Jie Hu

Plasma MicroRNA Panel to Diagnose Hepatitis B Virus–Related Hepatocellular Carcinoma

PURPOSEnMore than 60% of patients with hepatocellular carcinoma (HCC) do not receive curative therapy as a result of late clinical presentation and diagnosis. We aimed to identify plasma microRNAs for diagnosing hepatitis B virus (HBV) -related HCC.nnnPATIENTS AND METHODSnPlasma microRNA expression was investigated with three independent cohorts including 934 participants (healthy, chronic hepatitis B, cirrhosis, and HBV-related HCC), recruited between August 2008 and June 2010. First, we used microarray to screen 723 microRNAs in 137 plasma samples for diagnosing HCC. Quantitative reverse-transcriptase polymerase chain reaction assay was then applied to evaluate the expression of selected microRNAs. A logistic regression model was constructed using a training cohort (n = 407) and then validated using an independent cohort (n = 390). Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy.nnnRESULTSnWe identified a microRNA panel (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a and miR-801) that provided a high diagnostic accuracy of HCC (AUC = 0.864 and 0.888 for training and validation data set, respectively). The satisfactory diagnostic performance of the microRNA panel persisted regardless of disease status (AUCs for Barcelona Clinic Liver Cancer stages 0, A, B, and C were 0.888, 0.888, 0.901, and 0.881, respectively). The microRNA panel can also differentiate HCC from healthy (AUC = 0.941), chronic hepatitis B (AUC = 0.842), and cirrhosis (AUC = 0.884), respectively.nnnCONCLUSIONnWe found a plasma microRNA panel that has considerable clinical value in diagnosing early-stage HCC. Thus, patients who would have otherwise missed the curative treatment window can benefit from optimal therapy.

Expression of aquaporin 5 increases proliferation and metastasis potential of lung cancer

Water channel aquaporin 5 (AQP5) is highly expressed at the apical membrane of alveolar type I epithelial cells and confers high osmotic water permeability. AQP5 is also expressed in lung cancer tissue. Previous studies showed there was an up‐regulation of AQP5 expression in cancer tissue compared to surrounding normal tissue. In addition, expression of AQP5 in lung cancer tissue was associated with poor prognosis. Herein, we tested the role of AQP5 in lung cancer oncogenesis and development. Lung cancer cells with different expression of AQP5 were used to study cell proliferation and migration, two important parameters for tumour cell biology. We found enhanced proliferation and migration potential in cancer cells with high AQP5 expression, while reduced proliferation and metastasis potential in cancer cells with low AQP5 expression. Oncogene analysis showed significantly increased PCNA and c‐myc expression in AQP5 transfected cells. AQP5 transfected cells also showed significant increased MUC5AC mucin expression, which might contribute to the enhanced metastasis potential of lung cancer. AQP5 overexpression resulted in enhanced activation of the epidermal growth factor receptor (EGFR), extracellular receptor kinase (ERK1/2), and p38 mitogen‐activated protein kinase (p38 MAPK) pathway in cancer cells. Moreover, deletion of AQP5 demonstrated decreased activation of the EGFR/ERK/p38 MAPK pathway in AQP5 knockout mice lungs, while deletion of AQP1 or AQP3 did not exhibit significant changes on activation of the EGFR/ERK/p38 MAPK pathway in lung tissue. In conclusion, our results provide evidence for AQP5‐facilitated lung cancer cell proliferation and migration, possibly through activation of the EGFR/ERK/p38 MAPK signalling pathway, but why AQP5 but not other aquaporin expression affects the EGFR/ERK/p38 MAPK pathway still needs further exploration. Copyright

Two MicroRNA Panels to Discriminate Three Subtypes of Lung Carcinoma in Bronchial Brushing Specimens

RATIONALEnEffective treatment for lung cancer requires accuracy in subclassification of carcinoma subtypes.nnnOBJECTIVESnTo identify microRNAs in bronchial brushing specimens for discriminating small cell lung cancer (SCLC) from non-small cell lung cancer (NSCLC) and for further differentiating squamous cell carcinoma (SQ) from adenocarcinoma (AC).nnnMETHODSnMicroarrays were used to screen 723 microRNAs in laser-captured, microdissected cancer cells from 82 snap-frozen surgical lung specimens. Quantitative reverse-transcriptase polymerase chain reaction was performed on 153 macrodissected formalin-fixed, paraffin-embedded (FFPE) surgical lung specimens to evaluate seven microRNA candidates discovered from microarrays. Two microRNA panels were constructed on the basis of a training cohort (n = 85) and validated using an independent cohort (n = 68). The microRNA panels were applied as differentiators of SCLC from NSCLC and of SQ from AC in 207 bronchial brushing specimens.nnnMEASUREMENTS AND MAIN RESULTSnTwo microRNA panels yielded high diagnostic accuracy in discriminating SCLC from NSCLC (miR-29a and miR-375; area under the curve [AUC], 0.991 and 0.982 for training and validation data set, respectively) and in differentiating SQ from AC (miR-205 and miR-34a; AUC, 0.977 and 0.982 for training and validation data set, respectively) in FFPE surgical lung specimens. Moreover, the microRNA panels accurately differentiated SCLC from NSCLC (AUC, 0.947) and SQ from AC (AUC, 0.962) in bronchial brushing specimens.nnnCONCLUSIONSnWe found two microRNA panels that accurately discriminated between the three subtypes of lung carcinoma in bronchial brushing specimens. The identified microRNA panels may have considerable clinical value in differential diagnosis and optimizing treatment strategies based on lung cancer subtypes.

Silencing the epidermal growth factor receptor gene with RNAi may be developed as a potential therapy for non small cell lung cancer

Lung cancer has emerged as a leading cause of cancer death in the world. Non-small cell lung cancer (NSCLC) accounts for 75–80% of all lung cancers. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. Double stranded RNA (dsRNA) -mediated RNA interference (RNAi) has shown promise in gene silencing, the potential of which in developing new methods for the therapy of NSCLC needs to be tested. We report here RNAi induced effective silencing of the epidermal growth factor receptor (EGFR) gene, which is over expressed in NSCLC. NSCLC cell lines A549 and SPC-A1 were transfected with sequence- specific dsRNA as well as various controls. Immune fluorescent labeling and flow cytometry were used to monitor the reduction in the production of EGFR protein. Quantitative reverse-transcriptase PCR was used to detect the level of EGFR mRNA. Cell count, colony assay, scratch assay, MTT assay in vitro and tumor growth assay in athymic nude mice in vivo were used to assess the functional effects of EGFR silencing on tumor cell growth and proliferation. Our data showed transfection of NSCLC cells with dsRNA resulted in sequence specific silencing of EGFR with 71.31% and 71.78 % decreases in EGFR protein production and 37.04% and 54.92% in mRNA transcription in A549 and SPC-A1 cells respectively. The decrease in EGFR protein production caused significant growth inhibition, i.e.: reducing the total cell numbers by 85.0% and 78.3 %, and colony forming numbers by 63.3% and 66.8%. These effects greatly retarded the migration of NSCLC cells by more than 80% both at 24 h and at 48 h, and enhanced chemo-sensitivity to cisplatin by four-fold in A549 cells and seven-fold in SPC-A1. Furthermore, dsRNA specific for EGFR inhibited tumor growth in vivo both in size by 75.06 % and in weight by 73.08 %. Our data demonstrate a new therapeutic effect of sequence specific suppression of EGFR gene expression by RNAi, enabling inhibition of tumor proliferation and growth. However, in vivo use of dsRNA for gene transfer to tumor cells would be limited because dsRNA would be quickly degraded once delivered in vivo. We thus tested a new bovine lentiviral vector and showed lentivector-mediated RNAi effects were efficient and specific. Combining RNAi with this gene delivery system may enable us to develop RNAi for silencing EGFR into an effective therapy for NSCLC.

Early Detection of Lung Cancer in Serum by a Panel of MicroRNA Biomarkers

INTRODUCTIONnThe objective of the study was to develop a panel of microRNAs (miRNAs) as highly sensitive and specific biomarkers for lung cancer early detection.nnnMATERIALS AND METHODSnThe study contained 2 phases: first, preliminary marker selection based on previous reports on the serum of 24 early stage lung cancer patients and 24 healthy control subjects by TaqMan probe-based real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR); and second, validation of miRNA markers on 94 early stage lung cancer, 48 stage III to IV lung cancer, and 111 healthy control serum samples.nnnRESULTSnA total of 3 miRNAs (miR-125a-5p, miR-25, and miR-126) were selected for further analysis in this study. The combination of the 3 miRNAs could produce 0.936 area under the receiver operating characteristic curve value in distinguishing early stage lung cancer patients from control subjects with 87.5% sensitivity and 87.5% specificity, respectively. The diagnostic value of the miRNA panel in an independent set of lung cancer patients confirmed the sensitivity and specificity.nnnCONCLUSIONnThe results demonstrated that the panel of miRNA biomarkers had the potential for the early detection of lung cancer.

Reply to G. Cai et al

We would like to thank Cai et al for their comments on our article. In the study by Gao et al, microRNA (miRNA) was extracted from formalin-fixed, paraffin-embedded dysplastic nodules, hepatocellular carcinomas (HCCs), and the adjacent nontumorous liver tissues. This study showed that miRNA deregulation at the tissue level was an early event and accumulated throughout the various steps of hepatitis B virus–associated hepatocarcinogenesis. In our study, the panel with seven miRNAs (miR-122, miR-192, miR-21, miR-223, miR-26a, miR-27a, and miR-801) was identified in the plasma of patients with HCC. The discrepancy between the two studies would be primarily a result of the different categories of the study specimens (tissue v plasma). To our knowledge, the expression profiles of plasma miRNA are different from that of tissue miRNA. Plasma circulating miRNA was reported to have diverse origins. It is well known that most hepatitis B virus–related HCCs involve two disease processes: the HCC as well as liver damage as a result of either hepatitis or cirrhosis. Thus, liver damage, tumor immune response, and/or hepatitis may also influence the miRNA profiles in the plasma of patients with HCC. Therefore, the expression profiles of plasma miRNAs in patients with HCC may not trend according to the expression profiles in HCC tissue. For example, studies by Wang et al and Starkey Lewis et al found miR-122 and miR-192 to be associated with liver damage, and the two miRNAs expressed opposite regulations in the tissue and plasma specimens. This indicates that the miRNA profiles in the plasma specimens are more complicated compared with those in pure tumor tissue in patients with HCC. At the tissue level, two studies revealed the differences in miRNA dysregulation in early versus advanced HCCs. Such differences have not been reported in the current plasma studies of miRNA profiles. It would be interesting to further investigate the differences in miRNA spectrum and expression level between early and advanced HCCs in the plasma of patients with HCC.

Role of a serum-based biomarker panel in the early diagnosis of lung cancer for a cohort of high-risk patients

This study applied a combined cancer biomarker panel to clinically identify small cell lung cancer (SCLC) and non–small cell lung cancer (NSCLC) in a high‐risk population.

Integrin αvβ5 as a biomarker for the assessment of non-small cell lung cancer metastasis and overall survival

There is growing interest in how integrins play a role in cancer disease biology and what applications these may have in anti‐cancer therapeutic development. This study investigates integrin αvβ5 expression in non‐small cell lung carcinoma (NSCLC) and its correlations with clinical information.

Knockdown of annexin A5 restores gefitinib sensitivity by promoting G2/M cell cycle arrest

BackgroundEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, including gefitinib, are first-line drugs against advanced non-small cell lung cancer with activating EGFR mutations. However, the development of resistance to such drugs is a major clinical challenge.MethodsThe role of annexin A5 in resistance to EGFR tyrosine kinase inhibitors was investigated by qPCR and western blot of relevant molecules, by CCK8 and EdU assay of cell proliferation and viability, by annexin V/propidium iodide assay of apoptosis and cell cycle distribution, by JC-1 assay of mitochondrial integrity, and by xenograft assay of tumorigenicity.ResultsWe found that annexin A5 is upregulated in gefitinib-resistant cell lines, as well as in clinical specimens resistant to EGFR tyrosine kinase inhibitors. Accordingly, knockdown of the gene from gefitinib-resistant cells restores gefitinib sensitivity in vitro and in vivo by downregulating polo-like kinase 1 signal pathway, thereby inducing mitochondrial damage, caspase activation, cell cycle arrest at G2/M, and, finally, apoptosis.ConclusionsThe data indicate that annexin A5 confers gefitinib resistance in lung cancer by inhibiting apoptosis and G2/M cell cycle arrest, and is thus a potential therapeutic target in non-small cell lung cancers resistant to EGFR tyrosine kinase inhibitors.

Quantitative CT analysis of pulmonary nodules for lung adenocarcinoma risk classification based on an exponential weighted grey scale angular density distribution feature

BACKGROUND AND OBJECTIVESnTo improve lung nodule classification efficiency, we propose a lung nodule CT image characterization method. We propose a multi-directional feature extraction method to effectively represent nodules of different risk levels. The proposed feature combined with pattern recognition model to classify lung adenocarcinomas risk to four categories: Atypical Adenomatous Hyperplasia (AAH), Adenocarcinoma In Situ (AIS), Minimally Invasive Adenocarcinoma (MIA), and Invasive Adenocarcinoma (IA).nnnMETHODSnFirst, we constructed the reference map using an integral image and labelled this map using a K-means approach. The density distribution map of the lung nodule image was generated after scanning all pixels in the nodule image. An exponential function was designed to weight the angular histogram for each component of the distribution map, and the features of the image were described. Then, quantitative measurement was performed using a Random Forest classifier. The evaluation data were obtained from the LIDC-IDRI database and the CT database which provided by Shanghai Zhongshan hospital (ZSDB). In the LIDC-IDRI, the nodules are categorized into three configurations with five ranks of malignancy (1 to 5). In the ZSDB, the nodule categories are AAH, AIS, MIA, and IA.nnnRESULTSnThe average of Students t-test p-values were less than 0.02. The AUCs for the LIDC-IDRI database were 0.9568, 0.9320, and 0.8288 for Configurations 1, 2, and 3, respectively. The AUCs for the ZSDB were 0.9771, 0.9917, 0.9590, and 0.9971 for AAH, AIS, MIA and IA, respectively.nnnCONCLUSIONnThe experimental results demonstrate that the proposed method outperforms the state-of-the-art and is robust for different lung CT image datasets.