Yunchao Huang

Down-regulation of microRNA-144 in air pollution-related lung cancer

Air pollution has been classified as a group 1 carcinogen in humans, but the underlying tumourigenic mechanisms remain unclear. In Xuanwei city of Yunnan Province, the lung cancer incidence is among the highest in China, owing to severe air pollution generated by the combustion of smoky coal, providing a unique opportunity to dissect lung carcinogenesis. To identify abnormal miRNAs critical for air pollution-related tumourigenesis, we performed microRNA microarray analysis in 6 Xuanwei non-small cell lung cancers (NSCLCs) and 4 NSCLCs from control regions where smoky coal was not used. We found 13 down-regulated and 2 up-regulated miRNAs in Xuanwei NSCLCs. Among them, miR-144 was one of the most significantly down-regulated miRNAs. The expanded experiments showed that miR-144 was down-regulated in 45/51 (88.2%) Xuanwei NSCLCs and 34/54 (63%) control region NSCLCs (p = 0.016). MiR-144 interacted with the oncogene Zeb1 at 2 sites in its 3′ untranslated region, and a decrease in miR-144 resulted in increased Zeb1 expression and an epithelial mesenchymal transition phenotype. Ectopic expression of miR-144 suppressed NSCLCs in vitro and in vivo by targeting Zeb1. These results indicate that down-regulation of miR-144 is critical for air pollution-related lung cancer, and the miR-144-Zeb1 signalling pathway could represent a potential therapeutic target.

Long non-coding RNA stabilizes the Y-box-binding protein 1 and regulates the epidermal growth factor receptor to promote lung carcinogenesis

Indoor and outdoor air pollution has been classified as group I carcinogen in humans, but the underlying tumorigenesis remains unclear. Here, we screened for abnormal long noncoding RNAs (lncRNAs) in lung cancers from patients living in Xuanwei city which has the highest lung cancer incidence in China due to smoky coal combustion-generated air pollution. We reported that Xuanwei patients had much more dysregulated lncRNAs than patients from control regions where smoky coal was not used. The lncRNA CAR intergenic 10 (CAR10) was up-regulated in 39/62 (62.9%) of the Xuanwei patients, which was much higher than in patients from control regions (32/86, 37.2%; p=0.002). A multivariate regression analysis showed an association between CAR10 overexpression and air pollution, and a smoky coal combustion-generated carcinogen dibenz[a,h]anthracene up-regulated CAR10 by increasing transcription factor FoxF2 expression. CAR10 bound and stabilized transcription factor Y-box-binding protein 1 (YB-1), leading to up-regulation of the epidermal growth factor receptor (EGFR) and proliferation of lung cancer cells. Knockdown of CAR10 inhibited cell growth in vitro and tumor growth in vivo. These results demonstrate the role of lncRNAs in environmental lung carcinogenesis, and CAR10-YB-1 represents a potential therapeutic target.

Tobacco smoke induces production of chemokine CCL20 to promote lung cancer

Tobacco kills nearly 6 million people each year, and 90% of the annual 1.59 million lung cancer deaths worldwide are caused by cigarette smoke. Clinically, a long latency is required for individuals to develop lung cancer since they were first exposed to smoking. In this study, we aimed to identify clinical relevant inflammatory factors that are critical for carcinogenesis by treating normal human lung epithelial cells with tobacco carcinogen nicotine-derived nitrosaminoketone (NNK) for a long period (60 days) and systematic screening in 84 cytokines/chemokines. We found that a chemokine CCL20 was significantly up-regulated by NNK, and in 78/173 (45.1%) patients the expression of CCL20 was higher in tumor samples than their adjacent normal lung tissues. Interestingly, CCL20 was up-regulated in 48/92 (52.2%) smoker and 29/78 (37.2%) nonsmoker patients (p = 0.05), and high CCL20 was associated with poor prognosis. NNK induced the production of CCL20, which promoted lung cancer cell proliferation and migration. In addition, an anti-inflammation drug, dexamethasone, inhibited NNK-induced CCL20 production and suppressed lung cancer in vitro and in vivo. These results indicate that CCL20 is crucial for tobacco smoke-caused lung cancer, and anti-CCL20 could be a rational approach to fight against this deadly disease.

Down‐regulated SOX4 Expression Suppresses Cell Proliferation, Metastasis and Induces Apoptosis in Xuanwei Female Lung Cancer Patients

The transcription factor SOX4 has functional importance in foetal lung maturation and tumorigenesis in a number of cancers. However, its biological functions in the progression of lung tumorigenesis remain unclear. In this study, we found that the expression levels of SOX4 mRNA and protein were significantly higher in Xuanwei female lung cancer tissues than in benign lung lesions. The patients with high expression of the SOX4 protein had a higher pathological grade, lymph node (LN) metastasis, poor tumor differentiation and worse prognosis than those patients with low expression of SOX4. Knockdown of the SOX4 gene in the Xuanwei female lung cancer cell line XWLC‐05 resulted in apoptotic morphological changes, decreased cell proliferation, invasion and migration. Furthermore, knockdown of the SOX4 gene resulted in obvious sub‐G1 peaks and induction of apoptosis through upregulation of caspase‐3 expression, while in cells treated with a caspase‐3 inhibitor, apoptosis induced by silencing SOX4 expression was inhibited. In vivo analysis in nude mice further confirmed that knockdown of SOX4 suppressed tumor growth. In conclusion, SOX4 appears to be an important tumor suppressor gene in the regulation of Xuanwei female lung cancer cell proliferation, apoptosis and metastases, and it may be a potential target for effective lung cancer therapy. J. Cell. Biochem. 116: 1007–1018, 2015.

The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

More than 90% of lung cancers are caused by cigarette smoke and air pollution, with polycyclic aromatic hydrocarbons (PAHs) as key carcinogens. In Xuanwei City of Yunnan Province, the lung cancer incidence is among the highest in China, attributed to smoky coal combustion-generated PAH pollution. Here, we screened for abnormal inflammatory factors in non-small cell lung cancers (NSCLCs) from Xuanwei and control regions (CR) where smoky coal was not used, and found that a chemokine CXCL13 was overexpressed in 63/70 (90%) of Xuanwei NSCLCs and 44/71 (62%) of smoker and 27/60 (45%) of non-smoker CR patients. CXCL13 overexpression was associated with the region Xuanwei and cigarette smoke. The key carcinogen benzo(a)pyrene (BaP) induced CXCL13 production in lung epithelial cells and in mice prior to development of detectable lung cancer. Deficiency in Cxcl13 or its receptor, Cxcr5, significantly attenuated BaP-induced lung cancer in mice, demonstrating CXCL13’s critical role in PAH-induced lung carcinogenesis. DOI: http://dx.doi.org/10.7554/eLife.09419.001

Air pollution and DNA methylation alterations in lung cancer: A systematic and comparative study

The lung cancer incidence in the Xuanwei and neighboring region, Yunnan, China, is among the highest in China and is attributed to severe air pollution with high benzo(a)pyrene levels. We systematically and comparatively analyzed DNA methylation alterations at genome and gene levels in Xuanwei lung cancer tissues and cell lines, as well as benzo(a)pyrene-treated cells and mouse samples. We obtained a comprehensive dataset of genome-wide cytosine-phosphate-guanine island methylation in air pollution-related lung cancer samples. Benzo(a)pyrene exposure induced multiple alterations in DNA methylation and in mRNA expressions of DNA methyltransferases and ten-11 translocation proteins; these alterations partially occurred in Xuanwei lung cancer. Furthermore, benzo(a)pyrene-induced DKK2 and EN1 promoter hypermethylation and LPAR2 promoter hypomethylation led to down-regulation and up-regulation of the genes, respectively; the down-regulation of DKK2 and EN1 promoted the cellular proliferation. Thus, DNA methylation alterations induced by benzo(a)pyrene contribute partially to abnormal DNA methylation in air pollution-related lung cancer, and these DNA methylation alterations may affect the development and progression of lung cancer. Additionally, vitamin C and B6 can reduce benzo(a)pyrene-induced DNA methylation alterations and may be used as chemopreventive agents for air pollution-related lung cancer.

[Serum and lung tissue selenium measurements in subjects with lung cancer from Xuanwei, China].

BACKGROUND AND OBJECTIVE Xuanwei is an area of the highest incidence and mortality with lung cancer in China. The aim of this study is to determine serum selenium concentrations in lung cancer patients from Xuanwei as well as selenium levels of cancerous tissues, cancer-adjacent pulmonary tissues, and normal pulmonary lung tissues from lung cancer patients, and the relationship between selenium and the high incidence of lung cancer in Xuanwei. METHODS One hundred and twenty female adults from Xuanwei were enrolled in the study (60 lung cancer patients and 60 with non-tumor and non-respiratory diseases, respectively) and blood samples were collected. Sixty fresh cancerous tissues and their adjacent as well as normal tissues were collected (31 samples from lung cancer patients living in Xuanwei for more than 2 years and 29 from patients in other regions of Yunnan Province outside of Xuanwei, respectively). Serum and tissue selenium concentrations were assayed using a fluorometric method. RESULTS Women with lung cancer had a mean serum selenium value (55.22 μg/L±13.34 μg/L) of averagely 8.47%, significantly lower than that in subjects with non-tumor and non-respiratory disease controls (60.33 μg/L±13.82 μg/L)(P < 0.05). Selenium concentrations in the tumor tissues (0.105 μg/g±0.034 μg/g) were statistically lower than that of normal ones (0.140 μg/g±0.048 μg/g)(P < 0.05) from lung cancer patients in Xuanwei. Statistical differences had not been found between the cases from Xuanwei and non-Xuanwei district, adenocarcinoma and squamous cell carcinoma, among Stage I, Stage II, stage III groups. CONCLUSIONS Lower serum selenium state was negatively related to the incidence of lung cancer in Xuanwei. It was likely that lower selenium level of lung tissues was potential risk factor to lead to lung cancer.

The clinical use of circulating microRNAs as non-invasive diagnostic biomarkers for lung cancers

Many studies have investigated the diagnostic role of circulating microRNAs (miRNAs) in patients with lung cancer; however, the results still remain inconclusive. An updated system review and meta-analysis was necessary to give a comprehensive evaluation of diagnostic role of circulating miRNAs in lung cancer. Eligible studies were searched in electronical databases. The sensitivity and specificity were used to plot the summary receiver operator characteristic (SROC) curve and calculate the area under the curve (AUC). The between-study heterogeneity was evaluated by Q test and I2 statistics. Subgroup analyses and meta-regression were further performed to explore the potential sources of heterogeneity. A total of 134 studies from 65 articles (6,919 patients with lung cancer and 7,064 controls) were included for analysis. Overall analysis showed that circulating miRNAs had a good diagnostic performance in lung cancers, with a sensitivity of 0.83, a specificity of 0.84, and an AUC of 0.90. Subgroup analysis suggested that combined miRNAs and Caucasian populations may yield relatively higher diagnostic performance. In addition, we found serum might serve as an ideal material to detecting miRNA as good diagnostic performance. We also found the diagnostic role of miRNAs in early stage lung cancer was still relatively high (the sensitivity, specificity and an AUC of stage I/II was 0.81, 0.82 and 0.88; and for stage I, it was 0.80, 0.81, and 0.88). We also identified a panel of miRNAs such as miR-21-5p, miR-223-3p, miR-155-5p and miR-126-3p might serve as potential biomarkers for lung cancer. As a result, circulating miRNAs, particularly the combination of multiple miRNAs, may serve as promising biomarkers for the diagnosis of lung cancer.

Epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) of Yunnan in southwestern China

To investigate the Epidermal Growth Factor Receptor (EGFR) mutation status in non-small cell lung cancer (NSCLC) in Yunnan province in southwestern China, we detected EGFR mutation by Amplification Refractory Mutation System (ARMS) polymerase chain reaction (PCR) using DNA samples from 447 pathologically confirmed NSCLC specimens (175 tissue, 256 plasma and 16 cytologic samples). The relationship between EGFR mutations and demographic and clinical factors were further explored. Subgroup analyses according to sample type (tissue and plasma) and histological type (adenocarcinoma) were done. We found the mutation rate was 34.9% in overall patients (42.3%, 29.7%, and 37.5% for tissue, plasma, and cytologic samples respectively). We found female (p < 0.0001), no smoking (p = 0.001), adenocarcinoma (p < 0.0001), and tissue specimen (p = 0.026) were associated with higher EGFR mutation rate. The most common mutations were exon 19 deletions (40%) and L858R point (30%) mutation. Interestingly, NSCLC patients from Xuanwei harbored a strikingly divergent mutational pattern for EGFR when compared with non-Xuanwei patients (higher G719X, G719X+S768I mutations, but lower 19 deletion and L858R mutations). Generally, EGFR mutation rate and pattern in Yunnan province was in accord with other Asian populations. However, Xuanwei subgroup showed strikingly divergent EGFR mutation spectrum from other general population. Our analysis also indicated that cftDNA analysis for EGFR mutations detection was feasibility for the patients lacking sufficient tissue for molecular analyses.

Tree shrew as a new animal model for the study of lung cancer

Animal models play a key role in identifying treatments for various types of cancer, including lung cancer. The aim of the present study was to develop a new animal model for lung cancer induction using tree shrews from the Yunnan region in China. Tree shrews are suitable for a full simulation of human disease because their structure, function and metabolism are adequately close to human. This animal may offer a new experimental animal model to be used in the study of lung cancer. In the present study, 80 healthy tree shrews were distributed in experimental and control groups. Animals in the experimental group received different concentrations of iodized oil suspension of 3-methylcholanthrene (3-MC) and diethylnitrosamine (DEN) while animals in the control groups received saline or lipiodol solvent via endotracheal instillation. In the 3rd, 5th, 7th, 9th and 11th weeks the body weights of the animals were measured and chest X-ray examinations were conducted. Pathological studies on the lung tissues were also performed and the pathological changes occurring in bronchial epithelium in all the groups were examined. Animals in the experimental group gradually lost their body weight. For tree shrews in the blank control and solvent control groups the survival rates were 100 and 80%, respectively while the survival rate for the experimental group was 0%. Results from the chest X-ray conducted on animals in the blank control and solvent control groups revealed no obvious abnormalities while in the experimental group high-density shadow spots within the perfusion sites were observed. Pathological studies performed on these high-density areas confirmed changes in the bronchial epithelium. In the experimental groups we also detected bronchial epithelial atypical hyperplasia, and apparent changes in carcinoma in situ. In conclusion, lung cancer was successfully induced in tree shrews by a one-time endotracheal introduction of iodized oil suspension of 3-MC and DEN.