Identification of potential urine proteins and microRNA biomarkers for the diagnosis of pulmonary tuberculosis patients

Figures & data

Fig. 1 Representative 2-DE gel image obtained from pooled protein samples.

Comparison of (a) healthy individuals and (b) pulmonary tuberculosis patient proteins in pH 3–10 separated on 2-DE gels. Isoelectric points are indicated at the top and molecular weight markers in kDa on the left. The data are representative of two separate experiments. A p-value <0.05 indicates statistical significance using the two-tailed t-test

Characteristics of proteins identified in this study

Rank	Protein names	Accession no.	Protein score	Protein score C.I. %	Protein MW	Protein PI	Pep. Count
N1	Amylase,alpha2A; pancreatic variant (Fragment)	tr|Q53F26|Q53F26	501	100	58,368.3	6.6	17
N2	Albumin (fragment)	tr|F6KPG5|F6KPG	500	100	68,483.7	5.73	28
N2	Serum albumin	sp|P02768|ALBU_	189	100	71,317	6	5
N2	Cadherin-11OS = homo sapiens GN = CDH11 PE = 2	tr|H3BUU9|H3BUU	186	100	74,032.8	4.53	12
N3	Α-1-microglobulin/bikunin precursor. (AMBP)	gi|122801	423	100	39,886.3	5.95	7
N3	Thyroid receptor-interacting protein 11 isoform X3	gi|530404933	76	99	110,014	5	66
N4	Retinol-binding protein 4, plasma, isoform	gi|119570453	371	100	23,301	6	12
N4	Ataxin-7-like protein 2	gi|119588598	81	100	250,066	6	38
N4	Early endosome antigen 1, 162 kd	gi|93277115	67	92	78,785	9	19
N4	CD59 antigen, complement regulatory protein	gi|119617877	57	24	163,337	6	31
N5	Transferrin variant (fragment)	tr|Q53H26|Q53H2	334	100	79,311	7	8
N6	Lectin, mannose-binding 2, isoform	gi|119605421	318	100	26,812.3	5.12	8
N7	Basement membrane-specific heparansulfate proteoglycan core protein precursor variant	gi|62089288	314	100	249,966	7	17
N7	Chain a, crystal structure of protein hc from homo	gi|482677111	285	100	22,757.1	7.81
N7	Prostaglandin-H2 d-isomerase	sp|P41222|PTGDS	130	100	21,243	8	10
N8	Mannan-binding lectin serine protease 2 isoform 1 preproprotein	gi|21264363	276	100	77,193	5	16
N9	35 kDa Inter-alpha-trypsin inhibitor H4	tr|B2RMS9|B2RM	225	100	103,526	7
N10	Non-secretory ribonuclease precursor	tr|A0PJA6|A0PJA6	199	100	20,522	9	21
N10	TF protein (fragment)	gi|4506549	190	100	18,855	9	3
N11	Pro-epidermal growth factor EGF OS = homo sapiens	sp|P01133|EGF_H	176	99.668	17,983.4	7.42	5
N12	Alpha-1B-glycoprotein	sp|P04217|A1BG_	148	100	54,789.8	5.56	13
N13	Keratin, type II cytoskeletal 1	gi|119395750	148	100	66,170.1	8.15	20
N14	α-kinase anchor protein 9 isoform X2	gi|57015308	108	100	534,808.9	6.01	71
N14	Dynein, cytoplasmic 1, heavy chain 1	gi|530385399	75	98.765	458,794.5	4.96	63
N15	Gelsolin	tr|Q5T0H7|Q5T0H	101	100	20,827	5	13
N16	ALB protein	tr|Q8IUK7|Q8IUK7	89	99.984	46,441.9	5.77	14
N17	Microtubule-actin cross-linking factor 1	gi|578798796	75	99	878,150	5
N18	NADH dehydrogenase [ubiquinone] complex I,	tr|C9JS27|C9JS27	62	91.289	29,973.4	5.63	9
N19	Acyl-coa-binding protein	sp|P07108|ACBP_	58	79.104	10,038	6.12	7

Protein score calculated by the Mascot search engine is the conformity between each protein with the MS peak list; confidence interval (C.I. %) for the protein score is statistical calculation of correlation between the acquired data and previous database under differing conditions using normal probability distribution mathematics by Mascot engine

N means number

Fig. 2 Data mining of the set of PTB urinary biomarker candidates.

a Gene ontology (GO) enrichment analysis; b KEGG pathway mapping; c biological processes; d cellular component; and e molecular function. A p-value <0.05 indicates statistical significance using a binomial test

Fig. 3 mRNA levels of three urine proteins in PTB patients and healthy controls.

The relative contents of three identified urine proteins from tuberculosis patients and healthy controls determined using a qRT-PCR assay (a–c) and ROC curve analyses (d–f). The contents of the miRNAs were normalized to U6 and calculated using the 2^−ΔΔCt method. Each point represents the mean of triplicate samples. A p-value <0.05 indicates statistical significance with a non-parametric analysis using the two-tailed unpaired t-test. **p < 0.01, ***p < 0.001

Fig. 4 Comparison of candidate differential proteins using immunoblotting.

Samples were analyzed using immunoblotting to facilitate comparisons of RBP4, ITIH4-35k, and MBL2 levels (a). Twenty-five pulmonary tuberculosis (PTB) patients and 25 healthy controls (HC) were randomly divided into five subgroups (PTB₁ to PTB₅; HC₁ to HC₅), each containing five individuals. The samples of each subgroup were pooled and detected in one lane, and each pair consisted of PTB patients and healthy control subgroups. b The densitometry quantification of the results of (a) using Image J software; p-values between the two groups with the two-tailed unpaired t-test. **p < 0.01, ***p < 0.001

Fig. 5 Integrative proteomics and transcriptomics analyses.

Interactions between proteins are based on the reported literature. Interactions between proteins and miRNAs using four websites. The ellipse represents miRNA, and the rectangle represents proteins

Fig. 6 Urine levels of two miRNAs in Sn-PTB and Sp-PTB patients and healthy controls.

a The expression of the positive control miR-155 in the PTB group and healthy controls; b the relative contents of miR-625-3p in Sn-PTB and Sp-PTB patients and healthy controls determined using qRT-PCR assay and ROC curve analysis between healthy controls compared with Sp-PTB patients (c); Sn-PTB patients compared with healthy controls (d); (e) the ROC analysis of Y (binary logistic regression model with miR-625-3p, ITIH4-35k, and MBL2), miR-625-3p, ITIH4-35k, and MBL2. The contents of the miRNAs were normalized to RNA U6. A p-value <0.05 indicates significance using the two-tailed unpaired t-test

Information of TB patients and healthy control

Characteristic	Active pulmonary PTB (n = 167)			Healthy control (n = 118)
	2-DE screening	Validation tests		2-DE screening	Validation tests
		WB	qRT-PCR		WB	qRT-PCR
Total Number	45	25	97	45	25	48
Age (y)	32 ± 9.2	40 ± 5.1	35 ± 6.3	29 ± 10.3	28 ± 10.4	32 ± 8.7
Sex (M/F)	30/15	16/9	52/45	28/17	14/11	26/22
Fluorescent sputum smear(±)	23/22	15/10	46/51	/	/	/
Biochip tests
LAM(±)	22/23	19/6	89/8	/	/	/
16 kDa protein(±)	33/12	15/10	84/13	/	/	/
38 kDa protein(±)	34/11	19/6	90/7	/	/	/
Chest X-ray and CT-proven	36/9	23/2	93/4	—	—	—
PPD(±)	35/10	21/4	88/14	4/41	6/19	8/40
IGRAs(±)	29/16	23/2	92/5	1/44	3/22	4/44

Values expressed as means ± SD, number of patients, or number (percent)

PTB pulmonary tuberculosis, WB western blot, 2-DE 2-dimensional electrophoresis, LAM lipoarabinomannan, PPD purified protein derivative of tuberculin, IGRAs IFN-γ release assays