Enterovirus 71 induces pyroptosis of human neuroblastoma SH-SY5Y cells through miR-146a/ CXCR4 axis

Abstract

Enterovirus 71 (EV71) is a predominant causative pathogen of hand-foot-and-mouth disease (HFMD) in children. Compared with other HFMD-associated viruses, EV71 tends to induce more severe neurological complications and even death. However, the detailed mechanism of EV71 causes nervous system disorder is still unclear. In this study, we found that EV71 induced the GSDMD/NLRP3-mediated pyroptosis of SH-SY5Y cells through up-regulated miR-146a. Through bioinformatic analysis, we identified C-X-C chemokine receptor type 4 (CXCR4) as the potential target of miR-146a. We noticed that the expression of CXCR4 was regulated by miR-146a during EV71 infection. Moreover, our results show that over-expression of CXCR4 attenuated EV71-induced pyroptosis of SY-SY5Y cells. These results reveal a previously unrecognized mechanism in which EV71 induces nervous system cells damage through regulating miR-146a/CXCR4 mediated pyroptosis.

Keywords: CXCR4; Enterovirus 71 (EV71); Pyroptosis; miR-146a; miRNA.

Fig. 1

EV71 induced pyroptosis of SH-SY5Y cells. (A) SH-SY5Y cells were treated with indicated doses of EV71 for 48 h. Scale bar indicates 50 μm and magnification is 200 × . (B) SH-SY5Y cells were treated with indicated doses of EV71 for different times. The cell viability rate was calculated as described as method. (C and E) Total protein was extracted and analyzed by Western blot using the antibodies as indicated. The expression of GAPDH was the internal control. (D and F) The expression level of protein was presented by relative expression which was normalized by GAPDH. Each group compared with the 0 TCID50 group (D) or 0 h group (F).

Fig. 2

EV71 induced pyroptosis of SH-SY5Y cells through the up-regulation of miR-146a. Cells were infected with EV71 at indicated doses for 48 h (A) or treated with 1 × 10⁵TCID50 EV71 for indicated time (B). The expression of miR-146a was analyzed by qRT-PCR and U6 as the internal standard. (C) Cells were transfected with 50 nM miRNA-146a inhibitors or miRNA inhibitor negative control (NC-inhibitor). ns. Means no statistical difference compared with the NC-inhibitor group. (D) SH-SY5Y cells were treated as indicated, and then cellular proteins were extracted and analyzed by Western blot. The expression levels of pyroptosis related proteins were presented as relative expression to GAPDH.

Fig. 3

miR-146a targeted CXCR4 during EV71 infection. (A) miR-146a targeted the 3′-UTR of CXCR4 (nucleotides 2784–2790). Total RNA (B) and protein (D) of EV71 infected cells were extracted, the expressions of CXCR4 mRNA and protein were evaluated respectively. (C and E) Cells were treated with 1 × 10⁵ TCID50 EV71 for indicated time. (F and G) SH-SY5Y cells were transfected with 50 nM miRNA-146a inhibitor or NC-inhibitor, then treated with 1 × 10⁵ TCID50 EV71 for 48 h. The expression level of CXCR4 was evaluated by qRT-PCR (F) and Western blot (G).

Fig. 4

The effects of CXCR4 on cell pyroptosis in EV71 infection. (A and B) HEK 293T cell was transfected with 2 μg (Phace-CXCR4 or Phace-vector) plasmid for 48 h, then total RNA and protein were extracted to evaluate CXCR4 expression. (C) Transfected cells treated with 1 × 10⁵TCID50 EV71 for 48 h. The expression of pyroptosis related proteins were evaluated. (D) The expression levels of those proteins were presented as relative to GAPDH. Each group compared with the Phace-vector group.

Fig. 5

The schematics of EV71 induces miR-146a-mediated pyroptosis of SH-SY5Y cells. The infection of EV71 increases the expression of cellular miR-146a which leads to down-regulate the expression of CXCR4 through binding to the 3′-UTR of CXCR4. Then the increased inflammasome NLRP3 will cleave pro-caspase-1 into activated caspase-1. Activated caspase-1 further cleaves GSDMD into the N-terminal GSDMD fragments which directly form the pores in cell membrane to initiate SH-SY5Y cell pyroptosis. Pro-IL-1β is also cleaved by activated caspase-1, and mature IL-1β releases during pyroptosis.