Effect of miR-17 on Polygonum Cillinerve polysaccharide against transmissible gastroenteritis virus

Abstract

Transmissible gastroenteritis virus (TGEV) could cause diarrhea, vomiting, dehydration and even death in piglets, miRNA played an important role in the interaction between virus and cell. The study aimed to investigate the impact of miR-17 on the polysaccharide of Polygonum Cillinerve (PCP) in combating TGEV. miR-17 was screened and transfection validation was performed by Real-time PCR. The function of miR-17 on PK15 cells infected with TGEV and treated with PCP was investigated by DCFH-DA loading probe, JC-1 staining and Hoechst fluorescence staining. Furthermore, the effect of miR-17 on PCP inhibiting TGEV replication and apoptosis signaling pathways during PCP against TGEV infection was measured through Real-time PCR and Western blot. The results showed that miR-17 mimic and inhibitor could be transferred into PK15 cells and the expression of miR-17 significantly increased and decreased respectively compared with miR-17 mimic and inhibitor (P < 0.05). A total 250 μg/mL of PCP could inhibit cells apoptosis after transfection with miR-17. PCP (250 μg/mL and 125 μg/mL) significantly inhibited the decrease in mitochondrial membrane potential induced by TGEV after transfection with miR-17 (P < 0.05). After transfection of miR-17 mimic, PCP at concentrations of 250 μg/mL and 125 μg/mL significantly promoted the mRNA expression of P53, cyt C and caspase 9 (P < 0.05). Compared with the control group, the replication of TGEV gRNA and gene N was significantly inhibited by PCP at concentrations of 250 μg/mL and 125 μg/mL after transfection of both miR-17 mimic and inhibitor (P < 0.05). PCP at 62.5 μg/mL significantly inhibited the replication of gene S following transfection with miR-17 inhibitor (P < 0.05). These results suggested that PCP could inhibit the replication of TGEV and apoptosis induced by TGEV by regulating miR-17.

Keywords: Polygonum Cillinerve polysaccharide; apoptosis; miR-17; transmissible gastroenteritis virus; virus replication.

Figure 1

The relative expression of differential miR-17. P < 0.05 was statistically significant, ^*P < 0.05, ^**P < 0.01.

Figure 2

The relative expression of miR-17. The PK15 cells were treated with miR-17 mimic, mimic NC, inhibitor and inhibitor NC, respectively, and without using PCP and TGEV. (A) The expression level of miR-17 in PK15 cells transfected with miR-17 mimic (n = 3); (B) The expression level of miR-17 in PK15 cells transfected with miR-17 inhibitor The PK15 cells were treated with miR-17 mimic, mimic NC, inhibitor and inhibitor NC, respectively, and without using PCP and TGEV (n = 3). P < 0.05 was statistically significant, ^***P < 0.001.

Figure 3

Cell morphology ( × 200). The arrow referred to the PK15 cells that have swollen, ruptured and shed into clusters. (A) Cell morphology of PCP groups (250, 125, 62.5 μg/mL) transfected with miR-17 mimic, mimic group, mimic NC group and mimic blank group ( × 200); (B) Cell morphology of PCP groups (250, 125, 62.5 μg/mL) transfected with miR-17 inhibitor, inhibitor group, inhibitor NC group and inhibitor blank group ( × 200). The scale was 20 μm.

Figure 4

ROS production in PK15 cells. The level of ROS production in PK15 cells was detected by DCFH-DA. The green part represented ROS levels. PCP, mimic, mimic NC, inhibitor and inhibitor NC groups were treated with TGEV while blank group was untreated. (A) ROS level of PCP groups (250, 125, 62.5 μg/mL) transfected with miR-17 mimic, mimic group, mimic NC group and mimic blank group ( × 200); (B) ROS level of PCP groups (250, 125, 62.5 μg/mL) transfected with miR-17 inhibitor, inhibitor group, inhibitor NC group and inhibitor blank group ( × 200).

Figure 5

Mitochondrial membrane potential. Mitochondrial membrane potential of PK15 cells was detected by JC-1. The red part represented normal mitochondria, and the green part represented mitochondria with decreased membrane potential. The PCP, mimic, mimic NC, inhibitor and inhibitor NC groups were treated with TGEV while blank group was untreated. (A) Mitochondrial membrane potential of PCP groups (250, 125, 62.5 μg/mL) transfected with miR-17 mimic, mimic NC group, mimic group and blank group ( × 200); (B) Mitochondrial membrane potential of PCP groups (250, 125, 62.5 μg/mL) transfected with miR-17 inhibitor, inhibitor group, inhibitor NC group and inhibitor blank group ( × 200); (C) The relative ratio of red and green fluorescence of PCP groups transfected with miR-17 mimic; (D) The relative ratio of red and green fluorescence of PCP groups transfected with miR-17 inhibitor. P < 0.05 was statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Hoechst 33258 detected the effect on cell apoptosis ( × 200). The white part represented apoptotic cells. The PCP, mimic, mimic NC, inhibitor and inhibitor NC groups were treated with TGEV while blank group was untreated. (A) Apoptosis level of PCP groups (250, 125, 62.5 μg/mL) transfected with miR-17 mimic, mimic group, mimic NC group and mimic blank group ( × 200); (B) Apoptosis level of PCP groups (250, 125, 62.5 μg/mL) transfected with miR-17 inhibitor, inhibitor group, inhibitor NC group and inhibitor blank group ( × 200).

Figure 7

Effect of expression of key signaling molecule after transfection of miR-17 mimic. The mRNA expression levels of P53, cyt C and caspase-9 were measured by RT-qPCR. The PCP, mimic and mimic NC groups were treated with TGEV. (A) The mRNA expression level of P53 (n = 3); (B) The mRNA expression level of cyt C (n = 3); (C) The mRNA expression level of caspase-9 (n = 3). P < 0.05 was statistically significant to the mimic group, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

Effect of expression of key signaling molecule after transfection of miR-17 inhibitor. The mRNA expression levels of P53, cyt C and caspase-9 were measured by RT-PCR. The PCP, inhibitory and inhibitor NC groups were treated with TGEV. (A) The mRNA expression level of P53 (n = 3); (B) The mRNA expression level of cyt C (n = 3); (C) The mRNA expression level of caspase-9 (n = 3). P < 0.05 was statistically significant to the inhibitory group, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9

Effect of transfected miR-17 mimic on protein expression in cells. The protein expression levels of cyt C, caspase-9 and P53 were measured by Western blot. The PCP, mimic and mimic NC were treated with TGEV. (A) Protein bands; (B) The protein expression levels of cyt C (n = 3); (C) The protein expression levels of cleaved caspase-9 (n = 3); (D) The protein expression levels of P53 (n = 3). P < 0.05 was statistically significant to the mimic group, ^*P < 0.05, ^**P < 0.01. The data and analysis results were plotted as bar graphs using GraphPad Prism 7.00.

Figure 10

Effect of transfected miR-17 inhibitor on protein expression in cells. The protein expression levels of cyt C, caspase-9 and P53 were measured by Western blot. The PCP, inhibitory and inhibitor NC groups were treated with TGEV. (A) Protein bands; (B) The protein expression levels of cyt C (n = 3); (C) The protein expression levels of cleaved caspase-9 (n = 3); (D) The protein expression levels of P53 (n = 3). P < 0.05 was statistically significant to the inhibitory group, *P < 0.05, **P < 0.01. The data and analysis results were plotted as bar graphs using GraphPad Prism 7.00.

Figure 11

The effect of miR-17 mimic on TGEV replication. The mRNA expression levels of TGEV gRNA, gene N and gene S were measured by RT-qPCR. The PCP, mimic and mimic NC were treated with TGEV. (A) The mRNA expression levels of TGEV gRNA (n = 3); (B) The mRNA expression levels of TGEV gene N (n = 3); (C) The mRNA expression levels of TGEV gene S (n = 3). P < 0.05 was statistically significant to the mimic group, *P < 0.05, **P < 0.01, ***P < 0.001. The data and analysis results were plotted as bar graphs using GraphPad Prism 7.00.

Figure 12

The effect of miR-17 inhibitor on TGEV replication. The mRNA expression levels of TGEV gRNA, gene N and gene S were measured by RT-qPCR. The PCP, inhibitory and inhibitor NC were treated with TGEV. (A) The mRNA expression levels of TGEV gRNA (n = 3); (B) The mRNA expression levels of TGEV gene N (n = 3); (C) The mRNA expression levels of TGEV gene S (n = 3). P < 0.05 was statistically significant to the inhibitory group, *P < 0.05, **P < 0.01, ***P < 0.001. The data and analysis results were plotted as bar graphs using GraphPad Prism 7.00.