Zhong-Jie Li

Screening and Identification of Six Serum microRNAs as Novel Potential Combination Biomarkers for Pulmonary Tuberculosis Diagnosis

Background It is very difficult to prevent pulmonary tuberculosis (TB) due to the lack of specific and diagnostic markers, which could lead to a high incidence of pulmonary TB. We screened the differentially expressed serum microRNAs (miRNAs) as potential biomarkers for the diagnosis of pulmonary TB. Methods In this study, serum miRNAs were screened using the Solexa sequencing method as the potential biomarkers for the diagnosis of pulmonary TB. The stem-loop quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay was used to verify differentially expressed serum miRNAs. The receiver operating characteristic (ROC) curve and logistic regression model were used to analyze the sensitivity and specificity of the single miRNA and a combination of miRNAs for diagnosis, respectively. Using the predicted target genes, we constructed the regulatory networks of miRNAs and genes that were related to pulmonary TB. Results The Solexa sequencing data showed that 91 serum miRNAs were differentially expressed in pulmonary TB patients, compared to healthy controls. Following qRT-PCR confirmation, six serum miRNAs (hsa-miR-378, hsa-miR-483-5p, hsa-miR-22, hsa-miR-29c, hsa-miR-101 and hsa-miR-320b) showed significant difference among pulmonary TB patients, healthy controls (P<0.001) and differential diagnosis groups (including patients with pneumonia, lung cancer and chronic obstructive pulmonary disease) (P<0.05). The logistic regression analysis of a combination of six serum miRNAs revealed that the sensitivity and the specificity of TB diagnosis were 95.0% and 91.8% respectively. The miRNAs-gene regulatory networks revealed that several miRNAs may regulate some target genes involved in immune pathways and participate in the pathogenesis of pulmonary TB. Conclusion Our study suggests that a combination of six serum miRNAs have great potential to serve as non-invasive biomarkers of pulmonary TB.

Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ‐coupled two‐dimensional LC‐MS/MS

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis is a chronic disease. Currently, there are no sufficiently validated biomarkers for early diagnosis of TB infection. In this study, a panel of potential serum biomarkers was identified between patients with pulmonary TB and healthy controls by using iTRAQ‐coupled 2D LC‐MS/MS technique. Among 100 differentially expressed proteins screened, 45 proteins were upregulated (>1.25‐fold at p < 0.05) and 55 proteins were downregulated (<0.8‐fold at p < 0.05) in the TB serum. Bioinformatics analysis revealed that the differentially expressed proteins were related to the response to stimulus, the metabolic and immune system processes. The significantly differential expression of apolipoprotein CII (APOCII), CD5 antigen‐like (CD5L), hyaluronan‐binding protein 2 (HABP2), and retinol‐binding protein 4 (RBP4) was further confirmed using immunoblotting and ELISA analysis. By forward stepwise multivariate regression analysis, a panel of serum biomarkers including APOCII, CD5L, and RBP4 was obtained to form the disease diagnostic model. The receiver operation characteristic curve of the diagnostic model was 0.98 (sensitivity = 93.42%, specificity = 92.86%). In conclusion, APOCII, CD5L, HABP2, and RBP4 may be potential protein biomarkers of pulmonary TB. Our research provides useful data for early diagnosis of TB.

Serum protein S100A9, SOD3, and MMP9 as new diagnostic biomarkers for pulmonary tuberculosis by iTRAQ-coupled two-dimensional LC-MS/MS

This study aimed to discover the novel noninvasive biomarkers for the diagnosis of pulmonary tuberculosis (TB). We applied iTRAQ 2D LC‐MS/MS technique to investigate protein profiles in patients with pulmonary TB and other lung diseases. A total of 34 differentially expressed proteins (24 upregulated proteins and ten downregulated proteins) were identified in the serum of pulmonary TB patients. Significant differences in protein S100‐A9 (S100A9), extracellular superoxide dismutase [Cu‐Zn] (SOD3), and matrix metalloproteinase 9 (MMP9) were found between pulmonary TB and other lung diseases by ELISA. Correlations analysis revealed that the serum concentration of MMP9 in the pulmonary TB was in moderate correlation with SOD3 (r = 0.581) and S100A9 (r = 0.471), while SOD3 was in weak correlation with S100A9 (r = 0.287). The combination of serum S100A9, SOD3, and MMP9 levels could achieve 92.5% sensitivity and 95% specificity to discriminate between pulmonary TB and healthy controls, 90% sensitivity and 87.5% specificity to discriminate between pulmonary TB and pneumonia, and 85% sensitivity and 92.5% specificity to discriminate between pulmonary TB and lung cancer, respectively. The results showed that S100A9, SOD3, and MMP9 may be potential diagnostic biomarkers for pulmonary TB, and provided experimental basis for the diagnosis of pulmonary TB.

Association of the miR-146a, miR-149, miR-196a2 and miR-499 polymorphisms with susceptibility to pulmonary tuberculosis in the Chinese Uygur, Kazak and Southern Han populations

BackgroundSingle nucleotide polymorphisms (SNPs) within precursor microRNAs (miRNAs) can affect miRNAs expression, and may be involved in the pathogenesis of pulmonary tuberculosis (TB). This study aimed to investigate potential associations between the four precursor miRNA SNPs (miR-146a C > G, miR-149 T > C, miR-196a2 T > C, and miR-499 T > C) and susceptibility to pulmonary TB in the Chinese Uygur, Kazak, and Southern Han populations.MethodsA case-control study was performed on Chinese Uygur (n = 662), Kazak (n = 612), and Southern Han (n = 654) populations using the PCR-PFLR method. The allele and genotype frequencies for all populations were analyzed. Linkage disequilibrium was performed, and different models of inheritance were tested.ResultsThe allele and genotype frequencies of the miR-499 SNP were significantly different between the TB patients group and the healthy control group in the Uygur population, and were found to be codominant, dominant, recessive and additive models in association with pulmonary TB. The haplotype CTCC showed significant correlation with pulmonary TB. The allele and genotype frequencies of miR-146a and miR-196a2 SNPs were significantly different between the two groups in the Kazak population. The miR-146a SNP was found to be codominant, recessive and additive models, whereas, the miR-196a2 SNP was found to be codominant, dominant, and additive models in association with pulmonary TB. The haplotypes TCCC and CCCT showed significant correlation with pulmonary TB.ConclusionsThe results suggested that susceptibility to pulmonary TB may be closely related to individual differences caused by genetic factors among different ethnic groups in China.

Screening and identification of five serum proteins as novel potential biomarkers for cured pulmonary tuberculosis

Rapid and efficient methods for the determination of cured tuberculosis (TB) are lacking. A total of 85 differentially expressed serum proteins were identified by iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) analysis (fold change >1.50 or <0.60, Pu2009<u20090.05). We validated albumin (ALB), Rho GDP-dissociation inhibitor 2 (ARHGDIB), complement 3 (C3), ficolin-2 (FCN2), and apolipoprotein (a) (LPA) using the enzyme-linked immunosorbent assay (ELISA) method. Significantly increased ALB and LPA levels (Pu2009=u20090.036 and Pu2009=u20090.012, respectively) and significantly reduced ARHGDIB, C3, and FCN2 levels (Pu2009<u20090.001, Pu2009=u20090.035, and Pu2009=u20090.018, respectively) were observed in cured TB patients compared with untreated TB patients. In addition, changes in ALB and FCN2 levels occurred after 2 months of treatment (Pu2009<u20090.001 and Pu2009=u20090.030, respectively). We established a cured TB model with 87.10% sensitivity, 79.49% specificity, and an area under the curve (AUC) of 0.876. The results indicated that ALB, ARHGDIB, C3, FCN2, and LPA levels might serve as potential biomarkers for cured TB. Our study provides experimental data for establishing objective indicators of cured TB and also proposes potential markers for evaluating the efficacy of anti-TB drugs.

Association of CTLA4 gene polymorphisms with susceptibility and pathology correlation to pulmonary tuberculosis in Southern Han Chinese.

The cytotoxic T lymphocyte antigen-4 (CTLA4) gene is a key negative regulator of the T lymphocyte immune response. It has been found that CTLA4 +49A>G (rs231775), +6230G>A (rs3087243), and 11430G>A (rs11571319) polymorphisms are associated with susceptibility to many autoimmune diseases, and can down-regulate the inhibition of cellular immune response of CTLA4. Three SNPs in CTLA4 were genotyped by using the PCR and DNA sequencing methods in order to reveal the susceptibility and pathology correlation to pulmonary tuberculosis in Southern Han Chinese. We found that the frequency of CTLA4 +49AG genotype in the pulmonary tuberculosis patients (38.42%) was significantly lower than that of the healthy controls (49.77%), (Pcor=0.038, OR 0.653, 95% CI 0.436-0.978). But, no associations were found between the other 2 SNPs (+6230G>A, 11430G>A) and tuberculosis (P>0.05). Haplotype analysis showed that the frequency of haplotype AGG in the healthy controls group (6.9%) was significantly higher than the pulmonary tuberculosis patients group (1.4%), (global P=0.005, Pcor=0.0002, OR 0.183, 95% CI 0.072-0.468). In addition, haplotype GGA was found to be significantly related to tuberculosis with double lung lesion rather than single lung lesion (Pcor=0.042). This study is the first to report that genetic variants in the CTLA4 gene can be associated with pulmonary tuberculosis in Southern Han Chinese, and CTLA4 +49AG genotype as well as haplotype AGG may reduce the risk of being infected with pulmonary tuberculosis. The GGA haplotype was related to tuberculosis with double lung lesion, which provides a new experimental basis to clarify the pathogenesis of pulmonary tuberculosis.

The novel human MRC1 gene polymorphisms are associated with susceptibility to pulmonary tuberculosis in Chinese Uygur and Kazak populations

The MRC1 gene, encoding the human mannose receptor (MR), is a member of the C-type lectin receptors family. MR can recognize and bind to Mycobacterium tuberculosis by the extracellular structure, and play a role in antigen-presenting and maintaining a stable internal environment. This study aimed to investigate potential associations of SNPs in exon 7 of the MRC1 gene with pulmonary tuberculosis (TB). G1186A, G1195A, T1212C, C1221G, C1303T and C1323T were genotyped using PCR and DNA sequencing in 595 Chinese Uygur and 513 Kazak subjects. In the Uygur, the frequency of allele G (Pxa0=xa00.031, ORxa0=xa01.29, 95xa0% CIxa0=xa01.02–1.62) and AA genotype (Pxa0=xa00.033, ORxa0=xa01.64, 95xa0% CIxa0=xa01.04–2.60) for G1186A was lower in the pulmonary TB than healthy control and were significantly correlated with pulmonary TB. After adjustment for age and gender, G1186A was found to be additive models in association with pulmonary TB (Pxa0=xa00.04, ORxa0=xa01.27, 95xa0% CIxa0=xa01.01–1.60). By calculating linkage disequilibrium, the frequency of haplotype GGTCCT (Pxa0=xa00.032, ORxa0=xa00.75, 95xa0% CIxa0=xa00.57–0.97) and GGTCCC (Pxa0=xa00.044, ORxa0=xa00.57, 95xa0% CIxa0=xa00.33–0.99) was significantly associated with pulmonary TB. No association was found between other SNPs and pulmonary TB. In the Kazak, all SNPs were not associated with pulmonary TB. Our results suggest that genetic factors play an important role in susceptibility to pulmonary TB at the individual level, and provide an experimental basis to clarify the pathogenesis of pulmonary TB.

A Group of Novel Serum Diagnostic Biomarkers for Multidrug-Resistant Tuberculosis by iTRAQ-2D LC-MS/MS and Solexa Sequencing

The epidemic of pulmonary tuberculosis (TB), especially multidrug-resistance tuberculosis (MDR-TB) presented a major challenge for TB treatment today. We performed iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) and Solexa sequencing among MDR-TB patients, drug-sensitive tuberculosis (DS-TB) patients, and healthy controls. A total of 50 differentially expressed proteins and 43 differentially expressed miRNAs (fold change >1.50 or <0.60, P<0.05) were identified in the MDR-TB patients compared to both DS-TB patients and healthy controls. We found that 22.00% of differentially expressed proteins and 32.56% of differentially expressed miRNAs were related, and could construct a network mainly in complement and coagulation cascades. Significant differences in CD44 antigen (CD44), coagulation factor XI (F11), kininogen-1 (KNG1), miR-4433b-5p, miR-424-5p, and miR-199b-5p were found among MDR-TB patients, DS-TB patients and healthy controls (P<0.05) by enzyme-linked immunosorbent assay (ELISA) and SYBR green qRT-PCR validation. A strong negative correlation, consistent with the target gene prediction, was found between miR-199b-5p and KNG1 (r=-0.232, P=0.017). Moreover, we established the MDR-TB diagnostic model based on five biomarkers (CD44, KNG1, miR-4433b-5p, miR-424-5p, and miR-199b-5p). Our study proposes potential biomarkers for MDR-TB diagnosis, and also provides a new experimental basis to understand the pathogenesis of MDR-TB.

Serum complement C4b, fibronectin, and prolidase are associated with the pathological changes of pulmonary tuberculosis

BackgroundMycobacterium tuberculosis infection can activate the immune system, leading to characteristic pathological changes such as inflammatory granuloma, caseous necrosis, and cavity formation.MethodsClinical data of 187 cases of pulmonary tuberculosis (PTB) were analyzed using statistical methods, while serum levels of complement C4b (C4b), fibronectin (FN), and prolidase (PEPD) were detected using the ELISA method among the control, minimal PTB, moderate PTB, and advanced PTB groups.ResultsWe found significantly higher levels of serum C4b and PEPD (Pu2009=u20090.018, Pu2009=u20090.003), and significantly lower levels of serum FN (Pu2009<u20090.001) in PTB patients. Furthermore, the serum levels of 3 proteins were significantly different among 3 PTB groups. FN level was significantly higher in the moderate PTB group, compared with patients in the minimal and advanced PTB groups (Pu2009<u20090.05, Pu2009<u20090.01). PEPD level was significantly higher in the moderate PTB group, compared with the minimal PTB group (Pu2009<u20090.05). Analysis of clinical data showed that serum albumin, C-reactive protein (CRP), prealbumin, and C4 were significantly higher (Pu2009<u20090.05), while serum globulin was significantly lower in patients with PTB (Pu2009<u20090.001). A significant negative correlation was found between C4b and albumin, prealbumin. On the other hand, a significant positive correlation was found between C4b and globulin, CRP, PEPD, as well as between PEPD and CRP (Pu2009<u20090.05).ConclusionsOur study showed that C4b, FN, and PEPD are associated with tissue damage, granuloma formation, and cavity formation, respectively, in patients with PTB. The present study provides a new experimental basis to understand the pathogenesis and pathological changes of PTB.

NOD2 polymorphisms and pulmonary tuberculosis susceptibility: a systematic review and meta-analysis.

The association between NOD2 and tuberculosis (TB) risk has been reported widely, but the results of previous studies remained controversial and ambiguous. To assess the association between NOD2 polymorphisms and TB risk, a meta-analysis was performed. A literature search was conducted by using the PubMed, Ovid, ISI Web of Knowledge, Elsevier ScienceDirect, and Chinese National Knowledge Infrastructure (CNKI). We identified the data from all articles estimating the association between NOD2 polymorphisms and TB risk. In total, 2,215 cases and 1,491 controls in 7 case-control studies were included. In meta-analysis, we found significant association between the Arg702Trp polymorphism and TB risk (OR = 0.43, 95% CI = 0.20-0.90, P = 0.02). However, no significant association was found between the Arg587Arg (OR = 1.31, 95% CI = 0.83-2.07, P = 0.25) and Gly908Arg (OR = 0.78, 95% CI = 0.21-2.87, P = 0.71) polymorphisms and TB risk. The present meta-analysis suggested that NOD2 Arg702Trp polymorphism was likely to be a protective factor for TB. However, the Arg587Arg and Gly908Arg polymorphisms might not be the genetic risk factors for TB susceptibility.