Journal: Clinical Cancer Research

Article Title: Epigenetic Inactivation of microRNA-34b/c Predicts Poor Disease-Free Survival in Early-Stage Lung Adenocarcinoma

doi: 10.1158/1078-0432.ccr-13-0736

Figure Lengend Snippet: Figure 1. Methylation status of miR-34b/c in lung adenocarcinoma cell lines and primary tumors. A and B, representative examples of MCA for miR-34b/c. Melting curves and derivative peaks from two cell lines (H2087 and H1838) are shown in the left (A) and from a lung adenocarcinoma and its corresponding nonmalignant lung tissue in the right (B). Differences in the GC content after bisulfite treatment results in variation of the melting temperature that make possible to discriminate the methylation status of samples assessed. While homogeneously unmethylated or methylated samples yielded a sharply defined melting curve, heterogeneously methylated samples resulted in a double-peak pattern. This bimodal pattern may correspond to the presence of nontumoral DNA from stromal cells within the tumoral samples or also may correspond to methylation that affects only one allele. C and D, representative chromatograms from bisulfite genomic sequencing of the miR-34b/c promoter. CpG sites are depicted as pink bars and the transcription start sites as blue bars. Five clones of each sample were sequenced and each row represents one sequenced allele. Black and white squares represent methylated and unmethylated cytosines at CpG sites, respectively.

Article Snippet: Chemicals and demethylation treatment of lung adenocarcinoma cell lines Stock solutions of 1 mmol/L cis-diammine-dichloroplatinum (Sigma-Aldrich), 1 mmol/L pemetrexed (Lilly), 10 mmol/L erlotinib hydrochloride (Selleckchem), and 25 mmol/L 5-aza-20-desoxycytidine (5-aza-dC; Sigma-Aldrich) were stored at 20 C and freshly dissolved in culture medium before use.

Techniques: Methylation, Genomic Sequencing, Clone Assay

Journal: Clinical Cancer Research

Article Title: Epigenetic Inactivation of microRNA-34b/c Predicts Poor Disease-Free Survival in Early-Stage Lung Adenocarcinoma

doi: 10.1158/1078-0432.ccr-13-0736

Figure Lengend Snippet: Figure 2. MiR-34b/c expression in lung adenocarcinoma cell lines (A) and primary tumors (B). We observed a strong correlation between DNA methylation, TP53 status and miR-34b/c expression. These boxplots show miR-34b/c expression (log2) in a set of 15 lung adenocarcinoma cell lines (A) and 49 lung adenocarcinoma tumors (B) according to DNA methylation (M, methylated; U, unmethylated) and TP53 status (mut, mutated; WT, wild-type). P values correspond to Kruskal–Wallis test among all 4 categories.

Article Snippet: Chemicals and demethylation treatment of lung adenocarcinoma cell lines Stock solutions of 1 mmol/L cis-diammine-dichloroplatinum (Sigma-Aldrich), 1 mmol/L pemetrexed (Lilly), 10 mmol/L erlotinib hydrochloride (Selleckchem), and 25 mmol/L 5-aza-20-desoxycytidine (5-aza-dC; Sigma-Aldrich) were stored at 20 C and freshly dissolved in culture medium before use.

Techniques: Expressing, DNA Methylation Assay, Methylation

Journal: Clinical Cancer Research

Article Title: Epigenetic Inactivation of microRNA-34b/c Predicts Poor Disease-Free Survival in Early-Stage Lung Adenocarcinoma

doi: 10.1158/1078-0432.ccr-13-0736

Figure Lengend Snippet: Figure 3. Kaplan–Meier plots of DFS according to miR-34b/c methylation level. Lung adenocarcinoma tumors were divided according to their source into training (Bellvitge Hospital, A) and test set (University of Michigan, B).

Article Snippet: Chemicals and demethylation treatment of lung adenocarcinoma cell lines Stock solutions of 1 mmol/L cis-diammine-dichloroplatinum (Sigma-Aldrich), 1 mmol/L pemetrexed (Lilly), 10 mmol/L erlotinib hydrochloride (Selleckchem), and 25 mmol/L 5-aza-20-desoxycytidine (5-aza-dC; Sigma-Aldrich) were stored at 20 C and freshly dissolved in culture medium before use.

Techniques: Methylation

Journal: Clinical Cancer Research

Article Title: Epigenetic Inactivation of microRNA-34b/c Predicts Poor Disease-Free Survival in Early-Stage Lung Adenocarcinoma

doi: 10.1158/1078-0432.ccr-13-0736

Figure Lengend Snippet: Figure 4. Impact of miR-34b/c on lung adenocarcinoma cell proliferation, migration and invasion. A, cell proliferation of miR-34b/c transfected cells was assessed using WST-1 assay. Values are expressed as the means SD of 3 experiments. Cell proliferation was significantly lower in cells expressing miR-34b/c as compared with cells transfected with the empty vector (ev; , P < 0.05; , P < 0.01; , P < 0.001). B, wound-healing assay in SK-LU-1 cells. Quantitative values indicate the mean distance SD between the migration fronts measured at different time-points per 3 wells and are representative of 3 experiments. Cells expressing miR-34b/c closed the induced cell wound significantly slower than cells transfected with ev. C, invasion experiments using Boyden chamber in SK-LU-1 cells. Invasive cells stained by Diff-Quick were counted. The quantitative values represent the mean SD of 5 microscopic fields per 2 wells and are representative of 2 experiments. Cells expressing miR-34b/c were significantly less invasive as compared with control empty vector-transfected cells.

Article Snippet: Chemicals and demethylation treatment of lung adenocarcinoma cell lines Stock solutions of 1 mmol/L cis-diammine-dichloroplatinum (Sigma-Aldrich), 1 mmol/L pemetrexed (Lilly), 10 mmol/L erlotinib hydrochloride (Selleckchem), and 25 mmol/L 5-aza-20-desoxycytidine (5-aza-dC; Sigma-Aldrich) were stored at 20 C and freshly dissolved in culture medium before use.

Techniques: Migration, Transfection, WST-1 Assay, Expressing, Plasmid Preparation, Wound Healing Assay, Staining, Diff-Quik, Control