Yong-Kui Zhang

High expression of serum miR-21 and tumor miR-200c associated with poor prognosis in patients with lung cancer

Serum microRNAs have been identified as potential cancer biomarkers. However, the detailed mechanism by which expression of microRNAs contributes to the development and diagnosis of NSCLC remains unknown. This study was to identify specific miRNAs for diagnosing or predicting the prognosis of NSCLC patients and their correlation between miRNA expression in tissues and serums. Six matched cancer and noncancerous tissues from NSCLC patients were analyzed by miRNA microarray. Among these, three miRNAs (miR-21, miR-141, and miR-200c) were examined in 70 NSCLC paired samples (cancer, normal tissue, and serum) and 44 serum samples of normal volunteers by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Consisting with the microarray results, the expression levels of miR-21, miR-141, and miR-200c in NSCLC were higher than those in normal tissues. While the level of serum miR-21 was increased in cancer patients as compared with that in normal counterpart, expression of miR-141 and miR-200c showed lower levels in serums from cancer patients. Overexpression of serum miR-21 was strongly associated with lymph node metastasis and advanced clinical stage of NSCLC. Finally, log-rank and Cox regression tests demonstrated that high expressions of tumor miR 21 and miR-200c or serum miR-21 were associated with a poor survival in NSCLC patients. Our results suggest that tumor miR-21, miR-141, miR-200c, and serum miR-21 may be potential novel biomarkers for the diagnosis of NSCLC. In addition, this study, for the first time, identifies a significant role of the tumor miR-200c played in predicting prognosis in patients with NSCLC.

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.

An Integrated Analysis of miRNA and mRNA Expressions in Non-Small Cell Lung Cancers

Using DNA microarrays, we generated both mRNA and miRNA expression data from 6 non-small cell lung cancer (NSCLC) tissues and their matching normal control from adjacent tissues to identify potential miRNA markers for diagnostics. We demonstrated that hsa-miR-96 is significantly and consistently up-regulated in all 6 NSCLCs. We validated this result in an independent set of 35 paired tumors and their adjacent normal tissues, as well as their sera that are collected before surgical resection or chemotherapy, and the results suggested that hsa-miR-96 may play an important role in NSCLC development and has great potential to be used as a noninvasive marker for diagnosing NSCLC. We predicted potential miRNA target mRNAs based on different methods (TargetScan and miRanda). Further classification of miRNA regulated genes based on their relationship with miRNAs revealed that hsa-miR-96 and certain other miRNAs tend to down-regulate their target mRNAs in NSCLC development, which have expression levels permissive to direct interaction between miRNAs and their target mRNAs. In addition, we identified a significant correlation of miRNA regulation with genes coincide with high density of CpG islands, which suggests that miRNA may represent a primary regulatory mechanism governing basic cellular functions and cell differentiations, and such mechanism may be complementary to DNA methylation in repressing or activating gene expression.

Increased Tim-3 expression in peripheral NK cells predicts a poorer prognosis and Tim-3 blockade improves NK cell-mediated cytotoxicity in human lung adenocarcinoma.

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been shown to play an important role in mediating NK-cell function in human diseases. However, the relationship between Tim-3 expression in natural killer (NK) cells and human lung adenocarcinoma remains unclear. We therefore investigated the expression of Tim-3 in NK cells and explored the effect of Tim-3 blockade on NK cell-mediated activity in human lung adenocarcinoma. Upregulated expression of Tim-3 on CD3-CD56+ cells (P<0.05) and CD3-CD56(dim) cells (P<0.05) of patients with lung adenocarcinoma was detected by flow cytometry. Moreover, Tim-3 expression in CD3-CD56+ NK cells was higher in patients with lung adenocarcinoma with lymph node metastasis (LNM) (P<0.05) or with tumor stage T3-T4 (P<0.05). Tim-3 expression in CD56(dim) NK-cell subset was higher in patients with tumor size ≥3cm (P<0.05), or LNM (P<0.05) or with tumor stage T3-T4 (P<0.05). Further analysis showed that higher expressions of Tim-3 on both CD3-CD56+ NK cells and CD56(dim) NK-cell subset were independently correlated with shorter overall survival of patients with lung adenocarcinoma (log-rank test, P=0.0418, 0.0406, respectively). Importantly, blockade of Tim-3 signaling with anti-Tim-3 antibodies resulted in the increased cytotoxicity and IFN-γ production of peripheral NK cells from patients with lung adenocarcinoma. Our data indicate that Tim-3 expression in NK cells can function as a prognostic biomarker in human lung adenocarcinoma and support that Tim-3 could be a new target for an immunotherapeutic strategy.

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.

Prognostic Evaluation of CapG, Gelsolin, P‐gp, GSTP1, and Topo‐II Proteins in Non‐Small Cell Lung Cancer

Metastasis and multidrug resistance (MDR) are the main reasons for the poor prognosis of non‐small cell lung cancer (NSCLC) patients. The use of biomarkers may contribute to a more accurate prediction of tumor metastasis, a better response to chemotherapy, and better patient survival. Gelsolin‐like actin‐capping protein (CapG) and gelsolin have been identified as playing important roles in tumor invasion and metastasis. Permeability glycoprotein (P‐gp), glutathione S‐transferase pi (GSTP1), and topoisomerase‐II (Topo‐II) are proteins that are closely related to MDR. In this study, we assessed the prognostic significance of CapG and gelsolin (both markers of tumor motility), and of P‐gp, GSTP1, and Topo‐II (markers of MDR) in NSCLC patients. One hundred and twenty‐one patients with pathologically confirmed, resectable NSCLC were included in the study. The expression levels of the five kinds of proteins mentioned above were determined by immunohistochemistry (IHC). The correlation between the clinical characteristics and IHC findings were analyzed. Expression of CapG, gelsolin, and P‐gp was found to be associated with an increased risk of death (Hazard Ratio (HR) = 2.799, 95% Confidence Interval (CI) = 1.2705–6.169, P = 0.011; HR = 3.968, 95% CI = 1.811–8.693, P = 0.001; HR = 3.251, 95% CI = 1.456–7.260, P = 0.004, respectively), whereas expression of GSTP1 and Topo‐II was not. These results suggest that higher tumor motility and MDR may be important in NSCLC prognosis. Anat Rec, 2012.

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.

Downregulation of miR-21 increases cisplatin sensitivity of non–small-cell lung cancer

Recent studies have shown that plasma miR-21 is a biomarker of chemotherapeutic response in lung cancer, but the influence of miR-21 on the sensitivity of non-small-cell lung cancer (NSCLC) to cisplatin (DDP) has not been confirmed. The aim of this study was to evaluate the role of miR-21 in NSCLC sensitivity to DDP in vitro and in vivo. Real-time quantitative PCR was used to detect miR-21 expression in lung cancer cell lines. Synthesized locked nucleic acid (LNA) anti-miR-21 was transiently transfected into A549 cells and pre-miR-21 was transfected into SK-MES-1 cells. We also investigated the effects of miR-21 downregulation and upregulation on growth and colony formation in DDP-treated cells. Finally, the effect of miR-21 downregulation on in vivo sensitivity of A549 cells to DDP was determined in BALB/c nude mice. miR-21 expression was significantly higher in A549 than in other lung cancer cell lines. LNA-based knockdown of miR-21 significantly inhibited growth and induced death in A549 cells, possibly via apoptotic signaling. Pre-miR-21 significantly promoted growth and inhibited death in SK-MES-1 cells. Moreover, ectopic suppression of miR-21 sensitized A549 cells to DDP in vivo. Our findings demonstrate that miR-21 suppression enhances the sensitivity of lung cancer cells to DDP in vitro and in vivo.

Membrane palmitoylated protein 3 promotes hepatocellular carcinoma cell migration and invasion via up-regulating matrix metalloproteinase 1

Membrane associated guanylate kinase (MAGUK) family, has been extensively studied in cellular adhesion and signal transduction at sites of cell–cell contact. Recently, growing attention has been paid to its role in the initiation and progression of various cancers. However, its role in hepatocellular carcinoma (HCC) has been rarely investigated. In this study, we found that membrane palmitoylated protein 3 (MPP3), a member of MAGUK family, was significantly up-regulated in both high metastatic potential cell lines and clinical tissue samples of HCC, and the most significant increase was observed in the tumors invading the portal veins. Higher level of MPP3 correlated with poorer survival of patients with HCC. Forced expression of MPP3 significantly enhanced HCC cell migration and invasion, whereas knockdown of this gene inhibited this oncogenic effect. Mechanismly, we found that MPP3 promoted HCC cell migration and invasion via up-regulating matrix metalloproteinase 1 (MMP1). These findings indicate that MPP3 play an important role in HCC metastasis by promoting cell migration and invasion, suggesting that it may serve as a novel prognostic marker and molecular target for therapy of HCC.