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. 2017 Dec 1;196(11):1443–1455. doi: 10.1164/rccm.201610-2157OC

Figure 2.

Figure 2.

MicroRNA-1 (miR-1) is down-regulated in tumor endothelium and regulates angiogenesis. (A) Endothelial cells (CD31+, CD45−) were isolated from non–small cell lung cancer tumors (Tumor) and adjacent cancer-free tissues (Normal). miR-1/18s levels were normalized to the median of the control (Normal) group and expressed as 2−∆∆Ct (n = 28; *P = 0.0351). Red horizontal lines indicate medians of the groups. (B) Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant/transformation-related protein 53 (P53) (KP) knockout mice received intranasal Cre recombinase (or control [ctrl]) vector and their lungs harvested after 6 months. miR-1/18s levels were measured in the whole lungs, normalized to the mean of the control group, and expressed as 2−∆∆Ct (n = 12 in each group from two experiments; *P = 0.01533). (C) Endothelial (CD31+, CD45−) cells were isolated from the lungs of KP and ctrl mice and miR-1 levels were measured and normalized as described in B (n ≥ 5 in each group from two experiments; *P = 0.016). (D) Vascular-specific miR-1 expression vector (V-miR-1). Vascular endothelial cadherin (VECad) promoter was cloned upstream of primary (pri)-miR-1 sequence in a lentiviral transfer vector containing enhanced green fluorescent protein as a marker gene driven by elongation factor 1 α subunit (EF1α) promoter. (E–G) Effect of vascular-specific miR-1 overexpression on tumor growth and angiogenesis. Lewis lung carcinoma implants were injected with V-miR-1 or scrambled control vector (V-scr) on Day 10, and harvested on Day 19 after implantation. The sizes of the tumors were measured and volumes calculated according to the following formula: volume = 0.52 × width2 × length. (E) Tumor volumes are graphed versus the time of measurement (number of days on the x-axis; data points are means of tumor volume) (n ≥ 13 in each group from three experiments; *P = 0.036013, **P = 0.016034). (F) Representative tumors from the two groups on Day 19 after implantation. (G) Tumor sections were stained with anti-CD31 antibody and DAPI (4',6-diamidino-2-phenylindole). (Left) Representative images. (Right) Quantification of vessel density based on the percentage of CD31-positive areas/whole area examined (n ≥ 7 in each group from two experiments; *P = 0.002645). Error bars represent SEM. ψ = packaging signal; EGFP = enhanced green fluorescent protein; LTR = long terminal repeat.