Mechanism of ELL-associated factor 2 and vasohibin 1 regulating invasion, migration, and angiogenesis in colorectal cancer

Figure 1 Expression of ELL-associated factor 2 and Vasohibin 1 proteins in tissues of patients with advanced colorectal cancer. A: Representative immunohistochemical staining of ELL-associated factor 2 (EAF2) and vasohibin 1 (VASH1) in colorectal adenocarcinoma tissue and the adjacent non-cancerous tissue (magnification 200 ×); B: The protein bands of EAF2 and VASH1 in 8-matched pairs of colorectal adenocarcinoma tissue and adjacent tissue were detected by Western blot assay. EAF2: ELL-associated factor 2; VASH1: Vasohibin 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; T: Colorectal adenocarcinoma tissue; N: Adjacent non-tumor tissue.

Figure 2 Kaplan-Meier survival curves. A and B: Kaplan-Meier survival curves for the protein expression of ELL-associated factor 2 and vasohibin 1 in colorectal cancer tissue. EAF2: ELL-associated factor 2; VASH1: Vasohibin 1.

Figure 3 Protein expression of pathway-related factors in a variety of human colorectal cancer cells and colorectal epithelial cells. A and B: The protein bands of ELL-associated factor 2, vasohibin 1, phosphorylated-signal transducer and activator of transcription 3, and transforming growth factor-β1 in colorectal cancer cell lines (SW480, RKO, HCT116, HT29, and LoVo) and normal colorectal epithelial cells (NCM460) by Western blot assay and the corresponding data. ^aP < 0.05, ^bP < 0.001. EAF2: ELL-associated factor 2; VASH1: Vasohibin 1; p-STAT3: Phosphorylated-signal transducer and activator of transcription 3; TGF-β1: Transforming growth factor-β1.

Figure 4 Silencing signal transducer and activator of transcription 3 phosphorylation significantly inhibits the expression of transforming growth factor-β1 protein in RKO cells. A: The protein bands of phospho-signal transducer and activator of transcription 3 (STAT3) in RKO cells transfected with STAT3 siRNA plasmids by Western blot assay and the corresponding data; B: The protein bands of pathway-related factors in RKO cells treated with STAT3 siRNA or exogenous transforming growth factor-β1 recombinant protein by Western blot assay. ^bP < 0.001. RKO-C: RKO cells were cultured in 1640 medium containing 10% fetal bovine serum (FBS). STAT3-si: RKO cells were transfected with STAT3 siRNA plasmids for 6 h and then cultured in 1640 medium containing 10% FBS for 24 h. RKO + NC: RKO cells were transfected empty vector for 6 h and then cultured in 1640 medium containing 10% FBS for 24 h. RKO + T: RKO cells were cultured for 24 h in 1640 medium containing 10% FBS in which 5 ng/mL TGF-β1 recombinant protein was added. VASH1: Vasohibin 1; STAT3: Signal transducer and activator of transcription 3; EAF2: ELL-associated factor 2; TGF-β1: Transforming growth factor-β1.

Figure 5 Overexpressing vasohibin 1 inhibits signal transducer and activator of transcription 3 phosphorylation (Tyr705) as well as transforming growth factor-β1 protein expression in RKO cells. A: The protein bands of pathway-related factors in RKO cells transfected with ELL-associated factor 2 (EAF2) overexpression plasmids, or vasohibin 1 (VASH1) overexpression plasmids, or VASH1 silencing plasmids by Western blot assay; B: The protein bands of VASH1 in RKO cells transfected with VASH1 siRNA plasmids by Western blot assay and the corresponding data. ^aP < 0.05, ^bP < 0.001. VASH1-si: RKO cells transfected with VASH1 siRNA plasmids for 6 h and then cultured in 1640 medium containing 10% fetal bovine serum (FBS) for 24 h. VASH1-OV: The group of RKO cells transfected with VASH1 overexpression plasmids for 6 h and then cultured in 1640 medium containing 10% FBS for 24 h. EAF2-OV: RKO cells transfected with EAF2 overexpression plasmids for 6 h and then cultured in 1640 medium containing 10% FBS for 24 h. VASH1: Vasohibin 1; EAF2: ELL-associated factor 2; TGF-β1: Transforming growth factor-β1.

Figure 6 Overexpressing ELL-associated factor 2 inhibits the invasion and migration of RKO cells by suppressing transforming growth factor-β1 related pathway. A: The invasion and migration abilities were evaluated by the transwelll assay; B: The migration ability was analyzed by wound healing assay after 72 h; C: The results of migration and invasion abilities of RKO cells were measured using ImageJ software. ^aP < 0.05, ^bP < 0.001. ELL-associated factor 2 (EAF2)-OV + T: The group of RKO cells were transfected with EAF2 overexpression plasmids for 24 h and then cultured with 5 ng/mL transforming growth factor-β1 recombinant protein for 24 h. EAF2: ELL-associated factor 2.

Figure 7 Overexpressing Vasohibin 1 inhibits the invasion and migration of RKO cells by suppressing transforming growth factor-β1 related pathway. A: The invasion and migration abilities were evaluated by the transwelll assay; B: The migration ability was analyzed by wound healing assay after 72 h; C: The results of migration and invasion abilities of RKO cells were measured using ImageJ software. ^bP < 0.001. vasohibin 1 (VASH1)-OV + T: The group of RKO cells were transfected with VASH1 overexpression plasmids for 24 h and then cultured with 5 ng/mL transforming growth factor-β1 recombinant protein for 24 h. VASH1: Vasohibin 1.

Figure 8 Overexpressing ELL-associated factor 2 inhibits tumor-induced angiogenesis in RKO cells via transforming growth factor-β1 related pathway. A: Conditioned medium from RKO cells transfected with ELL-associated factor 2-overexpressed plasmids or treated with transforming growth factor-β1 recombinant protein was collected to perform tube formation assay; B and C: The migration ability of Human Umbilical Vein Endothelial Cells was analyzed by transwell migration assay (B) and wound healing assay (C). And the results were measured by ImageJ software. ^aP < 0.05, ^bP < 0.001. EAF2: ELL-associated factor 2; TGF-β1: Transforming growth factor-β1.

Figure 9 Overexpressing Vasohibin 1 inhibits tumor-induced angiogenesis in RKO cells via transforming growth factor-β1 related pathway. A: Conditioned medium from RKO cells transfected with VASH1-overexpressed plasmids or treated with transforming growth factor-β1 recombinant protein was collected to perform tube formation assay; B and C: The migration ability of Human Umbilical Vein Endothelial Cells was analyzed by transwell migration assay (B) and wound healing assay (C). And the results were measured by ImageJ software. ^bP < 0.001. VASH1: Vasohibin 1; EAF2: ELL-associated factor 2.

Figure 10  Silencing Vasohibin 1 reverses the signal transducer and activator of transcription 3/transforming growth factor-β1 pathway inhibited by ELL-associated factor 2 overexpression. ^aP < 0.05, ^bP < 0.001. ELL-associated factor 2 (EAF2)-OV- Vasohibin 1 (VASH1-si): The group of RKO cells transfected with EAF2 overexpression plasmids and VASH1 siRNA plasmids. VASH1: Vasohibin 1; EAF2: ELL-associated factor 2.

Figure 11  Schematic depiction of ELL-associated factor 2 and vasohibin 1 regulating the signal transducer and activator of transcription 3/ transforming growth factor-β1 signaling pathway in this study. Overexpression of ELL-associated factor 2 may inhibit the activity of signal transducer and activator of transcription 3/transforming growth factor-β1 pathway by up-regulating the expression of vasohibin 1, thereby inhibiting cell invasion, migration and angiogenesis. EAF2: ELL-associated factor 2; TGF-β1: Transforming growth factor-β1; STAT3: Signal transducer and activator of transcription 3; CRC: Colorectal cancer; HUVECs: Human umbilical vein endothelial cells; VASH1: Vasohibin 1.