Background: Newcastle disease virus (NDV) is an oncolytic virus that can inhibit cancer cell proliferation and kill cancer cells. The NDV nonstructural V protein can regulate viral replication; however, whether the V protein contributes to NDV oncolysis is unclear. Results: This study revealed that NDV inhibited tumor cell proliferation and that V protein expression promoted the proliferation of HepG2 cells, as determined at the single-cell level. In addition, to identify the regulatory mechanism of the V protein in HepG2 cells, transcriptome sequencing was performed and indicated that the expression/activation of multiple cell proliferation-related genes/signaling pathways were changed in cells overexpressing the V protein. Hence, the MAPK and WNT signaling pathways were selected for verification, and after blocking these two signaling pathways with inhibitors, the V protein promotion of cell proliferation was found to be attenuated. Conclusions: The results showed that the V protein regulated the proliferation of cancer cells through multiple signaling pathways, providing valuable references for future studies on the mechanism by which the V protein regulates cancer cell proliferation.
Keywords: Newcastle disease virus; V protein; Cell proliferation; MAPK signaling pathway; WNT signaling pathway
Zhili chu and Sihui Yang contributed equally to this work.
Newcastle disease virus (NDV) is an oncolytic virus in the Paramyxoviridae family. NDV replicates in human tumor cells but largely fails to replicate in noncancerous human cells [[
Among the viral structural proteins, NP encapsulates genomic RNA and binds to the P and L proteins to form ribonucleoprotein complexes (RNPs), which are templates for viral genome transcription and replication [[
During oncolysis, NDVs are toxic to human tumor cell lines of ecto-, endo-, and mesodermal origin, and the cytotoxicity induced by these NDV strains in tumor cells is related to multiple caspase-dependent pathways of apoptosis, which are activated independent of IFN signaling [[
The present study evaluated the possible mechanisms underlying the antiapoptotic effects of the V protein, and the results suggested that the V protein may be involved in regulating cancer cell proliferation. Moreover, the role played by the V protein in regulating cancer cell proliferation was confirmed, and the results indicated that the V protein can activate multiple signaling pathways to promote cancer cell replication.
To determine the effect of NDV on cell proliferation at the single-cell level, we first infected HepG2 cells with NDV at a multiplicity of infection (MOI) of 1 for 24 h and then added 10 nM EdU to the cells and incubated these treated cells for 1 h. Then, the cells were subjected to immunofluorescence and EdU analyses, and the results showed that the proportion of EdU-positive and GFP-positive cells was significantly lower than the proportion of GFP-negative cells (Fig. 1A). Then, another dish of HepG2 cells was infected with NDV (MOI of 1) for 48 h, followed by immunofluorescence and EdU analyses (Fig. 1B). The single-cell level results were similar to those obtained after NDV infection for 24 h and suggested that NDV inhibited cell proliferation.
Graph: Fig. 1 NDV inhibits cell proliferation at the single-cell level. HepG2 cells were infected with NDV. Then, 24 h (A) and 48 h (B) postinfection, the cells were labeled with EdU (10 nM) for 1 h and then stained with chicken anti-NDV serum and goat anti-chicken IgY Alexa Fluor® 488 (green) secondary antibodies. EdU staining was carried out according to the EdU manufacturer's instructions. Nuclei were stained with Hoechst 33,342. Images were captured using a Leica fluorescence microscope (400 ×); bar = 50 μm
To confirm the function of the V protein in cell proliferation, we overexpressed the V protein in HepG2 cells. Western blot (Fig. 2A and Supplementary Fig. 1) and immunofluorescence (Fig. 2B-C) analyses showed that the V protein was overexpressed in HepG2 cells. After transfection with pCAGEN-Flag-V for 24 h (Fig. 2B) or 48 h (Fig. 2C), 10 nM EdU was added and incubated for 1 h. The immunofluorescence and EdU analyses revealed more V protein-positive cells than V protein-negative cells. To verify this finding at the general level, we used a real-time label-free cell recorder to detect cell proliferation, and the results showed that the V protein promoted cell proliferation (Fig. 2D). These results suggested that the V protein enhanced HepG2 proliferation. However, compared with detection at 48 h after overexpression of the V protein (* P < 0.05), the effect of the V protein on promoting cell proliferation was more significant at 24 h (* * P < 0.01).
Graph: Fig. 2 The V protein promotes cell proliferation at the single-cell level. HepG2 cells were transfected with pCAGEN-Flag-V (to overexpress the V protein). WB results showed that V protein was successfully overexpressed A. After 24 h (B) and 48 h (C), the cells were labeled with EdU (10 nM) for 1 h and then stained with rabbit anti-Flag antibody and goat anti-rabbit IgG Alexa Fluor.® 488 (green) secondary antibody. For EdU staining, the steps were carried out according to the EdU manufacturer's instructions. Nuclei were subsequently stained with Hoechst 33,342. Images were captured with a Leica fluorescence microscope (400 ×); bar = 50 μm. The results of the unlabeled cell recorder showed that the number index of cells in the overexpression of V protein group was significantly higher than that in the control group (E)
To determine the gene expression difference between the V protein overexpression group and the control group, sequencing was performed, and the results showed that the expression of genes was increased/downregulated (Fig. 3A). The green circle indicates the control group, and the red circle indicates the V protein overexpression group. A total of 561 genes were specifically expressed in V protein-overexpressing cells, and 501 genes were specifically expressed in control cells (Fig. 3B). qRT–PCR with β-actin as the reference revealed that the expression of the cell proliferation-related genes FOS and RRBP1 was increased and that the expression of ERLEC1 and LMAN1 was decreased (Fig. 3C-F); these results were consistent with the sequencing results.
Graph: Fig. 3 Differentially expressed genes (DEGs) in HepG2 cells overexpressing the V protein. RNA sequencing (RNA-seq) analyses of three independent experiments were repeated. A Volcano map showing differences in genetic transcription in HepG2 cells 48 h after transfection with pCAGEN-Flag-V. B Venn diagram analysis was performed to identify the genes with expression regulated through V protein overexpression. (C-H) qPCR detection of changes in gene expression after V protein overexpression. *P < 0.05
An ingenuity pathway analysis showed significant changes in signaling pathways following V protein expression, and the 20 most significantly changed pathways are shown (Fig. 4A). NDV is a single-stranded negative-stranded RNA virus. Among the enriched pathways, herpes simplex virus 1 infection, viral carcinogenesis and RNA transport are closely related to viral cycle life, and changes in these signaling pathways suggested that the V protein plays a regulatory role in the viral life cycle. However, prior to this study, whether the V protein regulates cell proliferation, which affects changes in the cell cycle, was unknown. This study suggests a strategy for exploring the mechanism by which the V protein regulates cell proliferation. A total of 166 cell cycle-related genes were in the cell cycle signaling pathway (Fig. 4B). After V protein overexpression, the expression of most of these genes was increased, and they are marked in red (Fig. 4B). These results further demonstrate that the V protein can regulate cell proliferation.
Graph: Fig. 4 Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showing enriched differentially expressed genes (DEGs). A The KEGG pathways with the most significantly enriched DEGs. B, RNA sequencing (RNA-seq) results showed the relationship and expression changes of 166 cell cycle-related genes after overexpression of the V protein in HepG2 cells
Many signaling pathways regulate cell proliferation. Although RNA-seq showed that the cell cycle was affected, no specific signaling pathways associated with cell proliferation regulation were enriched with the identified DEGs. Therefore, the mechanism through which the V protein regulates cell proliferation remains unclear. The MAPK and WNT signaling pathways are classical signaling pathways that affect cell proliferation, and variable genes caused by overexpression of V protein are partially concentrated in MAPK and WNT signaling pathways (Supplementary table). To verify that these signaling pathways affected the proliferation of cells after the V protein was overexpressed, we first used the ERK1/2 inhibitors U0126 (100 nM) and IWR-1 (200 nM) to examine the role played by the ERK1/2 and WNT pathways in HepG2 proliferation. The EdU results showed a reduction in the number of EdU-positive cells (red fluorescence) after treatment with 100 nM U0126 or 200 nM IWR-1 for 24 h compared with the control group (Fig. 5A).
Graph: Fig. 5 The MAPK and ERK1/2 signaling pathway inhibitor U0126 and WNT/β-catenin signaling pathway inhibitor IWR-1 interfere with the effect of V protein on cell proliferation. A After 6 h, U0126- and IWR-1-treated cells were labeled with EdU (10 nM) and incubated for 1 h. The EdU staining steps were carried out according to the EdU manufacturer's instruction manual. Nuclei were subsequently stained with Hoechst 33,342, and EdU-positive cell percentages are shown on the right. HepG2 cells were transfected with pCAGEN-Flag-V (to overexpress the V protein). After 24 h (A), U0126 (B) or IWR-1 (C) was added to cells, which were incubated for another 6 h, followed by labeling with EdU (10 nM) and incubation for 1 h. Then, the cells were stained with rabbit anti-Flag antibody and goat anti-rabbit IgG Alexa Fluor® 488 (green) secondary antibody. For EdU staining, the steps were carried out according to the EdU manufacturer's instructions. Nuclei were subsequently stained with Hoechst 33,342. Images were captured using a Leica fluorescence microscope (400 ×), bar = 50 μm
Then, to verify that the changes in these two signaling pathways affected cell proliferation because of V protein expression, plasmids (pCAGEN-Flag-V) overexpressing the V protein were transfected into HepG2 cells, and inhibitors were added 24 h after transfection. For the cells treated for 12 h, EdU and immunofluorescence staining analysis showed that both U0126 and IWR-1 (Fig. 5 B) inhibited cell proliferation, as determined at the single-cell level. These results indicated that both signaling pathways affected cell proliferation through the action of the V protein. Hence, it is suggested that the V protein regulates cell proliferation through multiple mechanisms.
This study demonstrated at the single-cell level that NDV inhibited the proliferation of cancer cells; however, the V protein of NDV promoted the proliferation of cancer cells. These findings suggested that oncolytic NDV viral proteins may be involved in reducing the oncolytic effect, which provides a new perspective on oncolytic viruses.
NDV infects poultry, which can affect bursa development and can proliferate in susceptible chicken lines [[
Abdullah et al. [[
NDV potentially led to reactive oxygen species production in a dose-dependent manner, which might have led to NDV action against cervical cancer cell proliferation [[
Our present study performed at the single-cell level showed that NDV infection resulted in cell proliferation inhibition, which may be detrimental to viral replication. However, we also found that at different times (24 h and 48 h) after NDV infection, there were proportional differences in the effect of the V protein on cell proliferation, suggesting that NDV may engage in other dynamic mechanisms to affect cell proliferation. Perhaps this difference gradually decreases with the extension of time, suggesting that V protein may promote cell proliferation at an early stage.
Vaccinia virus (VACV) has been reported to alter cell cycle regulation and trigger the host cell DNA damage response. Caroline K. Martin et al. concluded that VACV modulates host cell proliferation and host cell cycle progression through temporal expression of multiple VACV effector proteins [[
Many mechanisms affect cell proliferation [[
The MEK/ERK signaling pathway regulates cancer cell proliferation, apoptosis, inflammation, angiogenesis, metastasis and drug resistance. In our previous work, we found that the V protein promotes viral replication in HeLa cells through the activation of the MEK/ERK signaling pathways [[
Similar to the MEK/ERK signaling pathway, the Wnt signaling pathway exhibits diverse functions. It is involved in an evolutionarily conserved cell-to-cell coordination mechanism and is crucial for a variety of physiological processes in an organism's body, including stem cell regeneration, proliferation, division, and migration; cell polarity and fate determination; and specification of neural crest, neural symmetry and morphogenesis [[
Studies have shown that absence of the V protein may be an important measure to transform oncolytic viruses [[
A well-understood phenomenon is the ability of oncolytic NDV to inhibit cancer cell proliferation. As a nonstructural protein of NDV, the V protein mainly plays a role in promoting virus replication, but our results show that the V protein can promote cancer cell proliferation at the single-cell level. This finding may suggest the idea of oncolytic virus transformation. Moreover, through transcriptome sequencing, we initially found some mechanisms by which the V protein promotes the proliferation of cancer cells, which may provide a new solution for better use of Newcastle disease virus for oncolysis. When killing cancer cells with viruses, it may be a good choice to delete the V protein from the viruses.
The HepG2 cells used in this experiment had been previously obtained and were stored in our laboratory (Shanghai EK-Bioscience Biotechnology Co., Ltd.). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo, Waltham, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, USA) (2% FBS was used in the maintenance culture medium), 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mM/L-glutamine (Invitrogen, Carlsbad, CA, USA), and 1% nonessential amino acids (Invitrogen, Carlsbad, CA, USA).
La Sota is an attenuated NDV strain that had been previously obtained and was stored in our laboratory. Viruses were propagated in the allantoic cavities of 9–11-day-old embryo-specific-pathogen-free chicken eggs, and the allantoic fluid was harvested and stored at − 70 °C until further use [[
The full-length V gene was amplified from NDV-infected cells and inserted into pCAGEN-Flag to generate pCAGEN-Flag-V. The sequences of the primers used are listed in sequence Table 1.
Table 1 Primers sequence
Gene Primers Forward primer (5'to 3') Reverse primer (5'to 3') GGGGCAAGGTGGAACAGTTA AGTTGGTCTGTCTCCGCTTG GAGTGGCTGCCAAAAACTCG GAGTGGCTGCCAAAAACTCG CGCAAGGAGATGGCGAAAAC GAGGACAGTCACATTGGGGG GATCCAGGCAAAGGGGTCTC TGTACTCGAAACGGCGATGT CCAGACAGTCAGGTGCAAAGA CACTGGAGATTCAACCCCAAGA GTACGGATGGTCTGGCAACA GTCTCTGATCAGCGGGTGTC GGGCATGGGTCAGAAGGATT TCGATGGGGTACTTCAGGGT
Kemix-TRLIP was used for plasmid transfection of HepG2 cells. After reaching a density of 60%, the cells were transfected. To overexpress the V protein, cells were transfected with pCAGEN-Flag-V, and the control cells were transfected with an empty pCAGEN-Flag plasmid. To examine the influence of V on cell proliferation, 24–48 h after transfection of the cells with the plasmid encoding the V protein, samples were collected for further analysis. At the appropriate time, whole RNA was collected and stored until further analysis.
From 24 to 48 h posttransfection with pCAGEN-Flag-V, the number of apoptotic cells was detected by propidium iodide (PI) staining assay according to the manufacturer's protocol. Briefly, 1 × 10
Cells were transfected with pCAGEN-Flag-V. Forty-eight hours later, 10 Nm EdU was added to the cultures and incubated for 1 h. The assay steps were performed according to the respective manufacturer's instructions. Briefly, the cells were treated with paraformaldehyde solution, 0.3% Triton X-100, primary antibody (anti-Flag, CST, # 14793S, 1:200 for immunofluorescence; anti-NDV (chicken immune serum) polyclonal antibody, prepared in our laboratory, 1:1000 for immunofluorescence), Click additive solution, secondary antibody (goat anti-rabbit IgG H&L (Alexa Fluor® 488), Abcam, ab150077, 1:300 for immunofluorescence; goat anti-chicken IgY H&L (Alexa Fluor® 488), Abcam, ab150169, 1:300 for immunofluorescence) and Hoechst 33,342. Between every step, the cells were washed with PBS three times.
After overexpressing the V protein in HepG2 cells, total RNA was isolated with RNAiso Plus (Takara, Code No.: 9109). Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. The samples were sequenced with an Illumina HiSeq 4000 system (Illumina Inc., USA). Differential expression of transcripts in the treatment and control groups was measured. RNA-Seq was performed by BGI Co., and differentially expressed genes were identified with the linear models for microarray data (limma) package in GEO2R; the cutoff criteria were P < 0.05.
After overexpressing the V protein in HepG2 cells, the relative mRNA expression of C-Myc, ATF1, c-Fos, CCND1, NDV vRNA and C1QC was measured by qRT–PCR. The cells were lysed with TRIzol reagent (TaKaRa, Dalian, China) to obtain total cellular RNA. Subsequently, cDNA was synthesized by reverse transcription using the Prime Script RT reagent kit (TaKaRa, Dalian, China). qRT–PCR was performed with RealStar Green Fast Mixture (GenStar, Beijing, China) according to the manufacturer's protocol. β-Actin was the internal control, and the relative expression of each gene was normalized to that of β-actin. Relative transcript levels were analyzed using the 2-△△T method. The sequences of the qRT–PCR primers are listed in Table 1.
A total of 10,000 cells per well were seeded on 16-well E-pates, and cell growth was recorded every 15 min for 96 h. Two hours after seeding the cells, a mixture consisting of a protein V expression vector and transfection reagent was added to the cells. Then, 0.2 µg of plasmid and 0.5 µl of transfection reagent were added to each well. After continuous recording, the experiments were replicated, and the results were analyzed.
Statistical analysis was performed with GraphPad Prism 5 software (GraphPad Software, Inc., CA, USA). All values are expressed as the means ± SDs of three independent experiments. Student's t test and one-way ANOVA were used to evaluate the significance of differences; P < 0.05 was considered to be statistically significant.
The authors thank the instrument sharing platform at Xinxiang Medical University for assistance with technical support.
Zhili Chu designed research. Zhili Chu、SihuiYang、Qianru Li、Jianing Shang and Zilong Ren performed research. Zhili Chu and Feng Ren analyzed data. Zhili Chu and Feng Ren wrote the paper. All authors read and approved the final manuscript.
This work was supported by the Key Research and Development Projects of Colleges and Universities in Henan Province (No. 21A320016).
Xinxiang Medical College Talent (PhD) Support Program (No. XYBSKYZZ201919).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
The raw data from both the mRNA and miRNA sequencing were submitted to the GEO of NCBI with accession number PRJNA871355 for the mRNA datahttps://
Animal testing methods were performed in accordance with relevant guidelines and regulations and handled according to standard use protocols and animal welfare regulations. The protocol in this study was approved by the Committee on the Ethics Committee of Xinxiang Medical (Permit XYLL-2021077). In addition, we confirm that all animal methods are reported in accordance with ARRIVE guidelines (
Not applicable.
The authors declare no competing interests.
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By Zhili Chu; Sihui Yang; Qianru Li; Jianing Shang; Zilong Ren and Feng Ren
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