8-Oxoguanine DNA glycosylase modulates the cell transformation process in pulmonary fibrosis by inhibiting Smad2/3 and interacting with Smad7.

The DNA repair enzyme 8-oxoguanine DNA glycosylase-1 (OGG1) is involved in early embryonic development, as well as in multiple conditions, including cardiac fibrosis, diabetes, and neurodegenerative diseases. But, function of OGG1 in pulmonary fibrosis was not entirely clear. In this study, we identified a novel function of OGG1 in the cell transformation process in pulmonary fibrosis. We demonstrated that OGG1 and Smad7 co-localize and interact in A549 cells. Bleomycin-induced pulmonary fibrosis was established in wild-type (WT) and Ogg1-/- mice. Upon treatment with transforming growth factor (TGF)-ß1, increased OGG1 expression was observed in WT mice with pulmonary fibrosis as well as in A549 cells, MRC-5 cells, and primary rat type II alveolar epithelial cells. The increased expression of OGG1 promoted cell migration, while OGG1 depletion decreased migration ability. Expression of the transformation-associated markers vimentin and alpha-smooth muscle actin were also affected by OGG1. We also observed that OGG1 promoted TGF-ß1-induced cell transformation and activated Smad2/3 by interacting with Smad7. The interaction between OGG1 and the TGF-ß/Smad axis modulates the cell transformation process in lung epithelial cells and fibroblasts. Moreover, we demonstrated that Ogg1 deficiency relieved pulmonary fibrosis in bleomycin-treated mice. Ogg1 knockout decreased the bleomycin-induced expression of Smad7 and phosphorylation of Smad2/3 in mice. These findings suggest that OGG1 has multiple biological functions in the pathogenesis of pulmonary fibrosis.

Tetraspanin 1 as a mediator of fibrosis inhibits EMT process and Smad2/3 and beta-catenin pathway in human pulmonary fibrosis.

Tetraspanin 1(TSPAN1) as a clinically relevant gene target in cancer has been studied, but there is no direct in vivo or vitro evidence for pulmonary fibrosis (PF). Using reanalysing Gene Expression Omnibus data, here, we show for the first time that TSPAN1 was markedly down-regulated in lung tissue of patient with idiopathic PF (IPF) and verified the reduced protein expression of TSPAN1 in lung tissue samples of patient with IPF and bleomycin-induced PF mice. The expression of TSPAN1 was decreased and associated with transforming growth factor-ß1 (TGF-ß1 )-induced molecular characteristics of epithelial-to-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). Silencing TSPAN1 promoted cell migration, and the expression of alpha-smooth muscle actin, vimentin and E-cadherin in AECs with TGF-ß1 treatment, while exogenous TSPAN1 has the converse effects. Moreover, silencing TSPAN1 promotes the phosphorylation of Smad2/3 and stabilizes beta-catenin protein, however, overexpressed TSPAN1 impeded TGF-ß1 -induced activation of Smad2/3 and beta-catenin pathway in AECs. Together, our study implicates TSPAN1 as a key regulator in the process of EMT in AECs of IPF.

lncINS-IGF2 Promotes Cell Proliferation and Migration by Promoting G1/S Transition in Lung Cancer.

Long noncoding RNAs are capable of regulating gene expression at multiple levels. These RNA molecules are also involved in a variety of physiological and pathological processes. Emerging data demonstrate that a series of differentially expressed long noncoding RNAs are implicated in tumorigenesis. In the present study, we used microarray analysis to identify long noncoding RNAs that are dysregulated in non-small-cell lung cancer when compared to normal lung tissues. Accordingly, we performed quantitative real-time polymerase chain reaction to analyze the levels of long noncoding RNA and the cis target gene. We further found the oncogene property of long noncoding RNA that long noncoding RNA downexpression inhibits non-small-cell lung cancer cells proliferation and migration based on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and colony formation assays and wound healing as well as transwell assays. The influence of long noncoding RNA on cell cycle of non-small-cell lung cancer cells is also analyzed by flow cytometry. Among the dysregulated long noncoding RNAs, we identified INS-IGF2 readthrough, transcript variant 1, noncoding RNA (NR_003512.3) is upregulated in non-small-cell lung cancer tissues, the cis gene of which is insulin-like growth factor 2 gene hinted by bioinformatics analysis. We also observed that downregulation of INS-IGF2 readthrough, transcript variant 1, noncoding RNA reduces insulin-like growth factor 2 messenger RNA expression. Furthermore, INS-IGF2 readthrough, transcript variant 1, noncoding RNA downregulation suppresses non-small-cell lung cancer cell proliferation and migration. This downregulation results in a concomitant inhibition of the G1/S transition in non-small-cell lung cancer cells. Our findings suggest that INS-IGF2 readthrough, transcript variant 1, noncoding RNA may be an oncogene involved in the development of lung cancer. Therefore, we speculate that INS-IGF2 readthrough, transcript variant 1, noncoding RNA represents a potential therapeutic target for lung cancer.

Tetraspanin 1 inhibits TNFα-induced apoptosis via NF-κB signaling pathway in alveolar epithelial cells.

OBJECTIVE: Tetraspanin family plays an important role in the pathogenesis of cancer, but its role in lung fibrosis is unknown. To determine whether tetraspanin 1 (TSPAN1), a member of the family, may be involved in the pathogenesis of pulmonary fibrosis. METHODS: TNFα -stimulated human alveolar epithelial (A549) and alveolar epithelial type II cell (AT2) were treated in vitro. Murine pulmonary fibrosis model was generated by injection of bleomycin (BLM). The expression of TSPAN1 was examined in vivo using the bleomycin-induced lung fibrosis model and tissue sample of IPF patients. Then we transfected the cells with TSPAN1 siRNA or plasmid and detected the expression changes of related proteins and cell apoptosis. RESULTS: In our study, we found that TSPAN1 was markedly down-regulated in lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and in bleomycin-induced pulmonary fibrosis in mice. We also found that TSPAN1 was significantly down-regulated in A549 and primary (AT2) cells following exposure to TNFα. Meanwhile, TSPAN1 inhibited p-IκBα, which attenuated nuclear NF-κB translocation and activation and inhibited apoptosis. We demonstrated that TSPAN1 reduced Bax translocation and caspase-3 activation, inhibited the apoptosis by regulating the NF-κB pathway in response to TNFα. CONCLUSIONS: We conclude that TSPAN1 mediated apoptosis resistance of alveolar epithelial cells by regulating the NF-κB pathway. TSPAN1 may be a potential therapeutic target for pulmonary fibrosis or acute lung injury.

A highly significant association between Cathepsin S gene polymorphisms rs12068264 and chronic obstructive pulmonary disease susceptibility in Han Chinese population.

Cathepsin S (CTSS) and Sirtuin-1 (SIRT1) played crucial roles in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the associations between the polymorphisms of CTSS as well as SIRT1 and COPD in Asian population remain elusive. In the present study, one single nucleotide polymorphism (SNP) in rs12068264 was discovered (in 385 individuals) to be associated with the susceptibility of COPD in a Chinese Han population. The genotyping was performed using improved multiplex ligase detection reaction (iMLDR) technique. Subjects with T allele of rs12068264 in CTSS gene had an increased risk of COPD (T compared with C: odds ratio (OR) = 1.351, 95% confidence interval (95% CI): 1.008-1.811, P=0.044) compared with C allele. Subjects with TT genotype at rs12068264 had a higher risk of COPD in a recessive model (TT compared with TC + CC: OR = 2.30, 95% CI: 1.06-4.989, P=0.035). Compared with the C variant of rs12068264, the homozygous carriers of the TT genotype had higher procalcitonin (PCT) levels. Finally, haplotype analysis demonstrated that the SNPs in the CTSS and SIRT1 gene had no statistical differences between patients with COPD and the controls. In conclusion, the genetic polymorphisms of CTSS were associated with the susceptibility of COPD in a Chinese Han population, which may be helpful in understanding genetic mechanisms underlying the pathogenesis of COPD.

ß-8-Oxoguanine DNA Glycosylase Overexpression Reduces Oxidative Stress-Induced Mitochondrial Dysfunction and Apoptosis Through the JNK Signaling Pathway in Human Bronchial Epithelial Cells.

8-Oxoguanine DNA glycosylase (OGG1) is responsible for repairing 8-oxo-7,8-dihydroguanine (8-oxoG). Our previous study demonstrated that α-OGG1 protects cells from oxidative damage-induced apoptosis and mitochondrial dysfunction in human lung cancer cells. However, the function of ß-OGG1 remains to be elucidated. In this study, we demonstrated that overexpressed ß-OGG1 has the same role as α-OGG1 in protecting human bronchial epithelial cells from apoptosis and mitochondrial dysfunction. Furthermore, flow cytometry, confocal microscopy, and western blotting showed that the overexpression of ß-OGG1 could block oxidant-induced apoptosis in human bronchial epithelial cells. Additionally, knocking down OGG1 enhanced oxidative damage-induced apoptosis and mitochondrial dysfunction, whereas the overexpression of ß-OGG1 had the opposite effects and led to the downregulation of Bax and PARP. The antiapoptotic function of ß-OGG1 involved the JNK signaling pathway. These findings suggest that ß-OGG1 and α-OGG1 have a similar function on preventing oxidative damage-mediated apoptosis and mitochondrial dysfunction; these effects might be important in the molecular events underlying oxidant-induced cytotoxicity.

MiR-5100 targets TOB2 to drive epithelial-mesenchymal transition associated with activating smad2/3 in lung epithelial cells.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease and the pathogenesis of IPF remains unclear. Our previous study indicated that miR-5100 promotes the proliferation and metastasis of lung epithelial cells. In this study, we investigated the effect and mechanism of miR-5100 on bleomycin (BLM)-induced mouse lung fibrosis and transforming growth factor ß (TGF-ß1) or epidermal growth factor (EGF) induced EMT-model in A549 and Beas-2B cells. The elevated level of miR-5100 was observed in both the mouse lung fibrosis tissues and EMT cell model. Furthermore, the exogenous expression of miR-5100 promoted the EMT-related changes, enhanced TGF-ß1 or EGF-induced EMT and activated the smad2/3 in lung epithelial cells, while silencing miR-5100 had the converse effects. In addition, transwell assay showed that miR-5100 can enhance cell migration. Using target prediction software and luciferase reporter assays, we identified TOB2 as a specific target of miR-5100 and miR-5100 can decrease the accumulation of endogenous TOB2 in A549 and Beas-2B cells. Moreover, the exogenous expression of TOB2 relieves the promotion of miR-5100 on EMT process and migration ability. Taken together, our results indicate that miR-5100 promotes the EMT process by targeting TOB2 associated with activating smad2/3 in lung epithlium cells. Our findings may provide novel insights into the pathogenesis of IPF.

TFPI2AS1, a novel lncRNA that inhibits cell proliferation and migration in lung cancer.

Accumulating evidence demonstrates that a series of differentially expressed lncRNAs is important in tumorigenesis. However, the function of many of the lncRNAs in lung cancer remains elusive. In the present study, we used microarray analysis to identify lncRNAs that are dysregulated in non-small-cell lung cancer (NSCLC) as compared with normal tissues. Among the dysregulated lncRNAs, we identified TFPI2AS1, an antisense transcript of the tumor suppressor TFPI2 (tissue factor pathway inhibitor 2). TFPI2AS1 was shown to be markedly upregulated in NSCLC patient tumors as compared to paired non-tumor samples. TFPI2AS1 knockdown increased NSCLC cell proliferation and migration, which was associated with enhanced G1/S transition and downregulation of cyclin D1 and cyclin-dependent kinases 2 (CDK2), while TFPI2AS1 overexpression had the opposite effect. Knockdown and overexpression experiments also suggested that TFPI2AS1 regulates NSCLC cell migration and AKT activation. Moreover, TFPI2AS1 is a positive regulator of TFPI2. Our findings bring new insights for understanding the role of TFPI2AS1 in mediating the proliferation and migration of NSCLC cells by regulating TFPI2 expression.