miR-27b attenuates apoptosis induced by transmissible gastroenteritis virus (TGEV) infection via targeting runt-related transcription factor 1 (RUNX1)

Abstract

Transmissible gastroenteritis virus (TGEV), belonging to the coronaviridae family, is the key cause of the fatal diarrhea of piglets and results in many pathological processes. microRNAs (miRNAs) play a key role in the regulation of virus-induced apoptosis. During the process of apoptosis induced by TGEV infection in PK-15 cells, the miR-27b is notably down-regulated. Thus, we speculate that miR-27b is involved in regulating the process of apoptosis in PK-15 cells. In this study we demonstrated that the over-expression of miR-27b led to the inhibition of TGEV-induced apoptosis, reduction of Bax protein level, and decrease of caspase-3 and -9 activities. Conversely, silencing of miR-27b by miR-27b inhibitors enhanced apoptosis via up-regulating Bax expression and promoting the activities of caspase-3 and -9 in TGEV-infected cells. Subsequently, the runt-related transcription factor 1 (RUNX1) is a candidate target of miR-27b predicted by bioinformatics search. We further identified that the miR-27b directly bound to the 3' UTR of RUNX1 mRNA and suppressed RUNX1 expression, which indicates RUNX1 is the direct target gene of miR-27b. The over-expression of RUNX1 increased apoptosis and knockdown RUNX1blocked apoptosis of viral-infected cells via regulating Bax expression and the activities of caspase-3 and -9. Our data reveal that miR-27b may repress the mitochondrial pathway of apoptosis by targeting RUNX1, indicating that TGEV may induce apoptosis via down-regulating miR-27b and that miR-27b may act as a target for therapeutic intervention.

Keywords: Apoptosis; RUNX1; Transmissible gastroenteritis virus; miR-27b.

Figure 1. The effect of miR-27b on apoptosis induced by TGEV.

(A) Effect of the miR-27b mimics and inhibitors on apoptosis induced by TGEV. The apoptosis of PK-15 was analyzed via flow cytometry at 24 and 48 hpi. (B) Detection of caspase-9 activities in PK-15 cells at 24 and 48 hpi. (C) Detection of caspase-3 activities in cells at 24 and 48 hpi. ^∗P < 0.05 in comparison with the control. ^∗∗P < 0.01 in comparison with the control.

Figure 2. miR-27b directly targets the 3′ UTR of RUNX1 mRNA.

(A) Bioinformatic prediction of interaction between miR-27b and the 3′ UTRs of swine RUNX1. For each schematic, the upper sequence is the binding site of miR-27b in 3′ UTRs of swine RUNX1, the middle sequence is the mature miR-27b, and the lower sequence is the mutated sequence of 3′ UTR. The seed sequence is underlined. (B) Schematic drawing of the putative binding sites or mutations of miR-27b binding-sites in 3′ UTR of RUNX1 mRNA. The locations of the potential binding sites or their mutations are presented by blank boxes. (C) The RUNX1 luciferase reporter construct was co-transfected with miR-27b mimics (or negative control) or miR-27b inhibitors (or negative control) into PK-15 cells (normalized to the firefly luciferase activity). Data are expressed as relative luciferase activities to control. (D) Western blot analysis of RUNX1 in cells transfected with miR-27b mimics or mimics control. Data represent means ± S.D. of three independent experiments. ^∗P < 0.05 in comparison with the control. ^∗∗P < 0.01 in comparison with the control.

Figure 3. miR-27b attenuates apoptosis via mitochondrial pathway.

(A) Western blot analysis of Bax in cells transfected with miR-27b mimics or miR-27b inhibitors. (B) Real-time PCR analysis of the expression of Bax in cells transfected with miR-27b mimics or miR-27b inhibitors. The Bax mRNA level was reduced at 24 and 48 hpi measured by real-PCR (normalized to β-actin). Data represent mean ± S.D. of three independent experiments. ^∗P < 0.05 in comparison with the control. ^∗∗P < 0.01 in comparison with the control.

Figure 4. RUNX1 enhances TGEV-induced apoptosis.

(A) Silencing effect of RUNX1 siRNAs on RUNX1 at mRNA level. PK-15 cells were transfected with RUNX1-specific siRNA or irrelevant siRNA and measured by real-time PCR (normalized to β-actin). (B) The silencing effect of siRUNX1-2 on RUNX1 expression. (C) The effect of siRUNX1-2 on PK-15-cell viability. The cells were incubated after transfecting with 100 nM siRUNX1-2 for 48 h. Cell viability was evaluated by CCK-8 assay. (D) The over-expression of RUNX1 using pCI-neo-RUNX1. PK-15 cells were transfected with pCI-neo- RUNX1 or pCI-neo vector, and the expression level of RUNX1 was assessed by western blot at 48 hpt. (E) The effect of RUNX1 on the expression of Bax. (F) The effect of RUNX1 on caspase-9 activity. (G) The effect of RUNX1 on caspase-3 activity. Data represent mean ± S.D. of three independent experiments. ^∗P < 0.05 in comparison with the control. ^∗∗P < 0.01 in comparison with the control.