Microarray expression profile and analysis of circular RNA regulatory network in malignant pleural effusion

Figures & data

Table 1. Demographics of the study participants

Group	No.	Age (years)	Sex	BMI (Kg/cm^2)	Accompanying diseases	TP (g/L)	ALB (g/L)	ADA (U/L)	LDH (U/L)
	1	72	F	28	HP, CHD	49.6	35	16.1	315
LA-MPE	2	83	M	21.9	CI	48.0	27.5	25.7	1286
	3	81	M	28.4	HP	49.2	29	5.5	175
	1	53	F	25.1	HP	49.6	33.3	60.7	515
TPE	2	45	M	20.9	No	48.4	25.6	41.7	522
	3	85	F	16.9	CHD, AF	47.3	24.5	40.9	102

LA-MPE: lung adenocarcinoma associated malignant pleural effusion; TPE: tuberculous pleural effusion; HP: hypertension; CHD: coronary heart disease; CI: cerebral infarction; AF: atrial fibrillation; TP: Total protein; ALB: Albumin; ADA: adenosine deaminase; LDH: lactic dehydrogenase.

Table 2. Specific circRNA primers used for quantitative RT-PCR analysis

Name	Sequence
hsa_circ_0001554	F: AGTTGGGGTGGTTTGAGGTT
	R: GCCAAACTCAGGTTCATTTCCT
hsa_circ_0003954	F: TGAGGTCATGCAGTGTCACC
	R: TAGGGTCGCTCCATGGTTGG
hsa_circ_0004086	F: GTGACCCAGGTGGCACATTC
	R: AGTTGTTCATGCTCGGAGAGT
hsa_circ_0050579	F: TGGCCTCCAGGTTCCAATGA
	R: AAGCCTGCCAGGAACACAAG
hsa_circ_0067705	F: CCACTATGTCTTGATTCCCTCG R: CCACCAAGAGCCAAGAGGAT
hsa_circ_0025542	F: GCCCACCTGAATGACCTTG
	R: GGTAGATCCGTGCTCCGAC
hsa_circ_0085386	F: CGACATCCTGCGGACCTACTC
	R: GCTGCTTCCAATAAAAAGGCTC
hsa_circ_0072793	F: GAAGAAAGATGGACAGGTATGACA
	R: CCACCGCTGCTGTAACGA
hsa_circ_0084927	F: GCGTTGAGGAAGCATAAAGAC
	R: GTGAACCCCATGAGTACGAATA
18SrRNA	F: GTAACCCGTTGAACCCCATT
	R: CCATCCAATCGGTAGTAGCG
HS-RPS18	BN61101
HS-GAPDH	BN61104

Figure 1. Differential expression of circRNAs and mRNAs in MPE and TPE. (a, b) Hierarchical clustering analysis of differentially expressed circRNAs (a) and mRNAs (b) between MPE group and TPE group; each group included three individuals (FC (absolute value) ≥ 2.0, P < 0.05). Each column represents one sample, and each row represents a transcript. “Red” indicates high relative expression, and “Green” indicates low relative expression. (c, d) Volcano plots showing differentially expressed circRNAs (c) and mRNAs (d) in MPE. Red and green points represent up-regulated and down-regulated transcripts in MPE, respectively (FC (absolute value) ≥ 2.0, P < 0.05). MPE, Malignant Pleural Effusion; TPE, Tuberculous Pleural Effusion.

Figure 2. Characterizations of circRNAs and mRNAs expression in MPE compared with TPE. (a) Pie chart of aberrantly expressed circRNAs and mRNAs in MPE. (b) The distribution of differentially expressed circRNAs in human chromosomes. MPE, Malignant Pleural Effusion; TPE, Tuberculous Pleural Effusion.

Figure 3. GO and KEGG analysis of differentially expressed mRNAs in MPE. (a, b) GO annotation of up-regulated (a) and down-regulated (b) mRNAs. BP, biological process; CC, cellular component; MF, molecular function. (c, d) KEGG pathway enrichment analysis for up-regulated (c) and down-regulated (d) mRNAs. The related pathways were ranked according to GeneRatio (Selection Counts/Selection Size).

Figure 4. The principle component analysis (PCA) on the microarray data. (a) PCA was performed on the differentially expressed circRNAs obtained from microarray data. The MPE samples can be separated from the TPE samples. (b) PCA plot of the differentially expressed mRNAs.

Figure 5. Validation of differentially expressed candidate circRNAs. The expression levels of circRNAs were detected using qRT-PCR. (a) Validation of the expression of has_circ_0001554 and has_circ_0004086. The heights of the columns represent the fold change (MPE/TPE) computed from the microarray data and the qPCR. (b, c, d, e, f) Validation of the expression of hsa_circ_0067705, hsa_circ_0025542, hsa_circ_0085386, hsa_circ_0072793, and hsa_circ_0084927. CircRNA expression was quantified using the 2^{−(ΔCt MPE – ΔCt TPE)} method with normalization to 18SrRNA expression levels. Values are expressed as the mean ± SEM (n = 9). qRT-PCR, quantitative reverse transcription real-time polymerase chain reaction; SEM, standard error of the mean; MPE, Malignant Pleural Effusion; TPE, Tuberculous Pleural Effusion; ND, non-detected.

Table 3. The top 5 up-regulated circRNAs targeted circRNA-miRNA-mRNA network

CircRNAs	MRE1	MRE2	MRE3	MRE4	MRE5	No. of mRNA targets
hsa_circ_0067705	hsa-miR-298	hsa-miR-3185	hsa-miR-544a	hsa-miR-4675	hsa-miR-6071	142
hsa_circ_0025542	hsa-miR-298	hsa-miR-607	hsa-miR-4710			163
hsa_circ_0072793	hsa-miR-4432	hsa-miR-4522	hsa-miR-1261	hsa-miR-6126	hsa-miR-3123	154
hsa_circ_0084927	hsa-miR-4319	hsa-miR-520e	hsa-miR-520b	hsa-miR-623	hsa-miR-302e	36
hsa_circ_0085386	hsa-miR-4692	hsa-miR-3612	hsa-miR-4698	hsa-miR-4514	hsa-miR-5695	180

MRE: miRNA response element.

Figure 6. GO and KEGG analysis of the top 5 up-regulated circRNA targeted mRNAs. (a) GO enrichment for targeted mRNAs. All significant enriched GO terms were listed. (b) KEGG pathway enrichment for targeted mRNAs. Top 10 significant enriched pathway terms were listed.

Figure 7. The circRNA-miRNA-mRNA network. 23 miRNA and 675 genes were targeted by the top 5 significantly up-regulated circRNAs. Diamond, circRNA; Rectangle, miRNA; Ellipse, mRNA; Red, up-regulated, Fold Change (FC) ≥ 2.0 and p-value < 0.05; Green, down-regulated, FC≤-2.0 and p-value < 0.05.