Fig. 6
CircPTK2 overexpression attenuates NSCLC cell metastasis in vivo, and circPTK2 levels were lower in metastatic NSCLC tissues than non-metastatic counterparts. a CircPTK2 expression in A549 cells stably overexpressing circPTK2. A549 stable cell line overexpressing circPTK2 was generated as described in Methods. pLCDH-circPTK2-copGFP(T2A)Puro lentiviral expression vector (upper) was used to stably overexpress circPTK2. The empty vector was served as negative control. CircPTK2 expression was determined by qRT-PCR (bottom left). CircPTK2 expression in circPTK2-overexpressed A549 cells was determined using northern blots. RNase R was used to digest linear RNA (bottom right). b Schematic flowchart of the in vivo metastasis experiments with A549 cells stably transfected with pLCDH-circPTK2 or vector (i.v.) and TGF-β1 (i.p.) injected into BALB/c nude mice (n = 6 mice per group in circPTK2 + TGF-β1 and vector + TGF-β1). c Representative images showing metastatic nodules established in lung taken from the mice injected with circPTK2-overexpressed A549 cells or vector control cells (upper). Scale bar, 4 mm. Haematoxylin and eosin (H&E) staining was performed for histological confirmation of metastasizing tumor cells in lung (bottom). Scale bar, 100 μm. d Gross view of metastatic nodules developed in liver (upper left) and dot plots showing the number of metastatic nodules in liver (upper right, n = 6 mice per group). Scale bar, 4 mm. Microscopic images of H&E staining for liver metastases (bottom left) and the distribution of the number of metastases in per section of liver (bottom right, n = 6 mice per group). Scale bar, 100 μm. Yellow and green arrowheads indicate metastatic nodules and micrometastases. e Representative images indicating metastatic nodules developed in pericardium (upper, n = 6 mice per group) and H&E staining of heart (bottom). Scale bar, 1 mm or 100 μm. f, g CircPTK2 and TIF1γ mRNA expression levels in human lung epithelial and NSCLC cells. β-actin was used as internal control. Each qRT-PCR analysis was performed in triplicate. h, i qRT-PCR analysis of circPTK2 and TIF1γ mRNA levels in 73 human NSCLC tissues and paired noncancerous lung tissues. Mean values are indicted by solid bars, and values are expressed as mean ± SEM. T, NSCLC tissues; N, paired noncancerous lung tissues. j, k Relative expression of circPTK2 and TIF1γ mRNA in 73 paired NSCLC tissues. Y-axis represents the log10 transformed fold change of T/N expression ratios of circPTK2 and TIF1γ mRNA. The number of each specimen is shown below x-axis. l Correlation between circPTK2 level and TIF1γ mRNA expression in 73 paired NSCLC tissues. X and y axes represent the T/N expression ratios of circPTK2 and TIF1γ mRNA, respectively. m Relative expression (T/N) of circPTK2 in metastatic (n = 41) and non-metastatic (n = 32) NSCLC tissues. Metastatic tissues were from NSCLC patients with lymph node metastasis or distant metastasis and non-metastatic tissues were from NSCLC patients without any metastasis, respectively. *P < 0.05; **P < 0.01; ***P < 0.001