The Study Of Association Between Laminin Beta 1 And Pneumoconiosis And Its Mechanisms

Pneumoconiosis is the most serious occupational disease in China, which developed after inhalation and deposition of occupational dust in the lungs. It is characterized by irreversible and diffuse lung fibrotic lesions. Coal workers’ pneumoconiosis(CWP) and silicosis are two types of the most common pneumoconiosis. In 2013, 87.72% of the total 23152 reported cases were attributed to pneumoconiosis and among them, CWP and silicosis accounted for 95.2%. A significant pathological hallmark of pneumoconiosis is excessive deposition of extracellular matrix(ECM). The ECM mainly contains collagens IV, nidogens, perlecan, agrin, and laminins. Laminins are a family of multidomain, heterotrimeric proteins which are composed of α, β, γ chains. Laminins contribute to the structure of ECM, and influence the biological behaviors of associated cells, such as adhesion, migration, differentiation, phenotype stability, and resistance to apoptosis. Laminin β1 chain(Lam B1), encoded by LAMB1, is widely expressed in tissues, and assembles with α, γ chains to form at least 7 laminin isoforms. Lam B1 is crucial for lung morphogenesis and physiological function. Increased Lam B1 expression has been reported to be associated with fibrosis in liver and kidney. However, studies on associations between Lam B1 and pneumoconiosis were few.The long-term occupational dust exposure and dust chemical and physical 6 properties are the most contributions to pneumoconiosis. However, the incidence, disease progression and the degree of fibrosis vary greatly among individuals with similar exposures, indicating an important role for genetic factors in the initiation and development of pneumoconiosis. Single nucleotide polymorphism(SNP) is an important approach for genetic susceptibility researches. In our previous GWAS on pneumoconiosis, a SNP rs4320486, located in the promoter region of human LAMB1 gene, was found significantly associated with the susceptibility of pneumoconiosis.Given the complexity of pathogenesis, there are yet no effective therapies and specific serum biomarkers. Researches on the relationship between genetic factors and pneumoconiosis will help to establish effective susceptibility screenings, and improve the level of health care for occupational individuals. ObjectiveA case-control study with enlarged sample was conducted to investigate the association between LAMB1 rs4320486 and risk for pneumoconiosis in a Chinese population. Cell experiments and serum samples of subjects were performed to evaluate the effect of rs4320486 mutation on LAMB1 expression. Additionally, the dynamic changes of Lam B1 expression during lung fibrosis were observed both in vivo and in vitro experiments, so as to provide new clues for pathogenesis and therapies of pneumoconiosis, or to function as a potential biomarker for susceptibility population. Method(1) 1091 CWP patients and 1042 controls were recruited in this study. Each subject completed a questionnaire, and donated a 5-ml venous blood for lab tests. The SNP rs4320486 was genotyped using Taq Man allelic discrimination assays.(2) We constructed two luciferase reporter plasmids by using p GL3-basic vector with either rs4320486 C or T allele to explore the effect of rs4320486 on gene expression. The concentration of Lam B1 in serum samples of subjects was detected using ELISA.(3) Both healthy and fibrosis lung tissues were collected. The expression differences in Lam B1 m RNA were evaluated with using q RT-PCR.(4) C57BL/6 mice were treated 7 with either Si O2 or BLM to produce experimental lung fibrosis models. Lung tissue samples were selected based on the typical pathological changes. Total RNA and protein were extracted to observe dynamic expressions of Lam B1, E-cadherin, and α-SMA.(5) HBE cells were treated with Si O2, while NIH-3T3 cells with TGF-β1, then the expressions of Lam B1, E-cadherin and α-SMA were detected by q RT-PCR, western blot and immunocytochemistry test, respectively. With specific inhibitors blocking the activation of signaling pathways, we explored the mechanisms underlying the expression of Lam B1.(6) All data arrangement and statistical analyses were performed with SPSS16.0 software, PLINK1.07 software and STATA12.0 software. P< 0.05 was considered a significant difference. Results(1) Logistic regression analysis revealed that CT/TT genotypes were associated with a significantly decreased risk of CWP(adjusted OR = 0.74, 95%CI = 0.62-0.88), compared with CC genotypes. And the decreased risk was more evident among subgroups of age >68, CDE ≤66 and smoking. Furthermore, COX regression analysis showed that CT/TT genotypes could delay the development of CWP, compared with CC genotype(P< 0.001).(2) The vectors with the p GL3-T allele had a significant decrease in the relative luciferase activities, compared with the p GL3-C allele in both HBE cells and MRC-5 cells(all P< 0.05). Accordingly, lower levels of circulating Lam B1 in serum were detected in subgroups of CT/TT genotypes and controls.(3) Compared with healthy people, the m RNA level of Lam B1 was significantly over-expressed in the lung tissues of IPF and CWP.(4) Experimental lung fibrosis models were successfully induced in C57BL/6 mice, and confirmed by pathological section examination. Expressions of Lam B1 and α-SMA-a mesenchymal biomarker were enhanced progressively, along with the development of lung fibrosis, while E-cadherin- an epithelial biomarker decreased.(5) The results were further validated in HBE and NIH-3T3 cell experiments. After treatment, Lam B1 expressions at both protein and m RNA levels were markedly increased. Immunofluorescence staining also showed that Lam B1 was up-regulated in treatment groups, compared with controls.(6) Molecules of PI3K-Akt and Smad signaling pathways were activated in HBE with Si O2 challenged. However, pre-treated HBE with specific inhibitors LY294002 and LY2157299, resulted in down-regulation of p-Akt, smad2 as well as p-smad2. Accordingly, the Lam B1 expression was reduced, especially in subgroups of LY2157299. ConclusionsIn the present study, the functional Lam B1 rs4320486 mutation was found associated with decreased risk of CWP in a Chinese population, and delayed the progression of CWP, probably owing to reduced activity of LAMB1 transcription. Additionally, Lam B1 expression was increased gradually in vivo and in vitro experiments, along with the development of lung fibrosis. These suggested that Lam B1 may play an important role in the initiation and progression of pneumoconiosis, or may function as a potential biomarker for diagnosis and genetic susceptibility.