MiR-21 in Substance P-induced exosomes promotes cell proliferation and migration in human colonic epithelial cells

Abstract

Exosomes are cellular vesicles involved in intercellular communication via their specialized molecular cargo, such as miRNAs. Substance P (SP), a neuropeptide/hormone, and its high-affinity receptor, NK-1R, are highly expressed during colonic inflammation. Our previous studies show that SP/NK-1R signaling stimulates differential miRNA expression and promotes colonic epithelial cell proliferation. In this study, we examined whether SP/NK-1R signaling regulates exosome biogenesis and exosome-miRNA cargo sorting. Moreover, we examined the role of SP/NK-1R signaling in exosome-regulated cell proliferation and migration. Exosomes produced by human colonic NCM460 epithelial cells overexpressing NK-1R (NCM460-NK1R) were isolated from culture media. Exosome abundance and uptake were assessed by Western blot analysis (abundance) and Exo-Green fluorescence microscopy (abundance and uptake). Cargo-miRNA levels were assessed by RT-PCR. Cell proliferation and migration were assessed using xCELLigence technology. Colonic epithelial exosomes were isolated from mice pretreated with SP for 3 days. Cell proliferation in vivo was assessed by K_i-67 staining. SP/NK-1R signaling in human colonic epithelial cells (in vitro) and mouse colons (in vivo) increased 1) exosome production, 2) the level of fluorescence in NCM460s treated with Exo-Green-labeled exosomes, and 3) the level of miR-21 in exosome cargo. Moreover, our results showed that SP/NK-1R-induced cell proliferation and migration are at least in part dependent on intercellular communication via exosomal miR-21 in vitro and in vivo. Our results demonstrate that SP/NK-1R signaling regulates exosome biogenesis and induces its miR-21 cargo sorting. Moreover, exosomal miR-21 promotes proliferation and migration of target cells.NEW & NOTEWORTHY Substance P signaling regulates exosome production in human colonic epithelial cells and colonic crypts in wild-type mice. MiR-21 is selectively sorted into exosomes induced by Substance P stimulation and promotes cell proliferation and migration in human colonocytes and mouse colonic crypts.

Keywords: NK-1R; Substance P; cell migration; cell proliferation; exosomes; miR-21; sorting.

Graphical abstract

Fig. 1.

Substance P (SP)/NK-1R signaling activation promotes RNA loading to exosomes in human colonocytes and regulates protein expression in colonic epithelial exosomes. A and B: exosomes were collected from culture medium of NCM460 cells and labeled with Exo-Green and incubated with naive NCM460 cells. Fluorescent signals from the exosomes uptaken by NCM460 cells were quantified by ImageJ (scale bar = 10 μm). C: total RNA was harvested from exosomes collected from cell culture media of NCM460-NK1R cells treated with SP (10⁻⁷ M, 6 h) or vehicle quantified by spectrometry. D: representative image from Western blot analysis examining CD9 expression in exosomes produced upon SP/NK-1R signaling activation. E: relative amount of exosome production under different treatments was analyzed using densitometry with actin from cell lysates as reference.

Fig. 2.

Substance P (SP)-induced exosomes are more readily taken up by human colonocytes. A: exosomes produced by human colonic epithelial NCM460 cells with or without SP stimulation were labeled with Exo-Green and incubated with naive NCM460 cells. B: uptake of exosomes was analyzed by quantification of fluorescence.

Fig. 3.

MiRNAs are differentially sorted into exosomes upon Substance P (SP)/NK-1R signaling activation. A: miRNAs identified in cytoplasm and exosomes from human colonic epithelial NCM460-NK-1R cells (red: increased expression relative to control; green: reduced expression relative to control; gray: not present). B: levels of miR-21 (left) and miR-320 (right) in exosomes produced by NCM460-NK-1R cells with or without SP treatment were measured using RT-PCR.

Fig. 4.

Substance P (SP)-exosomes promote cell proliferation and migration in naive NCM460 cells. Cell proliferation (A) and cell migration (B) of naive NCM460 cells treated with SP-exosomes or control exosomes were measured using xCELLigence Real-Time Cellular Analysis system.

Fig. 5.

Exosomal miR-21 promotes cell proliferation and migration in naive NCM460 cells. Exosomes were collected from NCM460-NK-1R cells transfected with antisense-miR-21 or scrambled control and incubated with naive NCM460 cells. Cell proliferation (A) and cell migration (B) of naïve NCM460 cells were measured using xCELLigence Real-Time Cellular Analysis system.

Fig. 6.

Substance P (SP) administration promotes exosome production in vivo. Male wild-type C57BL6/J mice (n = 5) were administered with SP (72 μg/dose, 2 doses/day) for 2 days. A and B: representative image from Western blot analysis assessing exosome production. Lysates from colonic epithelial cells with equal amounts of protein were collected for harvesting exosomes. C: miR-21 levels in colonic epithelial cells were measured with RT-PCR. D and E: representative image from K_i-67 immunohistochemistry measuring cell proliferation in colonic epithelial cells in vivo (scale = 50 μm).