Hanbo Le

Expression of miR-29c, miR-93, and miR-429 as Potential Biomarkers for Detection of Early Stage Non-Small Lung Cancer

Background Altered expression of miRNA expression contributes to human carcinogenesis. This study was designed to detect aberrant miRNA expressions as a potential biomarker for early detection and prognosis prediction of non-small cell lung cancer (NSCLC). Methods miRNA array was used to profile differentially expressed miRNAs and Taqman-based quantitative RT-PCR assays were used to analyze levels of miR-29c, miR-93, and miR-429 expression in NSCLC tissue samples, corresponding normal tissue samples, and serum samples from 70 NSCLC patients as well as in serum samples from 48 healthy controls. Results Levels of miR-29c and miR-93 expression were upregulated in NSCLC tissues, while serum levels of miR-29c were also upregulated, but levels of serum miR-429 were decreased in NSCLC. Moreover, the levels of miR-429 expression in NSCLC tissues were associated with those in serum samples. Receiver operating characteristic (ROC) curve analysis showed that at the optimal cut-off point, the areas under the ROC curve for serum levels of miR-29c and miR-429 were 0.723 and 0.727, respectively, levels which are higher than that of carcinoma embryonic antigen (0.534) in diagnosis of stage I NSCLC. In addition, serum levels of miR-429 were associated with poor overall survival of NSCLC patients. Both univariate and multivariate analyses showed that serum miR-429 level was an independent prognostic predictor for NSCLC. Conclusions The results of the current study suggest that detection of serum miR-29c and miR-429 expression should be further evaluated as a novel, non-invasive biomarker for early stage NSCLC.

Diagnostic Value of Serum miR-182, miR-183, miR-210, and miR-126 Levels in Patients with Early-Stage Non-Small Cell Lung Cancer

Blood-circulating miRNAs could be useful as a biomarker to detect lung cancer early. We investigated the serum levels of four different miRNAs in patients with non-small cell lung cancer (NSCLC) and assessed their diagnostic value for NSCLC. Serum samples from 112 NSCLC patients and 104 controls (20 current smokers without lung cancer, 23 pneumonia patients, 21 gastric cancer patients, and 40 healthy controls) were subjected to Taqman probe-based quantitative reverse transcription–polymerase chain reaction (RT-PCR). The data showed that the serum levels of miR-182, miR-183, and miR-210 were significantly upregulated and that the miR-126 level was significantly downregulated in NSCLC patients, compared with the healthy controls. Further receiver operating characteristic (ROC) curve analysis revealed that the serum miR-182, miR-183, miR-210, or miR-126 level could serve as a diagnostic biomarker for NSCLC early detection, with a high sensitivity and specificity. The combination of these four miRNAs with carcinoembryonic antigen (CEA) further increased the diagnostic value, with an area under the curve (AUC) of 0.965 (sensitivity, 81.3%; specificity, 100.0%; and accuracy, 90.8%) using logistic regression model analysis. In addition, the relative levels of serum miR-182, miR-183, miR-210, and miR-126 could distinguish NSCLC or early-stage NSCLC from current tobacco smokers without lung cancer and pneumonia or gastric cancer patients with a high sensitivity and specificity. Data from the current study validated that the four serum miRNAs could serve as a tumor biomarker for NSCLC early diagnosis.

Differential Expression of miR-125a-5p and let-7e Predicts the Progression and Prognosis of Non-Small Cell Lung Cancer

Aberrant expression of various microRNAs (miRNA) has shown diagnostic and prognostic significance in non-small cell lung cancer (NSCLC). qRT-PCR analysis confirmed altered expression of miR-125a-5p, let-7e, miR-30a, miR-30e and miR-30e-3p in 70 paired tissue and serum samples from NSCLC patients. The reduced expression of miR-125a-5p, let-7e and miR-30e was strongly associated with NSCLC dedifferentiation. The lost expression of miR-125a-5p and let-7e was associated with shorter overall survival and let-7e was an independent prognostic factor for NSCLC patients. These five miRNA expressions should be further evaluated as biomarkers for the early detection and prognosis of NSCLC patients.

Changes of CD4+CD25+FOXP3+ and CD8+CD28- regulatory T cells in non-small cell lung cancer patients undergoing surgery.

Little is known about the regulatory T cells (Tregs) in the peripheral blood after surgery of non-small cell lung cancer (NSCLC) patients. In this study, we investigated whether CD4+CD25+FOXP3+ and CD8+CD28- regulatory T cells are decreased in the peripheral blood of NSCLC patients undergoing surgery. The study group (n = 49) comprised NSCLC, and the control group (n = 24) consisted of age- and sex-matched nonmalignant diseases. The prevalence of CD4+CD25+FOXP3+ and CD8+CD28- Tregs was analyzed using flow cytometry. The study group showed significantly higher percentage of CD4+CD25+FOXP3+ and CD8+CD28- Tregs than control. The percentage of CD4+CD25+FOXP3+ and CD8+CD28- Tregs increased with tumor stage. One way ANOVA test shows the significant differences between all subgroups. LSD test shows that there was a statistical significance between each of the two subgroups except stage II in CD4+CD25+FOXP3+ Tregs and control vs. each stage, stage I vs. stage III, and stage IV in CD8+CD28- Tregs. There is no significant difference among stages II, III, and IV in CD8+CD28- Tregs. No differences were found between squamous carcinoma and adenocarcinoma. These levels were dropped significantly after operation. Furthermore postoperative Treg percentage in the early stages (stage I and stage II) was not statistically different from that of controls. Postoperative Treg percentage in advanced stage (III+IV) remained above the values shown by controls. Our findings indicate that the percentage of CD4+CD25+FOXP3+ and CD8+CD28- Tregs correlated with the pathological stage in NSCLC and tumor burden.

Microvessel density and expression of thrombospondin-1 in non-small cell lung cancer and their correlation with clinicopathological features.

Microvessel density and thrombospondin-1 (TSP-1) expression were analysed in 42 non-small cell lung cancer (NSCLC) specimens and 40 normal lung tissue specimens using immunohistochemistry. Microvessel density was significantly higher and TSP-1 expression significantly lower in NSCLC tissue compared with normal tissue. Significantly lower levels of TSP-1 expression and higher microvessel densities were found in late-stage NSCLC compared with early-stage NSCLC, and in those with lymph node metastasis compared with those without metastasis. A statistically significant inverse correlation was observed between TSP-1 expression and microvessel density in squamous cell carcinoma but not in adenocarcinoma. These results suggest a close relationship between microvessel density and NSCLC tumour progress, and that a high expression of TSP-1 may play an important role in inhibiting tumour occurrence and development. The lack of correlation between microvessel density and TSP-1 expression in adenocarcinoma suggests that the mechanism of tumour inhibition by TSP-1 varies according to histological type.

The role of CD133 expression in the carcinogenesis and prognosis of patients with lung cancer

Cancer stem cells (CSCs) are a small population of undifferentiated cancer cells within tumors, which contribute to tumorigenicity and relapse. In the current study, CD133 (also termed prominin‑1), a CSC marker, was investigated to determine its involvement in predicting carcinogenesis and prognosis in patients with non‑small cell lung carcinoma (NSCLC). CD133‑positive lung cancer cells were isolated to analyze self‑renewal, differentiation and tumorigenic abilities in vitro and in vivo. Quantitative polymerase chain reaction was used to detect the expression of CD133 and three other CSC‑associated markers, octamer‑binding transcription factor 4 (OCT4A), Nanog homeobox (NANOG) and multidrug resistance protein 1 (MDR1), in primary NSCLC and adjacent non‑cancer tissues. A series of statistical methods were used to analyze the correlation between mRNA expression levels, clinicopathological features and patient survival. The results showed that CD133‑positive NSCLC cells demonstrated clonogenic, tumorigenic and drug‑resistance properties compared with their CD133‑negative counterparts or parental cells. In addition, compared with the adjacent normal lung tissue, the levels of CSC‑associated biomarkers CD133, OCT4A, NANOG and MDR1 were significantly increased in NSCLC tissue. Elevated expression of CD133 was associated with stage, tumor size and differentiation of NSCLC; however, the cox hazard regression analysis showed no significant association between CD133 expression and overall patient survival. The present study supports the hypothesis that the stem cell population can be enriched in cells expressing the CD133 cell surface marker and that highly expressed CD133 is involved in the occurrence of NSCLC. However, CD133 may not be considered as an independent factor in predicting the prognosis of patients with NSCLC. Further studies are required to investigate the association between CD133 expression and overall patient survival.

Tim-3 Expression by Peripheral Natural Killer Cells and Natural Killer T Cells Increases in Patients with Lung Cancer - Reduction after Surgical Resection

BACKGROUND The purpose of this study was to investigate Tim-3 expression on peripheral CD3-CD56+ natural killer (NK) cells and CD3+CD56+ natural killer T (NKT) cells in lung cancer patients. MATERIALS AND METHODS We analyzed Tim-3+CD3-CD56+ cells, Tim-3+CD3-CD56dim cells, Tim-3+CD3-CD56bright cells, and Tim- 3+CD3+CD56+ cells in fresh peripheral blood from 79 lung cancer cases preoperatively and 53 healthy controls by flow cytometry. Postoperative blood samples were also analyzed from 21 members of the lung cancer patient cohort. RESULTS It was showed that expression of Tim-3 was significantly increased on CD3-CD56+ cells, CD3- CD56dim cells and CD3+CD56+ cells in lung cancer patients as compared to healthy controls (p=0.03, p=0.03 and p=0.04, respectively). When analyzing Tim-3 expression with cancer progression, results revealed more elevated Tim-3 expression in CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in cases with advanced stages (III/IV) than those with stage I and II (p=0.02, p=0.04 and p=0.01, respectively). In addition, Tim-3 expression was significantly reduced on after surgical resection of the primary tumor (p<0.01). CONCLUSIONS Tim-3 expression in natural killer cells from fresh peripheral blood may provide a useful indicator of disease progression of lung cancer. Furthermore, it was indicated that Tim-3 might be as a therapeutic target.

Suppressive effects of gemcitabine plus cisplatin chemotherapy on regulatory T cells in nonsmall-cell lung cancer

Objective To investigate the effect of gemcitabine plus cisplatin chemotherapy on the percentage of CD4+CD25+FOXP3+ and CD8+CD28– regulatory T cells (Tregs) in the peripheral blood of patients with nonsmall-cell lung cancer (NSCLC). Methods Peripheral blood was taken from patients with NCSLC (before and after chemotherapy) and control subjects with nonmalignant disease. The percentages of CD4+CD25+FOXP3+ and CD8+CD28– Tregs were analysed using flow cytometry. Results Patients (n = 40) had significantly higher CD4+CD25+FOXP3+ and CD8+CD28– percentages than control subjects (n = 24). CD4+CD25+FOXP3+ and CD8+CD28– percentages increased with tumour progression, fell significantly after chemotherapy, but remained significantly higher than control values. Conclusions CD4+CD25+FOXP3+ and CD8+CD28– Treg percentages were higher in patients with NSCLC than control subjects, and increased in line with tumour progression. Percentages of CD4+CD25+FOXP3+ and CD8+CD28– Tregs were significantly reduced following gemcitabine plus cisplatin chemotherapy.

Anticancer effects of cinnamic acid in lung adenocarcinoma cell line h1299-derived stem-like cells.

Lung cancer is a lethal solid tumor with poor prognosis because of its high metastasis and resistance to current therapies. Recently, cancer stem cells (CSCs) were suggested to be major contributors to tumorigenicity and cancer relapse. However, therapeutic targets for lung cancer-related CSCs remain undetermined. The objective of the current study was to investigate whether cinnamic acid (CINN) exerts an antitumor activity against sphere-derived lung CSCs. In this study, CSCs were isolated from the non-small cell lung cancer cell line H1299 as tumor spheres under CSC-selective conditions, and found to have increased tumorigenicity, chemoresistance, and higher expression of both embryonic stem cell-related and drug resistance-related genes compared with parental cells. These observations are consistent with the notion that CSCs are tumorigenic, display the ability to self-renew, and generate differentiated progeny that constitute the majority of cells in tumors. Treatment of sphere-derived stem cells with CINN could diminish their CSC-like abilities by decreasing their proliferation and invasive abilities and facilitating their differentiation into CD133-negative cells. Furthermore, CINN treatment increased the sensitivity of CSCs to chemotherapeutic drugs through apoptosis. Of note, xenotransplantation experiments revealed that CINN combined with cisplatin had a synergistic effect in inhibiting the tumorigenicity of CSCs. In summary, our study clearly revealed the presence of a population of sphere-forming cells with stem-like properties among H1299 cells and CINN can attenuate CSC properties of this stem-like cell population. The potential of CINN should be verified further in future studies of anti-CSC therapy.

Identification of Factors for the Preoperative Prediction of Tumour Subtype and Prognosis in Patients with T1 Lung Adenocarcinoma

Aims. Identification of factors that can predict the subtypes of lung adenocarcinoma preoperatively is important for selecting the appropriate surgical procedure and for predicting postoperative survival. Methods. We retrospectively evaluated 87 patients with lung adenocarcinomas ≤30 mm. Results. Preoperative radiological findings, serum CEA level, serum microRNA-183 (miR-183) level, and tumour size differed significantly between patients with adenocarcinoma in situ (AIS) or minimally invasive adenocarcinoma (MIA) and those with invasive adenocarcinoma (IAC). Receiver operating characteristic curves and univariate analysis revealed that patients who were older than 57 years or had a pure solid nodule or a tumour with mixed ground-glass opacity (mGGO), a tumour >11 mm, a serum CEA level >2.12 ng/mL, or a serum miR-183 level >1.233 (2−ΔΔCt) were more likely to be diagnosed with IAC than with AIS or MIA. The combination of all five factors had an area under the curve of 0.946, with a sensitivity of 89.13% and a specificity of 95.12%. Moreover, patients with a cut-off value >0.499 for the five-factor combination had poor overall survival. Conclusions. The five-factor combination enables clinicians to distinguish AIS or MIA from IAC, thereby aiding in selecting the appropriate treatment, and to predict the prognosis of lung adenocarcinoma patients.