Journal: PloS one

Article Title: Down-Regulated miR-30a in Clear Cell Renal Cell Carcinoma Correlated with Tumor Hematogenous Metastasis by Targeting Angiogenesis-Specific DLL4.

doi: 10.1371/journal.pone.0067294

Figure Lengend Snippet: Figure 1. DLL4 is a direct target of miR-30a. (A) Putative complementary site of DLL4 mRNA 39-UTR for the seed region of miR-30a identified by prediction programs. (B) Endothelial HUVEC-C cells were transfected with DLL4 39-UTR vector and miR-30a vector or negative control vector (pcDNA3.0) for 48 hours. Relative luciferase activities of DLL4 39-UTR vector was reduced by miR-30a. (C) HUVEC-C cells were transfected with miR-30a inhibitor or negative control (scramble miRNA) for 24 hours. Relative mRNA expression of DLL4 was increased by miR-30a inhibitor. (D) HUVEC-C cells were transfected with miR-30a or negative control (pcDNA3.0) for 24 hours. Relative mRNA expression of DLL4 was decreased by miR-30a. (E) Western blotting showed that DLL4 was decreased by miR-30a after 48 hours of transfection. Data are represented as the means 6 standard errors of the means (SEM). *statistics significant. doi:10.1371/journal.pone.0067294.g001

Article Snippet: Transfection and Luciferase Assay The full-length human DLL4 and its corresponding empty vector PCMV6-XL6 were purchased from the OriGene Company (USA). miR-30a plasmid and its negative control vector pcDNA3.0 were provided by Prof. Guo (Department of Gastroenterology of Chinese PLA General Hospital). miR-30a inhibitor and scramble miRNA were purchased from GenePharma (Shanghai, China). miR-30a inhibitor: 59-CUU CCA GUC GAG GAU GUU UAC A-39, scramble miRNA: 59-CAG UAC UUU UGU GUA GUA CAA-39.

Techniques: Transfection, Plasmid Preparation, Negative Control, Luciferase, Expressing, Western Blot

Journal: PloS one

Article Title: Down-Regulated miR-30a in Clear Cell Renal Cell Carcinoma Correlated with Tumor Hematogenous Metastasis by Targeting Angiogenesis-Specific DLL4.

doi: 10.1371/journal.pone.0067294

Figure Lengend Snippet: Figure 2. miR-30a and DLL4 played opposite roles in the proliferation and migration of HUVEC-C cells. (A) DLL4-transfected HUVEC-C cells gained more proliferation capacity than control cells detected by MTS assay (left panel), while miR-30a suppressed HUVEC-C cell proliferation relative to control cells (middle panel). HUVEC-C cells were co-transfecting with either miR-30a and DLL4 or DLL4 and pcDNA3.0 and showed that miR-30a partially inhibited the DLL4-stimulated proliferation (right panel). (B) HUVEC-C cells transfected with DLL4 vector and miRNAs control vector (pcDNA) gained more migration capacity than cells transfected with double negative control vectors (XL6+pcDNA). However, HUVEC-C cells transfected with DLL4 vector and miR-30a gained less migration capacity than cells transfected with DLL4 and pcDNA vectors. (C) The numbers of migrated cells in the four groups are shown in bars. Data are presented as the mean 6 SEM. *statistics significant. doi:10.1371/journal.pone.0067294.g002

Article Snippet: Transfection and Luciferase Assay The full-length human DLL4 and its corresponding empty vector PCMV6-XL6 were purchased from the OriGene Company (USA). miR-30a plasmid and its negative control vector pcDNA3.0 were provided by Prof. Guo (Department of Gastroenterology of Chinese PLA General Hospital). miR-30a inhibitor and scramble miRNA were purchased from GenePharma (Shanghai, China). miR-30a inhibitor: 59-CUU CCA GUC GAG GAU GUU UAC A-39, scramble miRNA: 59-CAG UAC UUU UGU GUA GUA CAA-39.

Techniques: Migration, Transfection, Control, MTS Assay, Plasmid Preparation, Negative Control

Journal: PloS one

Article Title: Down-Regulated miR-30a in Clear Cell Renal Cell Carcinoma Correlated with Tumor Hematogenous Metastasis by Targeting Angiogenesis-Specific DLL4.

doi: 10.1371/journal.pone.0067294

Figure Lengend Snippet: Figure 4. Association of miR-30a with ccRCC hematogenous metastasis and metastasis-free survival. (A) Real-time PCR analyses of miR- 30a expression (left panel), DLL4 density (middle panel), and MVD (right panel) in non–metastatic (NM; n = 65), lymphatic metastatic (n = 6) and hematogenous metastatic (HM; n = 19) ccRCC. *p,0.05 based on a comparison between the HM and NM groups. Each box represents the median and 75th and 25th percentile values, and the bars represent minimums and maximums. (B) CD34 staining MVD in ccRCC including NM, LM, HM. (C) Statistical result of MVD in the three groups. (D) A total of 65 cases of non-metastatic RCC were grouped into two, ‘‘low miR-30a’’ (n = 50) and ‘‘high miR-30a’’ (n = 15) according to miR-30a expression at the threshold giving the lowest p value of log-rank test comparing metastasis-free survival between the two groups. (E) Kaplan-Meier graph representing the probability of metastasis-free survival in ccRCC from the two groups. The status was defined as ‘‘occurred metastasis or not’’, ‘‘High’’ referred to ‘‘High-level of miR-30a expression’’, while ‘‘Low’’ referred to ‘‘Low-level of miR-30a expression’’. Log-rank test was used to estimate and compare the probability of metastasis-free survival of the two groups. The p value was from a log-rank test. doi:10.1371/journal.pone.0067294.g004

Article Snippet: Transfection and Luciferase Assay The full-length human DLL4 and its corresponding empty vector PCMV6-XL6 were purchased from the OriGene Company (USA). miR-30a plasmid and its negative control vector pcDNA3.0 were provided by Prof. Guo (Department of Gastroenterology of Chinese PLA General Hospital). miR-30a inhibitor and scramble miRNA were purchased from GenePharma (Shanghai, China). miR-30a inhibitor: 59-CUU CCA GUC GAG GAU GUU UAC A-39, scramble miRNA: 59-CAG UAC UUU UGU GUA GUA CAA-39.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Comparison, Staining