PK15 porcine kidney cells and BHK-21 hamster kidney cells were purchased from the American Type Culture Collection (ATCC, USA). PK15 and BHK-21 cells were both grown in high-glucose Dulbecco’s modified Eagle medium (hgDMEM; Gibco, USA) containing 10% foetal bovine serum; they were incubated at 37 °C (5% CO${}_2$ and 95% humidity).

JEV strain SA14-14-2 was amplified in BHK-21 cells. All infections were carried out by incubating the cells with virus at the MOI = 1, then the inoculum was removed, the cells were washed three times with PBS and fresh medium was added. The infection was performed and the infected PK15 cells were maintained in DMEM supplemented with 2% FBS without penicillin/streptomycin mixtures.

For Western blotting and/or immunofluorescence, primary antibodies were purchased with specificity for the following proteins: eIF2$\alpha$ (Bioworld, Cat:BS3651, Nanjing, Jiangsu, China); p-eIF2$\alpha$ (Abcam, Cat:ab32157, Cambridgeshire, UK); GRP78 (Proteintech, Cat:11587-1-AP, Chicago, IL, USA); NS3 (GeneTex, Cat:GTX125868, SAN Antonio, TX, USA); Hsp70 (Abcam, Cat:ab47454, Cambridgeshire, UK); IRE1 (Bioss, Cat:bs-8680R, Beijing, China); p-IRE1 (Abcam, Cat:ab48187, Cambridgeshire, UK); cleaved Caspase-3 (ABclonal, Cat:A11021, Wuhan, Hubei, China); NF-$\kappa$B (Proteintech, Cat:10745-1-AP, Chicago, IL, USA); p-NF-$\kappa$B (Cell Signaling Technology, Cat:#3033S, Boston, MA, USA); TBB5 (Abgent, AM1031A, Shanghai, China). The following secondary antibodies were purchased: IgG-horseradish peroxidase mouse anti-rabbit (Cat:sc-2357) and mouse IgG kappa binding protein (Cat:sc-516102) coupled with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA); QuickBlock™ Primary Antibody Dilution Buffer for Western Blot (Beyotime, Cat:P0256, Shanghai, China); Bovine serum albumin (Sangon Biotech, Cat:A500023, Shanghai, China).

PK15 cells were seeded on plates for 24 h. When the cell growth density reached 80–90%, using Lipofectamine 3000 (Invitrogen, USA), 2 $\mu$g pcDNA 3.1 lncRNA-SUSAJ1 (the sequence of lncRNA-SUSAJ1 can be found in Supplementary file) or empty plasmid (negative control; NC) was transfected into each well in accordance with the manufacturer’s instructions. Short interfering RNAs (siRNA) targeting lncRNA-SUSAJ1 was transfected along with si-NC and antisense oligonucleotides (ASO) [28] at a final concentration of 100 nM per well. JEV was used to infect the cells at 6 h after transfection.

PK15 cells were seeded on 12-well plates for 24 h, then transfected using Lipofectamine. After 6 h of transfection, JEV infection was conducted. At 24 and 48 h after infection, 195 $\mu$L radioimmunoprecipitation assay lysis buffer, 1 $\mu$L protease inhibitor cocktail, 2 $\mu$L phenylmethylsulphonyl fluoride, and 2 $\mu$L phosphatase inhibitor were added to each well for 30 min; the contents of each well were then collected. Subsequently, protein lysates were mixed with sodium dodecyl sulphate–polyacrylamide gel electrophoresis loading buffer and incubated at 100 ℃ for 10 min. Protein samples (10 $\mu$L each) were separated by electrophoresis on 12% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 1% bovine serum albumin in Tris-buffered saline-Tween-20 (TBST) at room temperature for 2 h. Membranes were then incubated overnight at 4 ℃ with primary antibodies. Three 10-min washes in TBST were performed; membranes were then incubated with secondary antibodies at room temperature for 45 min. Finally, three 10-min washes in TBST were performed; membranes were incubated with chromogenic solution for chemiluminescence analysis. Images were obtained using a chemiluminescence imaging system. The chemiluminescence intensity of each protein band was quantified using the Image J (1.4.3.67, Broken Symmetry Software, USA), and then protein levels were normalized by the amount of TBB5 protein.

Apoptosis of JEV-infected cells was detected by flow cytometry. Cells were stained using a fluorescein isothiocyanate annexin V apoptosis detection kit (CA1020; Solarbio, China) in accordance with the manufacturer’s instructions. The cells were analysed using a BD-FACS Canto-II flow cytometer (BD Biosciences, USA) and the percentage of apoptotic cells was quantified. Annexin V-fluorescein isothiocyanate-positive cells were classified as early apoptotic cells; PI-positive cells were classified as necrotic cells. Cells with both markers were classified as late apoptotic cells. All data were analysed by FlowJo software (TreeStar FlowJo 10.4.0, USA).

RNA was extracted from PK15 cells using TRIzol (Invitrogen); RNA quality was evaluated by spectrophotometry (Nano-300; Hangzhou Allsheng, China). RNA was reverse-transcribed to cDNA using PrimeScript RT Master Mix (Perfect Real Time; TaKaRa, Japan), in accordance with the manufacturer’s instructions. Glyceraldehyde 3-phosphate dehydrogenase was used as the internal control for both lncRNAs and mRNA. SYBR Green was used for RT-qPCR on a real-time fluorescent thermocycler (CFX96 Touch; Bio-Rad, USA). The primer sequences used in this study are listed in Supplementary Table 1.

A commercial one-step terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) apoptosis detection kit (C1088; Beyotime, China) was used for TUNEL staining in accordance with the manufacturer’s instructions. Cells grown in 12-well plates were incubated with 50 $\mu$L TUNEL solution per well. After incubation at 37 °C for 60 min, three 5-min washes with phosphate-buffered saline were conducted. For nuclear staining, cells were incubated with 4’,6-diamidino-2-phenylindole (DAPI) at 37 °C for 10 min; anti-fluorescence quenching agent was added after three 5-min washes with phosphate-buffered saline. Apoptosis was observed using an inverted fluorescence microscope.

A mitochondrial membrane potential detection kit (JC-1 cationic dye; M8650; Solarbio) was used to measure the mitochondrial membrane potential of PK15 cells, in accordance with the manufacturer’s instructions. The following fluorescence values were detected using the Synergy-4 multi-functional enzyme reader (Synergy H1; BioTek, USA): 490 nm excitation wavelength/530 nm emission wavelength, and 525 nm excitation wavelength/590 nm emission wavelength. The mitochondrial membrane potential was determined on the basis of the red/green fluorescence ratio. Red and green fluorescence intensities were analysed to determine the mitochondrial membrane potential.

The ROS-sensitive probe 2’,7’-dichlorodihydrofluorescein diacetate (H${}_2$DCFDA) was used for the detection of intracellular ROS levels. Cells in 12-well plates were washed twice with phosphate-buffered saline, and then incubated for 30 min at 37 °C with 0.5 mL of test solution per well (1:1000 H${}_2$DCFDA; HY-D0940; MCE, USA) in DMEM). Cells were then washed three times with phosphate-buffered saline to remove excess H${}_2$DCFDA. Furthermore, Hoechst 33342 trihydrochloride (HY-15559A; MCE) was used to stain the nuclei of PK15 cells; fluorescence intensities were observed directly under a fluorescence microscope to determine the levels of ROS.

Synthesis of lncRNA-SUSAJ1 was conducted using a MAXIscript Kit (K0441; Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions. The interaction between lncRNA-SUSAJ1 and unknown proteins was examined using the Pierce Magnetic RNA-Protein Pull-Down Kit (20164; Thermo Fisher Scientific, USA), in accordance with the manufacturer’s instructions.

The SimpleCh (R) Kit (Cell Signaling Technology) was used for RNA immunoprecipitation (RIP) assay, to assess the binding between the Hsp70 protein and lncRNA-SUSAJ1. Dithiothreitol (Beyotime) was added to the lysate to prevent RNA degradation; TRIzol was used to extract total RNA. Finally, RT-qPCR was performed on the Hsp70 protein-containing RNA mixture after adsorption with BeyoMag™ Protein A+G Magnetic Beads (P2108; Beyotime); this confirmed that the target lncRNA-SUSAJ1 was present in the pull-down mixture.

SPSS (mac 26.0.0.2; SPSS Inc., USA) was used for data analysis. All data are presented as means $\pm$ standard errors of the mean. The t-test was used to compare two groups; I 0.05 was considered to indicate statistical significance. GraphPad Prism 8.0 (GraphPad Software Inc., USA) was used to generate visual summaries of the data. Unless otherwise specified, the analytical data in all experiments were obtained from more than three independent experiments.

In a previous study, we found that lncRNA-SUSAJ1 resisted the proliferation of JEV [28], although the molecular mechanism remained unclear. ER stress is an important pathway for regulating virus assembly and proliferation [29, 30, 31, 32], and leads to the activation of the UPR pathway, which includes three main signalling pathways: protein kinase RNA-like ER kinase (PERK), IRE1, and activating transcription factor 6 (ATF6).

To investigate the effect of lncRNA-SUSAJ1 on ER stress, we performed Western blotting analysis. PK15 cells were transfected with plasmid for 6 h, and then infected with JEV for 24 and 48 h; total protein samples were extracted for Western blotting. LncRNA-SUSAJ1 overexpression promoted the expression of the ER stress marker protein GRP78 and induced phosphorylation of the eIF2$\alpha$ and ATF6 pathways but did not activate the phosphorylation of IRE1 (Fig. 1A). Phosphorylated eIF2$\alpha$ inhibits protein synthesis and promotes apoptosis, and NF-$\kappa$B is a crucial protein involved in ER stress-induced apoptosis [33, 34, 35]. In this study, we examined the expression of the anti-apoptotic protein NF-$\kappa$B, and found that lncRNA-SUSAJ1 overexpression inhibited the phosphorylation of NF-$\kappa$B subunit p65, whereas lncRNA knockdown promoted its phosphorylation (Fig. 1B).

Fig. 1.

The effect of lncRNA-SUSAJ1 on ER stress. (A) Overexpression of lncRNA-SUSAJ1 affects the expression level of ER stress-related proteins. Overexpression of lncRNA-SUSAJ1 in PK15 cells by pcDNA3.1. Whole-cell lysates were harvested from the treatment groups as indicated. Immunoblotting was performed to detect ER stress related signal protein levels using TBB5, eIF2$\alpha$ and IRE1 as an internal control. (B) Overexpression of lncRNA-SUSAJ1 in PK15 cells by pcDNA3.1. Whole-cell lysates were harvested from the treatment groups as indicated. Immunoblotting was performed to detect NF-$\kappa$B p65 phosphorylation and NF-$\kappa$B protein levels. (C) Knockdown of lncRNA-SUSAJ1 in PK15 cells by ASO. Whole-cell lysates were harvested from the treatment groups as indicated. Immunoblotting was performed to detect NF-$\kappa$B p65 phosphorylation and NF-$\kappa$B protein levels.

HSP70 inhibits cell apoptosis by inhibiting pro-apoptosis factors [36, 37, 38]. In this study, we investigated the relationship between lncRNA-SUSAJ1 and HSP70. An RNA pull-down assay was performed to explore interactions in PK15 cells infected with JEV for 36 h. In JEV-infected PK15 cells, the amount of Hsp70 protein pulled down by lncRNA-SUSAJ1 was higher than that in non-infected JEV cells (Fig. 2A). RNA samples from the experimental Hsp70-RNA complex group and control IgG-RNA complex group were isolated and purified using the RIP assay, and then verified by qRT-PCR. The level of lncRNA-SUSAJ1 expression was significantly higher in the Hsp70 group than in the IgG group (Fig. 2B). Then the qRT-PCR product was diluted 100-fold and used for nucleic acid electrophoresis to confirm the presence of lncRNA-SUSAJ1 (Fig. 2C).

Fig. 2.

The interaction between lncRNA-SUSAJ1 and HSP70. (A) The interaction of sense lcnRNA-SUSAJ1 with HSP70 was determined by RNA-pull down and western blot. (B) The interaction between HSP70 and lcnRNA-SUSAJ1 was determined by RNA-RIP. IgG was used as a negative control. (C) The agarose gel of lncRNA-SUSAJ1 after RNA-rip by HSP70. (D) Overexpression of lncRNA-SUSAJ1 in PK15 cells by pcDNA3.1. Whole-cell lysates were harvested from the treatment groups as indicated. Immunoblotting was performed to detect HSP70 protein levels using TBB5 as an internal control. *p 0.05 and **p 0.01 VS pcDNA3.1-NC. (E) Knockdown of lncRNA-SUSAJ1 in PK15 cells by ASO. Whole-cell lysates were harvested from the treatment groups as indicated. Immunoblotting was performed to detect HSP70 protein levels using TBB5 as an internal control. *p 0.05 and **p 0.01 VS si-NC.

PK15 cells were transfected with plasmid for 6 h, and then infected with JEV for 24 and 48 h; total protein samples were extracted for Western blotting. At 24 and 48 h after infection, pcDNA 3.1 lncRNA-SUSAJ1 inhibited Hsp70 protein expression compared to the control group NC, whereas lncRNA-SUSAJ1 knockdown promoted its expression (Fig. 2D,E). These results show that Hsp70 protein interacted with lncRNA-SUSAJ1 and that lncRNA-SUSAJ1 inhibited the expression of Hsp70 protein. Therefore, we conclude that lncRNA-SUSAJ1 may facilitate apoptosis in PK15 cells.

To study the effects of lncRNA-SUSAJ1 on apoptosis in JEV-infected PK15 cells, we transfected the pcDNA 3.1 lncRNA-SUSAJ1 and NC into the cells according to the manufacturer’s instructions. After 6 h of transfection, JEV infection was conducted for 24 and 48 h. qRT-PCR was performed to detect the mRNA expression levels of apoptosis-related genes. After the transfection of pcDNA 3.1 lncRNA-SUSAJ1, the expression level of lncRNA-SUSAJ1 was significantly higher than that of NC (Fig. 3A). At 48 h after infection, the expression levels of the pro-apoptotic genes caspase-3, caspase-8, caspase-9, BCL2L11, Bax, and TP53 were significantly increased (Fig. 3B–F,H); at 24 h after infection, the expression level of the anti-apoptotic gene BCL2 was significantly decreased (Fig. 3G).

Fig. 3.

lncRNA-SUSAJ1 overexpression elevates the mRNA levels of apoptosis-related genes. (A–H) PK15 cells were transfected with pcDNA3.1 lncRNA-SUSAJ1. Time course of lncRNA-SUSAJ1, Caspase-8, Caspase-9 et al. mRNA levels were determined by RT-qPCR after JEV infection (n = 3 per group). * p 0.05 and ** p 0.01 VS negative control (NC).

Mitochondrial membrane potential was measured using the JC-1 cationic dye staining method. After transfection with lncRNA-SUSAJ1 overexpression and NC plasmids for 6 h, cells were infected with JEV for 24 and 48 h; they were then subjected to JC-1 cationic dye staining analysis.

When the mitochondrial membrane potential is normal, direct observation using a fluorescence-inverted microscope shows accumulation of JC-1 cationic dye in the mitochondrial matrix, forming a polymer that emits strong red fluorescence; when depolarised, the dye exists in the cytoplasm as a monomer that produces green fluorescence. At 24 and 48 h after infection, red and green fluorescence intensities indicated promotion of mitochondrial membrane potential depolarisation (Fig. 4A).

Fig. 4.

Effects of lncRNA-SUSAJ1 overexpression on mitochondrial membrane potential (MMP). (A) PK15 cells were transfected with pcDNA3.1 lncRNA-SUSAJ1 and stained by JC-1 after JEV infected. Time course of MMP were stained by JC-1 dye, red bar = 100 $\mu$m. (B) Time course of MMP levels were determined by Automatic microplate reader (n = 3 per group), *p 0.05 VS NC.

Enzyme labelling analysis showed that the degree of mitochondrial membrane potential depolarisation at 48 h after infection was higher in the lncRNA-SUSAJ1 overexpression group than NC control group (Fig. 4B).

After lncRNA-SUSAJ1 overexpression, ROS levels in JEV-infected PK15 cells were examined by H2DCFDA and Hoechst 33342 staining. Blue-green fluorescence intensity was analysed in merged images. Compared with the uninfected group, the green fluorescence signal at 48 h was significantly increased in the infected group (Fig. 5A), indicating that lncRNA-SUSAJ1 overexpression significantly increased ROS levels in PK15 cells after JEV infection.

Fig. 5.

Effects of lncRNA-SUSAJ1 overexpression on ROS level and cell apoptosis. (A) PK15 cells were transfected with pcDNA3.1 lncRNA-SUSAJ1 and stained by the ROS reagent after JEV infected. Time course of ROS levels were determined by immunofluorescence, red bar = 100 $\mu$m. (B) The apoptosis of PK15 cells were determined by TUNEL assay. Time course of cell apoptosis levels were stained by TUNEL reagent, white bar = 50 $\mu$m.

TUNEL analysis was used to identify late apoptotic cells. After lncRNA-SUSAJ1 overexpression and 48 h of JEV infection, the proportion of late apoptotic PK15 cells significantly increased (Fig. 5B). The results of TUNEL analysis were consistent with the those of flow cytometry analysis. With increasing JEV infection time, the proportion of late apoptotic cells increased among PK15 cells with lncRNA-SUSAJ1 overexpression.

Apoptosis was investigated by flow cytometry analysis. Among cells overexpressing lncRNA-SUSAJ1, after 24 h of JEV infection, the proportion of viable cells decreased by 11.5% (p 0.01), the proportion of early apoptotic cells increased by 17.7%, and the proportion of late apoptotic cells decreased by 5.79% (Fig. 6A–C). After 48 h of JEV infection, the proportion of viable cells decreased by 15.6% (p 0.01), while the proportion of early apoptotic cells increased by 4% (p 0.01) (Fig. 6D,F) and the proportion of late apoptotic cells increased by 12.7% (p 0.01).

Fig. 6.

Effects of lncRNA-SUSAJ1 overexpression on cell Annexin V and Caspase-3 levels. (A–B,D–E) PK15 cells were transfected with pcDNA3.1 lncRNA-SUSAJ1 and stained by the annexin V and PI reagent after JEV infected. The annexin V and PI levels were determined by Flow cytometry. (C and F) The ratios of viable, early apoptosis, late apoptosis, and necrotic cells detected by Flow cytometry, C (24 h), D (48 h), **p 0.01 VS pcDNA3.1-NC. (G) PK15 cells were transfected with pcDNA3.1 lncRNA-SUSAJ1. Whole-cell lysates were harvested from the treatment groups as indicated. Immunoblotting was performed to detect Caspase-3 protein levels using TBB5 as an internal control. (H) Knockdown of lncRNA-SUSAJ1 in PK15 cells by ASO. Whole-cell lysates were harvested from the treatment groups as indicated. Immunoblotting was performed to detect Caspase-3 protein levels using TBB5 as an internal control.

Caspase-3 is closely associated with apoptosis [39]; levels of cleaved caspase-3 protein were detected by Western blotting in this study. lncRNA-SUSAJ1 strongly affected cleaved caspase-3 protein expression. At 24 and 48 h after infection, lncRNA-SUSAJ1 overexpression promoted the expression of cleaved caspase-3 protein (Fig. 6G), whereas lncRNA-SUSAJ1 knockdown inhibited the expression of cleaved caspase-3 protein (Fig. 6H).