100284 were determined by qRT-PCR assays (means ± SD, n = 3). h Colony formation in soft agar (upper, bars = 250 μm) and Transwell assays (lower, bars = 250 μm). i and (j) relative colony numbers and relative levels of cell invasion and migration were determined (means ± SD, n = 3). *P < 0.05, different from T-L-02 cells

Fig. 2. Medium from arsenite-transformed L-02 cells induces increases of circRNA_100284 levels, a disorder of the cell cycle, and proliferation in normal L-02 cells.

Basal basal medium, CM medium from passage-control L-02 cells, T-CM medium from arsenite-transformed L-02 cells. Densities of bands were quantified by Image J software. Tubulin levels, measured in parallel, served as controls. L-02 cells were treated with various conditioned media. L-02 cells were cultured with Basal, CM, or T-CM for 24 h. a circRNA_100284 levels in L-02 cells were determined by qRT-PCR assays (means ± SD, n = 3). b Western blots were performed, and (c) relative protein levels (means ± SD, n = 3) of EZH2 and cyclin-D1 were determined in L-02 cells. d Flow cytometry was performed to analyze the cell cycle, and (e) representative histograms were prepared. f Proliferation efficiency was measured by use of a Cell Counting Kit-8 assay, and the relative ratios of cell proliferation were determined by comparison with cells in basal medium (means ± SD, n = 3). *P < 0.05, different from cells in basal medium

Fig. 3. Transfer of circRNA_100284 derived from arsenite-transformed L-02 cells to normal L-02 cells via exosomes.

CM-exo exosomes derived from passage-control L-02 cells, T-CM-exo exosomes derived from arsenite-transformed L-02 cells. Exosomes from C-L-02 cells and T-L-02 cells were fractionated by Exoquick. a Representative electron micrographs of CM-exo or T-CM-exo (right, bars = 100 nm). b Particle number and size analysis of CM-exo or T-CM-exo were determined by dynamic light scattering using a ZetaView® nanoparticle tracker (ParticleMetrix GmbH; Meerbusch, Germany). c Western blots of CD9 and CD81 in cells, CM-exo or T-CM-exo. d circRNA_100284 levels in CM-exo or T-CM-exo were measured by qRT-PCR (means ± SD, n = 3). *P < 0.05, different from CM-exo. CM-exo and T-CM-exo were labeled with PKH67, a green fluorescent cell linker. e Normal L-02 cells after 3 h incubation of exosomes with fluorescently labeled PKH67. Green represents CM-exo or T-CM-exo staining by PKH67, and blue represents nuclear DNA staining by DAPI

Fig. 4. Exosomal circRNA_100284 derived from arsenite-transformed L-02 cells induces acceleration of the cell cycle and proliferation in normal L-02 cells.

CM-exo exosomes derived from passage-control L-02 cells, T-CM-exo exosomes derived from arsenite-transformed L-02 cells. circRNA_100284 siRNA-T-CM-exo, exosomes derived from arsenite-transformed L-02 cells transfected with circRNA_100284 siRNA for 24 h; con siRNA-T-CM-exo exosomes derived from arsenite-transformed L-02 cells transfected with control siRNA for 24 h. Densities of bands were quantified by Image J software. Tubulin levels, measured in parallel, served as controls. Normal L-02 cells were treated with CM-exo or T-CM-exo for 24 h. a Levels of circRNA_100284 were determined by qRT-PCR assays (means ± SD, n = 3). b Flow cytometry was performed to analyze the cell cycle, and c representative histograms were prepared. d Proliferation efficiency was measured by use of a Cell Counting Kit-8 assay, and the relative ratios of cell proliferation were determined by comparison with cells in basal medium (means ± SD, n = 3). *P < 0.05, different from cells treated with CM-exo. Normal L-02 cells were treated with 0, 10, 20, 50, or 100 μg/mL CM-exo or T-CM-exo for 24 h. e and g western blots were performed; f, h relative protein levels (means ± SD, n = 3) of EZH2 and cyclin-D1 were determined in normal L-02 cells. *P < 0.05, different from control L-02 cells. Normal L-02 cells were treated with CM-exo, T-CM-exo, circRNA_100284-T-CM-exo, or con siRNA-T-CM-exo for 24 h. i The levels of circRNA_100284 were determined by qRT-PCR assays (means ± SD, n = 3). j Western blots were performed, and k relative protein levels (means ± SD, n = 3) of EZH2 and cyclin-D1 were determined. l Flow cytometry was performed to analyze the cell cycle, and m representative histograms were prepared. *P < 0.05, different from cells treated with T-CM-exo

Fig. 5. Exosomal circRNA_100284 derived from arsenite-transformed L-02 cells induces acceleration of the cell cycle and promotes proliferation via miR-217 in normal L-02 cells.

T-CM-exo exosomes derived from arsenite-transformed L-02 cells, CircRNA_100284 siRNA-T-CM-exo exosomes derived from arsenite-transformed L-02 cells transfected with circRNA_100284 siRNA for 24 h, con siRNA-T-CM-exo exosomes derived from arsenite-transformed L-02 cells transfected with control siRNA for 24 h. Densities of bands were quantified by Image J software. Tubulin levels, measured in parallel, served as controls. a A schematic graph illustrating binding sites between circRNA_100284 and miR-217. After normal L-02 cells were transfected with control mimic or miR-217 mimic for 24 h, they were treated with CM-exo or T-CM-exo for 24 h. b Western blots were performed, and c relative protein levels (means ± SD, n = 3) of EZH2 and cyclin-D1 were determined. *P < 0.05, different from cells treated with T-CM-exo. After normal L-02 cells were transfected with control inhibitor or miR-217 inhibitor for 24 h, they were treated with T-CM-exo, circRNA_100284-T-CM-exo, or con siRNA-T-CM-exo for 24 h. d Western blots were performed, and relative protein levels (means ± SD, n = 3) of EZH2 and cyclin-D1 were determined. e Flow cytometry was performed to analyze the cell cycle, and f representative histograms were prepared. g Proliferation efficiency was measured by use of the Cell Counting Kit-8 assay, and the relative ratios of cell proliferation were determined by comparison with cells in basal medium (means ± SD, n = 3). *P < 0.05, different from cells treated with T-CM-exo, ^#P < 0.05, different from cells treated with circRNA_100284-T-CM-exo alone

Fig. 6. Effects of exosomal circRNA_100284 on the process of malignant transformation of As-L-02-p20 cells.

As-L-02-p20 cells L-02 cells exposed to 2 μM of arsenite for 20 passages, CM-exo exosomes derived from passage-control L-02 cells, T-CM-exo exosomes derived from arsenite-transformed L-02 cells. circRNA_100284 siRNA-T-CM-exo, exosomes derived from arsenite-transformed L-02 cells transfected with circRNA_100284 siRNA for 24 h; con siRNA-T-CM-exo exosomes derived from arsenite-transformed L-02 cells transfected with control siRNA for 24 h. As-L-02-p20 cells were treated with CM-exo, T-CM-exo, circRNA_100284-T-CM-exo, or con siRNA-T-CM-exo for 24 h. a Colony formation in soft agar (upper, bars = 250 μm) and Transwell assays (lower, bars = 250 μm). b, c Relative colony numbers and relative levels of cell invasion and migration were determined (means ± SD, n = 3). d Tumors were examined, and e their volumes were measured (means ± SD, n = 6) at 4 weeks after cells were inoculated into nude mice. *P < 0.05, different from As-L-02-p20 cells treated with T-CM-exo

Fig. 7. Levels of circulating exosomal circRNA_100284 are overexpressed in people exposed to arsenite.

Circulating Exosome exosomes isolated from the sera of individuals exposed or not exposed to arsenite. a Representative electron micrograph of circulating exosomes (bars = 100 nm). b The levels of circRNA_100284 in circulating exosomes were determined by qRT-PCR assays (means ± SD, n = 16). *P < 0.05, different from circulating exosomes isolated from the sera of individuals not exposed to arsenite

Fig. 8. A schematic representation of the proposed pathway by which exosomal circRNA_100284 causes proliferation of L-02 cells in arsenite carcinogenesis.

Chronic exposure to arsenite elevates circRNA_100284 levels, which is involved in the malignant transformation of normal L-02 cells. circRNA_100284 from arsenite-transformed cells transferred into normal L-02 cells via exosomes induce acceleration of the cell cycle and promotes proliferation via miR-217