| Part I Screening and Identification of Serum microRNAs as Novel Potential Biomarkers for Pulmonary TuberculosisBackground:Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb), that can invade many organs. Pulmonary infection is the most common accounting for80-90%of the total TB various organs, and then develop into pulmonary tuberculosis (pulmonary TB). Pulmonary TB is one of the diseases a serious threat to human health. It is very difficult to prevent pulmonary TB due to the lack of specific and diagnostic markers, which could lead to a high incidence of pulmonary TB. So, new rapid, sensitive and efficient biomarkers or methods for pulmonary TB diagnosis are urgently needed to prevent the spread of pulmonary TB, fibrosis and permanent damage to pulmonary TB. The present study demonstrated that a combination of serum microRNA (miRNAs) may have great potential to serve as non-invasive biomarkers for detection of pulmonary TB.Methods:In this study, serum miRNAs were collected from326cases, including108cases of healthy controls,128cases of pulmonary TB patients, and90cases of differential diagnosis group (30cases of lung cancer,30cases of chronic obstructive pulmonary disease (COPD) and30cases of pneumonia patients. The serum miRNAs of20pulmonary TB patients and20healthy controls was screened using the Solexa sequencing method. In the validation stage, the stem-loop quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay was used to verify differentially expressed serum miRNAs from108pulmonary TB patients,88healthy controls and90differential diagnosis groups. 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 that91serum 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-101and 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 pulmonary TB diagnosis were95.0%and91.8%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.Conclusions:(1) Solexa sequencing technology combined with qRT-PCR detection technology can quickly build a stable and sensitive detection of pulmonary TB serum miRNAs fingerprinting.(2) The six specific serum miRNAs were screened for the establishment of clinical pulmonary TB early rapid-specific diagnostic criteria and the foundation to improve the level of prevention pulmonary TB.(3) A combination of miRNAs were used as non-invasive markers for the diagnosis of pulmonary TB. This provided a new foundation for those miRNAs can involving in the pathogenesis, drug-resistant and treatment of pulmonary TB. Part II Association of the miR-146a, miR-149, miR-196a2and miR-499Polymorphisms with Susceptibility to Pulmonary Tuberculosis in the Chinese Uygur, Kazak and Southern Han PopulationsBackground: MicroRNAs (miRNAs) are class of non-coding RNAs, and can inhibit the translation of target mRNAs. Single nucleotide polymorphisms (SNPs) within precursor miRNAs can affect miRNAs expression and maturation, that led to functional changes, and modified miRNA regulation to affect the phenotypes and disease susceptibility. A number of SNPs in human miRNA genes may affect miRNA biogenesis and function, and may be involved in the pathogenesis of pulmonary TB. This study is the first to report potential associations between four precursor miRNA SNPs (miR-146a C﹥G, miR-149T﹥C, miR-196a2T﹥C and miR-499T﹥C) and susceptibility to pulmonary TB in the Chinese Uygur, Kazak, and Southern Han populations. The study revealed that the four SNPs in precursor miRNA genes may be candidate for pulmonary TB susceptibility.Methods: The frequency distribution of SNPs was analyzed by PCR-PFLR (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method. The study included662Uygur (361healthy controls and301pulmonary TB patients),612Kazak (362healthy controls and251pulmonary TB patients), and654Southern Han subjects (300healthy controls and354pulmonary TB patients). The allelic and genotypic frequencies were calculated, and linkage disequilibrium and the genetic models were analyzed in the cases and controls.Results: In the Uygur, the frequency of allele T (P=0.003; OR=1.563;95%CI,1.162-2.103) and CC genotype (P=0.001; OR=5.344;95%CI,1.803-15.83) for the miR-499T﹥C SNP were significantly different between the pulmonary TB patients and healthy controls. After adjustment for age and gender, the miR-499T﹥C SNP was found to be codominant (P=0.0014; OR=5.37;95%CI,1.81-15.92), dominant (P=0.029; OR=1.47;95%CI,1.04-2.09), recessive (P=7e-04; OR=4.95;95%CI, 1.68-14.55), and additive models (P=0.0027; OR=1.57;95%CI,1.16-2.11) in association with pulmonary TB. By calculating linkage disequilibrium, the frequency of the CTCC haplotype was significantly associated with pulmonary TB (P=0.044; OR=7.19;95%CI,1.01-51.14). No significant differences were observed in the distribution of allelic and genotypic frequencies of miR-146aC﹥G, miR-149T﹥C and miR-196a2T﹥C SNPs between the two groups (P﹥0.05). In the Kazak, the frequency of allele G (P=0.023; OR=1.33;95%CI,1.04-1.69) and CC genotype (P=0.013; OR=1.84;95%CI,1.14-2.98) for the miR-146a C﹥G SNP were significantly different between the pulmonary TB patients and healthy controls. The frequency of allele C (P=0.008; OR=0.73;95%CI,0.58-0.92) and TT genotype (P=0.013; OR=0.54;95%CI,0.33-0.88) for the miR-196a2T﹥C SNP were significantly different between the pulmonary TB patients and healthy controls. After adjustment for age and gender, the miR-146a C﹥G SNP was found to be codominant (P=0.019; OR=1.87;95%CI,1.15-3.03), recessive (P=0.005; OR=1.84;95%CI,1.20-2.81), and additive models (P=0.022; OR=1.33;95%CI,1.04-1.69) in association with pulmonary TB. The miR-196a2T﹥C SNP were found to be codominant (P=0.029; OR=0.54;95%CI,0.33-0.88), dominant (P=0.014; OR=0.65;95%CI,0.46-0.92), and additive models (P=0.0083; OR=0.73;95%CI,0.58-0.92) in association with pulmonary TB. No significant differences between patients and controls were observed in the distribution of allelic and genotypic frequencies of miR-149T﹥C and miR-499T﹥C SNPs (P﹥0.05). In the Southern Han, the frequency distributions of haplotypes TCCC (P=0.044; OR=0.23;95%CI,0.05-0.96) and CCCT (P=0.024; OR=0.03;95%CI,0.01-0.63) were significantly associated with protective affect against pulmonary TB.Conclusions:(1) This study is the first to report that miR-499A﹥G, miR-146a C﹥G, miR-149T﹥C and miR-196a2T﹥C SNPs can be associated with pulmonary tuberculosis in the Chinese Uygur, Kazak and Southest Han populations by a case-control study, combined with statistical analysis.(2) The miR-499T﹥C SNP can be associated with susceptibility to pulmonary TB in a Chinese Uygur population and may increase the risk of being infected with pulmonary TB. No association was found between the other SNPs (miR-146a C﹥G, miR-149T﹥C and miR-196a2T﹥C) and pulmonary TB in a Chinese Uygur population.(3) The miR-146a C﹥G and miR-196a2T﹥C SNPs were associated with pulmonary TB in a Chinese Kazak population. No association was found between the other SNPs (miR-149T﹥C and miR-499T﹥C) and pulmonary TB in a Chinese Kazak population.(4) The miR-146a C﹥G, miR-149T﹥C, miR-196a2T﹥C and miR-499T﹥C SNPs were not associated with susceptibility to pulmonary TB in a Southern Han population. The results suggested that there were racial differences between miRNA SNPs and susceptibility to pulmonary TB. Part III The Novel Human MRC1Gene Polymorphisms are Associated with Susceptibility to Pulmonary Tuberculosis in the Chinese Uygur, Kazak and Southest Han PopulationsBackground: The host identifying and immune response to Mtb relyed on pattern recognition receptors (PRRs), that were located on the surface of the T lymphocytes, macrophages and dendritic cells. The MRC1gene, encoding the human mannose receptor (MR), is a member of the C-type lectin receptors family and belongs to PRRs family. MR can recognize and bind to Mtb by the extracellular structure, and play a role in antigen-presenting and maintaining a stable internal environment. However, to date, MRCl polymorphisms associated with susceptibility to pulmonary TB have not yet been reported. The present study aimed to investigate potential associations of SNPs in the MRCl gene with pulmonary TB in the Chinese Uygur, Kazak and Southest Han populations. The study speculated that the MRC1gene may be an excellent candidate for susceptibility to pulmonary TB.Methods:Six SNPs (G1186A, G1195A, T1212C, C1221G, C1303T and C1323T) in exon7of the MRC1gene were genotyped using the PCR and DNA sequencing methods in in595Chinese Uygur (345cases of healthy control group,250cases of pulmonary TB group),513Kazak (325cases of healthy control group,188cases of pulmonary TB group) and454Southern Han (226cases of healthy control group,230cases of pulmonary TB group). Allele and genotype frequency were analysed in the study. Linkage disequilibrium analysis was performed between polymorphic sites.Results:In the Uygur, the frequency of allele G for G1186A was lower in the pulmonary TB (0.464) than healthy control (0.536) and there was a significant difference between the were significantly correlated with pulmonary two groups (P=0.029; OR=1.29;95%CI,1.02-1.63). The frequency of A/A genotype was lower in the control group than the pulmonary TB group and there was a significant difference between the two groups (P=0.037; OR=1.66;95%CI,1.05-2.61). After adjustment for age and gender, G1186A was found to be additive models in association with pulmonary TB (P=0.039; OR=1.27;95%CI,1.01-1.60). By calculating linkage disequilibrium, the frequency of haplotype GGTCCT (P=0.034; OR=0.75;95%CI,0.57-0.98) and GGTCCC (P=0.043; OR=0.57;95%CI,0.33-0.98) was significantly associated with pulmonary TB. No association was found between other SNPs (G1195A, T1212C, C1221G, C1303T and C1323T) and pulmonary TB. In the Kazak, The allele and genotype frequencies of six SNPs were not significantly different between patients and controls (P>0.05). In the Southern Han, the frequency of allele G for G1186A was higher in the pulmonary TB group than the healthy control group. There was a significant difference in frequency distribution between the two groups (P=0.023; OR=0.76;95%CI,0.57-0.95). Genotypic analysis also indicated that the AG genotypes were significantly correlated with pulmonary tuberculosis (P=0.0076; OR=0.55;95%CI,0.36-0.85). After adjustment for age and gender, G1186A sites were found to be dominant (P=0.0057; OR=0.57;95%CI,0.38-0.85), over-dominant (P=0.039; OR=0.68;95%CI,0.47-0.98) and additive models (P=0.03; OR=0.75;95%CI,0.58-0.97) in association with pulmonary tuberculosis. But, no association was found between the other5SNPs (G1195A, T1212C, C1221G, C1303T and C1323T) and tuberculosis (P﹥0.05).Conclusions:(1) This study is the first to report that six SNPs in exon7of the MRC1gene can be associated with pulmonary tuberculosis in the Chinese Uygur, Kazak and Southest Han populations by a case-control study, combined with statistical analysis.(2) The G1186A polymorphism of MRC1gene was associated with pulmonary TB in a Chinese Uygur population, and may increase the risk of being infected with pulmonary TB, the G1186A SNP can be associated with pulmonary TB in a Chinese Southest Han population, and may reduce the risk of infecting pulmonary TB. However, G1186A site in the exon7of MRC1gene had no significant association with pulmonary TB in a Chinese Kazak population.(3) G1195A, T1212C, C1221G, C1303T and C1323T SNPs in the MRC1gene were not associated with pulmonary TB in the Chinese Uygur, Kazak and Southest Han populations, In this study, 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 in a Chinese population. |
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