Gene expression profiling of human lung adenocarcinoma of A549 cells induced by Toxoplasma virulence-related effector ROP16Ⅲ

doi:10.12140/j.issn.1000-7423.2021.04.009

Abstract

Abstract:

Objective To analyze the gene expression profile in the A549 human lung adenocarcinoma cell line transfected with the expression vector of Toxoplasma gondii rhoptry protein 16Ⅲ (ROP16Ⅲ), using bioinformatics methods, in order to identify candidate pathogenic genes. Methods The eukaryotic expression vector pEGFP-N1-ROP16Ⅲ was constructed. The A549 cells were assigned to three groups: untransfected, pEGFP-N1 and pEGFP-N1-ROP16Ⅲ groups. Cells in the latter two groups were instantantaneously transfected with pEGFP-N1 and pEGFP-N1-ROP16 plasmid, respectively. After 48 h, the cells were harvested and RNA was extracted. After RNA purification, microarray hybridization analysis was performed to identified differentially expressed genes (DEGs) which were further analyzed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional enrichment classification and pathway analysis. ToppGene was used to screen for Toxoplasma ROP16Ⅲ pathogenic candidate genes from DEGs. The STRING online analysis software was used to analyze the protein-protein interaction (PPI) network for protein products of the candidate genes. Results A total of 566 genes were differentially expressed in A549 transfected with Toxoplasma ROP16Ⅲ, of which 382 genes were up-regulated and 184 genes down-regulated. Bioinformatics analysis of the DEGs revealed 997 GO annotations and 50 KEGG signaling pathways. The DEGs were significantly enriched in defense response, type I interferon signaling pathway, innate immune response, and response to external biotic stimulus. KEGG pathway enrichment analysis suggested the DEGs were most significantly enriched in antigen processing and presentation, cytosolic DNA-sensing pathway, Toll-like receptor signaling pathway, and NOD-like receptor signaling pathway. In addition, 14 candidate genes were screened out from the DEGs by the ToppGene Suite. Among them, C-X-C motif chemokine 11 (CXCL11), toll-like receptor 3 (TLR3), C-C motif chemokine ligand 26 (CCL26), human leukocyte antigen E(HLA-E), promyelocytic leukemia protein (PML) and signal transducer and activator of transcription 2 (STAT2) had not been reported in Toxoplasma studies. The PPI network of the DEGs comprised 376 nodes and 1 152 edges, in which STAT2, HLA-E and PML were just located at the core position of the PPI network. Deletion of these key nodes led to the PPI network structure disorganized. Pathway enrichment analysis of 6 genes including CXCL11 resulted in 12 signaling pathways, which were mainly related to chemokine signaling pathway, Toll-like receptor signaling pathway, necroptosis, endocytosis and cytokine-cytokine receptor interaction. These bio-signal pathway may be related to the pathogenic mechanism of Toxoplasma ROP16Ⅲ. Conclusion CXCL11, TLR3, CCL26, HLA-E, PML and STAT2 were identified as candidate pathogenic genes, providing theoretical basis for further investigation of pathogenic mechanism of toxoplasmosis.

Key words: Toxoplasma ROP16Ⅲ, Gene expression profiling, Bioinformatics, Protein-protein interaction network

CLC Number:

R382.5

SU Ya-jing, QIAO Xia, WANG Peng-tao, KANG Yu-ting, YANG Ning-ai, JIA Wei, ZHAO Zhi-jun. Gene expression profiling of human lung adenocarcinoma of A549 cells induced by Toxoplasma virulence-related effector ROP16Ⅲ[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2021, 39(4): 473-480.

Figures/Tables 4

Table 1

Genes with highest FC value（up-/down-regulated）of human lung adenocarcinoma of A549 cells transfected with pEGFP-N1-ROP16Ⅲ

基因标志Gene symbol	基因名称Gene name	变化倍数Fold change	表达情况Expression
CYP2C18	细胞色素P450家族2亚家族C成员18 Cytochrome P450 family 2 subf amily C member 18	18.70	上调 Up
CMPK2	胞苷/尿苷酸激酶2 Cytidine/uridine monophosphate kinase 2	18.40	上调 Up
IFITM1	干扰素诱导的跨膜蛋白1 Interferon induced transmembrane protein 1	17.41	上调 Up
IFIT2	三角形四肽重复干扰素诱导蛋白2 Interferon induced protein with tetratricopeptide repeats 2	17.17	上调 Up
TRIM22	三结构域分子22 Tripartite motif-containing 22	14.88	上调 Up
OAS2	2′,5′-寡腺苷酸合成酶 2 2′,5′-oligoadenylate synthetase 2	13.65	上调 Up
IFIT3	扰素诱导的三角形四肽重复蛋白3 Interferon induced protein with tetratricopeptide repeats 3	12.98	上调 Up
OASL	2′,5′-寡腺苷酸合成酶样蛋白 2′,5′-oligoadenylate synthetase like	12.67	上调 Up
RSAD2	含基S-腺苷蛋氨酸结构域的蛋白2 Radical S-adenosyl methionine domain containing 2	12.63	上调 Up
XAF1	X连锁凋亡抑制蛋白相关因子1 X-linked inhibitor of apoptosis-associated factor 1	12.57	上调 Up
OR51G2	嗅觉受体家族51亚家族G成员2 Olfactory receptor family 51 subfamily G member 2	-127.96	下调Down
RGS3	G蛋白信号转导调节因子3 Regulator of G protein signaling 3	-5.84	下调Down
LIPG	内皮脂肪酶 Lipase G, endothelial type	-5.71	下调Down
OR51F2	嗅觉感受器家族51亚家族F成员2 Olfactory receptor family 51 subfamily F member 2	-4.65	下调Down
DIO3	碘甲腺原氨酸脱碘酶3 Iodothyronine deiodinase 3	-4.63	下调Down
NCRNA00204B	非蛋白编码RNA 204B Non-protein coding RNA 204B	-4.61	下调Down
TGIF2LY	重组人TGF-β诱导的转录因子2样蛋白 TGF-β induced factor homeobox 2 like Y-linked	-4.51	下调Down
C18orf26	18号染色体开放阅读框26 Chromosome 18 open reading frame 26	-4.21	下调Down
ZFYVE9	锌指FYVE域含蛋白9 Zinc finger FYVE-type containing 9	-4.13	下调Down
APCDD1	腺瘤性息肉病大肠埃希菌下调蛋白1 Adenomatosis polyposis coli down-regulated 1	-3.92	下调Down

Table 1

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序号Rank	类别Category	GO号 GO ID	GO注释 GO annotation	基因数Count	P值P value
上调Up-regulated
1	BP	0051607	病毒防御反应 Defense response to virus	46	< 0.01
2	BP	0009615	对病毒的应答反应 Response to virus	52	< 0.01
3	BP	0006952	防御反应 Defense response	94	< 0.01
4	BP	0060337	I型干扰素信号通路 Type I interferon signaling pathway	32	< 0.01
5	BP	0071357	细胞对I型干扰素的反应 Cellular response to type I interferon	32	< 0.01
6	BP	0034340	对I型干扰素的反应 Response to type I interferon	32	< 0.01
7	BP	0045087	先天免疫反应 Innate immune response	73	< 0.01
8	BP	0098542	对其他生物的防御反应 Defense response to other organism	52	< 0.01
9	BP	0043207	对外界生物刺激的反应 Response to external biotic stimulus	65	< 0.01
10	BP	0051707	对其他有机体的反应 Response to other organism	65	< 0.01
下调Down-regulated
1	CC	0005886	细胞质膜 Plasma membrane	40	< 0.01
2	CC	0071944	细胞外围 Cell periphery	40	< 0.01
3	CC	0009986	细胞表面 Cell surface	12	< 0.01
4	CC	0005887	细胞膜的组成成分 Integral component of plasma membrane	16	< 0.01
5	BP	0006814	钠离子运输 Sodium ion transport	6	< 0.01
6	CC	0044459	细胞膜部分 Plasma membrane part	22	< 0.01
7	CC	0031226	细胞膜的固有成分 Intrinsic component of plasma membrane	16	< 0.01
8	CC	0016021	膜组成部分 Integral component of membrane	40	< 0.01
9	BP	0032501	多细胞生物过程 Multicellular organismal process	46	< 0.01
10	MF	0004872	受体活性Receptor activity	17	< 0.01

序号Rank	通路号 Pathway ID	信号通路 Signaling pathway	基因数Gene count	P值 P value
上调 Up-regulated
1	Hsa 05168	单纯疱疹病毒感染-智人（人类） Herpes simplex infection-Homo sapiens (human)	26	< 0.01
2	Hsa 05164	甲型流感-智人（人类） Influenza A-Homo sapiens (human)	21	< 0.01
3	Hsa 05162	麻疹-智人（人类） Measles-Homo sapiens (human)	15	< 0.01
4	Hsa 04612	抗原处理及呈递-智人（人类） Antigen processing and presentation-Homo sapiens (human)	11	< 0.01
5	Hsa 05160	丙型肝炎-智人（人类） Hepatitis C-Homo sapiens (human)	13	< 0.01
6	Hsa 04623	细胞质DNA传感通路-智人（人类） Cytosolic DNA-sensing pathway-Homo sapiens (human)	9	< 0.01
7	Hsa 04620	Toll样受体信号通路-智人（人类） Toll-like receptor signaling pathway-Homo sapiens (human)	11	< 0.01
8	Hsa 05332	移植物抗宿主病-智人（人类） Graft-versus-host disease-Homo sapiens (human)	7	< 0.01
9	Hsa 05330	同种异体移植排斥反应-智人（人类） Allograft rejection-Homo sapiens (human)	6	< 0.01
10	Hsa 04621	NOD样受体信号通路-智人（人类） NOD-like receptor signaling pathway-Homo sapiens (human)	7	< 0.01
下调 Down-regulated
1	Hsa 00020	柠檬酸循环(TCA循环)-智人（人类） Citrate cycle (TCA cycle)-Homo sapiens (human)	2	< 0.05
2	Hsa 04024	cAMP信号通路-智人（人） cAMP signaling pathway-Homo sapiens (human)	4	< 0.05
3	Hsa 04360	轴突导向-智人（人类）Axon guidance-Homo sapiens (human)	3	< 0.05

序号 Rank	ToppGene
序号 Rank	基因标志 Gene symbol	平均得分 Average score（× 10^-1）	P值/× 10^-5 P value/× 10^-5	基因标志 Gene symbol	相互作用基因数 Interactant count	得分/× 10^-4 Score/× 10^-4
1	IL6	9.84	0.16	CCL5	41	12.90
2	MYH11	7.30	2.43	CCL2	21	10.10
3	STAT1	8.79	2.75	STAT1	199	9.72
4	CXCR4	9.71	3.05	CCL4	11	8.90
5	B2M	8.25	3.60	CCL3	13	8.52
6	CCL3	9.13	3.91	CXCL11	5	6.76
7	CCL2	9.09	4.47	CCL26	3	6.68
8	HLA-A	8.76	4.97	BIRC3	102	5.04
9	CCL5	9.02	7.59	AMOT	104	4.05
10	CXCL11	8.98	7.62	HDAC5	355	3.94
11	NOD2	8.58	8.10	NMI	70	3.72
12	HLA-B	8.66	8.23	PML	257	3.61
13	TLR3	8.31	11.10	IFI16	358	3.48
14	HLA-C	8.32	12.20	IRF7	48	3.39
15	HLA-G	8.61	16.20	TNFAIP3	99	3.35
16	TNFSF10	8.90	16.50	STAT2	51	3.30
17	CCL4	8.96	16.90	CXCR4	53	3.21
18	FGA	7.94	17.70	TLR3	32	3.14
19	LTB	8.81	18.90	LMO2	208	3.02
20	IFNB1	8.90	20.10	PARP14	14	2.91
21	TNFAIP3	7.77	22.10	CISH	34	2.86
22	FGFR2	7.82	24.80	CD93	35	2.63
23	CCL26	8.88	28.90	OPTN	80	2.45
24	ERAP1	7.78	30.40	BST2	13	2.39
25	DDX58	7.98	36.90	TRIM21	99	2.20
26	HLA-E	8.44	37.70	HLA-E	54	2.14
27	IFNA16	8.68	48.60	ISG15	189	1.88
28	RET	7.13	56.90	PCDH7	40	1.82
29	PML	7.53	58.30	EIF2AK2	187	1.68
30	STAT2	6.56	62.70	IL6	13	1.65