Long noncoding RNA BC005927 upregulates EPHB4 and promotes gastric cancer metastasis under hypoxia

Figures

• 

  Figure 1. Aberrant expression of BC005927 long, non‐coding RNA (lncRNA) in hypoxia‐induced gastric cancer (GC) cells and GC tissues. A, lncRNA‐BC005927 is upregulated under hypoxic culture conditions (1% O2, 5% CO 2, 94% N2) in (A1) SGC7901 cells, (A2) MKN45 cells and (A3) MKN28 cells. B1, B2, C1, C2, lncRNA‐BC005927 expression was assessed by real‐time PCR in 95 pairs of human GC and adjacent tissues. β‐Actin was used as an internal control. Significant differences between samples were analyzed using the Wilcoxon signed‐rank test (P = .002, n = 95). (D) Kaplan ‐Meier's analysis of the correlation between BC005927 expression and overall survival (OS) of GC patients. In all panels, results are representative of at least 3 independent experiments *P < .05, **P < .01

• 

  Figure 2. BC005927 is induced by hypoxia inducible factor‐1 alpha (HIF‐1α), and HIF‐1α interacts with the HIF‐1α response element in the BC005927 promoter region. A, Schematic illustration of consensus HIF‐1α response element in BC005927 gene promoter. B, C1, C2, SGC7901, MKN45 cells expressing control shRNA, HIF‐1α shRNAs were cultured under hypoxic conditions for 24 hours. Cell lysates and total RNA were subjected to western blot and real‐time RT‐PCR analyses, respectively. Data shown are mean ± SD (n = 3). D1, D2, Cell lysates and total RNA of MKN28 cells expressing NC (negative control) or pc‐DNA‐HIF‐1α were subjected to western blot and real‐time RT‐PCR analyses, respectively. Data shown are mean ± SD (n = 3). E, SGC7901 cells were cultured under normoxic or hypoxic conditions for 24 hours. Lysates were then subjected to ChIP assay. ChIP products were amplified by PCR reaction. F1, F2, SGC7901 cells were cotransfected with the indicated reporter constructs and Renilla luciferase plasmid. Twenty‐four hours after transfection, cells were cultured under normoxic or hypoxic conditions for 24 hours. Reporter activity was then measured and plotted after normalizing with respect to Renilla…

• 

  Figure 3. BC005927 is involved in cell invasion of gastric cancer (GC) cells and mediates hypoxia‐induced invasion of GC cells. SGC7901 and MKN45 cells transfected with lenti/si‐BC005927. A1, B1, MKN28 cells transfected with lenti‐BC005927 or (C1) NC were confirmed by real‐time PCR. Transwell migration and invasion assays of (A2, A3) SGC7901 and (B2, B3) MKN45 cells were carried out after transfection with (C2, C3) BC005927 BC‐si, MKN28 cells after transfection with BC005927. D1, D2, D3, Cell mobility was examined with a Cellomics ArrayScan VTI 1700 Plus. In all panels, results are representative of at least 3 independent experiments. E1, E2, Representative images and number of metastatic or invasive BC005927‐downregulated SGC7901 and MKN45 cells under normoxic or hypoxic conditions. In all panels, results are representative of at least 3 independent experiments

• 

  Figure 4. BC005927 promotes the metastasis and invasion of gastric cancer (GC) cells in vivo. SGC7901 cells transfected with BC005927‐si1, BC005927‐si2 or a negative control, MKN28 cells transfected with the LV‐BC005927‐expression vector or the control vector were injected into nude mice by the tail vein for an in vivo metastasis assay. Animals were killed 6 weeks after injection. A, Incidence of lung and liver metastasis in each group of nude mice. B1, Number of metastatic lung foci observed in each group. B2, Number of metastatic liver foci observed in each group. C1, Images showing representative hematoxylin and eosin (HE) staining of lung tissue samples from the different experimental groups. C2, Images showing representative HE staining of liver tissue samples from the different experimental groups. In all panels, the results are representative of at least 3 independent experiments

• 

  Figure 5. Association of BC005927 with EPHB4. A, Diagram of genes located around BC005927. B1, B2, C1, C2, mRNA and protein levels of EPHB4 were assessed by RT‐PCR and western blotting in SGC7901 and MKN45 cells transfected with an BC005927 siRNA‐lentivirus, MKN28 cells transfected with BC005927 lentivirus or a negative control (NC). D, Correlation between relative mRNA levels of BC005927 and EPHB4 in 95 gastric cancer (GC) tissue specimens. E, Immunohistochemical analysis of EPHB4 in metastatic and nonmetastatic GC: (a) noncancerous region of GC, (b) primary site of nonmetastatic GC, and (c,d) primary site of metastatic GC. F, Methylation mapping of 18 CpG island in EPHB4 promoter region obtained from bisulfite sequencing in scrambled siRNA control MKN45 or SGC7901 cells, and MKN45 or SGC7901cells infected with BC005927 siRNA. Each circle in the figure represents a single CpG site. For each cell line, percentage methylation at a single CpG site is calculated from the sequencing results of 10 independent clones. Black circles, 100% methylated; white circles, 0% methylation. In all panels, results are representative of at least 3 independent experiments.*P < .05

• 

  Figure 6. Upregulation of EPHB4 is potentially involved in the oncogenic function of BC005927. A1, A2, B, EPHB4 mRNA and protein levels were detected by qRT‐ PCR and western blot analysis. C1, C2, C3, Transwell assays were conducted to determine the migratory and invasive abilities of si‐BC005927‐transfected SGC7901 and MKN45 cells, BC005927‐overexpression MKN28 cells. D1, D2, Transwell assays were conducted to determine the migratory and invasive abilities of si‐NC, si‐BC005927, or both si‐BC005927 and EPHB4‐transfected SGC7901 cells. D2, Western blot analysis of EPHB4 expression. In all panels, results are representative of at least 3 independent experiments.*P < .05

• 

  Figure 7. Upregulation of EPHB4 by BC005927 mediates hypoxia‐induced gastric cancer (GC) metastasis. A1, A2, Protein levels of EPHB4 in SGC7901 and MKN45 cells. The cells were transfected with a BC005927 siRNA‐lentivirus or negative control (NC) and then exposed to hypoxia after transfection. The cells were then collected and subjected to western blot analysis at specific time points, as indicated. β‐Actin served as an internal control. B1, C1, Transwell migration and invasion assays of SGC7901‐NC and SGC7901‐si‐EPHB4 cells and MKN45‐NC and MKN28‐si‐EPHB4 cells were carried out under normoxic or hypoxic conditions. B2, C2, Protein levels of EPHB4 were determined by western blot assays. D1, D2, Transwell migration and invasion assays of SGC7901 cells were carried out after transfection with a BC005927 siRNA‐lentivirus, EPHB4 expression vector, or negative control (hypoxia) under normoxic or hypoxic conditions. Protein levels of EPHB4 were determined by western blot assays. In all panels, results are representative of at least 3 independent experiments