LIM domain only protein 7 promotes metastasis in colorectal cancer by TGF-β/ZEB1 pathway

Figure 1 LIM domain only protein 7 mRNA and protein expression is elevated in colorectal cancer tissues. A: Real-time reverse-transcriptase PCR; B: Western blotting; C: LIM domain only protein 7 protein expression in clinical samples was detected by immunohistochemistry; D: The expression of the LIM domain only protein 7 protein in the tissue chip was detected by immunohistochemistry. Using the Wilcoxon rank-sum test. ^aP < 0.05. CRC: Colorectal cancer; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LMO7: LIM domain only protein 7.

Figure 2 Knockdown of LIM domain only protein 7 in SW480 and LoVo cells resulted in reduced cell proliferation and decreased migration and invasion abilities. A: The knockdown efficiency of sh-LIM domain only protein 7 transfection was evaluated by real-time reverse-transcriptase PCR; B: The knockdown efficiency of sh-LIM domain only protein 7 transfection was evaluated by western blotting; C: Cell proliferation was detected by a cholecystokinin-8 assay; D: The cell wound scratch assay; E: Statistical graph of SW480 cells; F: Statistical graph of LoVo cells; G: The cell invasion experiment; H: Statistical graph of SW480 cells; I: Statistical graph of LoVo cells. Using the kruskal-Wallis. ^aP < 0.05, ^bP < 0.01. CTL: Cells transfected with the black vector; LMO7: LIM domain only protein 7; shL1: Cells transfected with sh-LMO7-1; shL2: Cells transfected with sh-LMO7-2.

Figure 3 Overexpression of LIM domain only protein 7 in HCT116 cells enhanced cell proliferation, migration, and invasion. A: The transfection efficiency of HCT116 cells overexpressing LIM domain only protein 7 was detected by real-time reverse-transcriptase PCR; B: The transfection efficiency of HCT116 cells overexpressing LIM domain only protein 7 was detected by western blotting; C: Cell proliferation was detected by a cholecystokinin-8 assay; D: The cell wound scratch assay; E: Statistical analysis; F: The cell invasion experiment; G: Statistical graph. ^aP < 0.05, ^bP < 0.01. Using the independent samples t-test. CTL: Cells transfected with the black vector; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LMO7: LIM domain only protein 7; LMO7-OV: Cells overexpressing LIM domain only protein 7.

Figure 4 Knockdown of LIM domain only protein 7 can inhibit the proliferation and lung metastasis of colorectal cancer cells in nude mice. A: Subcutaneous tumor formation test; B: Statistical graph of SW480 cells; C: Statistical graph of LoVo cells; D: Pulmonary metastasis after tail vein injection; E: Statistical analysis. Using the independent samples t-test. ^aP < 0.05, ^bP < 0.01. CTL, mice injected with blank vector-transfected cells. CTL: Cells transfected with the black vector; LMO7: LIM domain only protein 7.

Figure 5 Expression of the E-cadherin, N-cadherin, and vimentin proteins was detected by Western blotting. A: Western blotting; B: Statistical graph for SW480 cells; C: Statistical graph for LoVo cells; D: Statistical graph for HCT116 cells. Using the Kruskal-Wallis. ^aP < 0.05. CTL: Cells transfected with the black vector; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LMO7-OV: Cells overexpressing LIM domain only protein 7; shL1: Cells transfected with sh-LMO7-1; shL2: Cells transfected with sh-LMO7-2.

Figure 6 Zinc finger E-box binding homeobox 1 regulated the expression of the LMO7 gene in colorectal cancer. A: Prediction analysis revealed that the LIM domain only protein 7 (LMO7) promoter contains zinc finger E-box binding homeobox 1 (ZEB1) transcription binding sites; B: Chromatin immunoprecipitation assay detected ZEB1 binding to the LMO7 promoter by real-time reverse-transcriptase PCR using the indicated LMO7 primers; C: Mutation site in the LMO7 promoter; D: ZEB1 binding to the LMO7 promoter was assayed via dual-luciferase reporter assay; E: ZEB1 and LMO7 expression in ZEB1-overexpressing/knockdown colorectal cancer cells was analyzed by Western blotting. Using the independent samples t-test. ^aP < 0.05. CTL: Cells transfected with the black vector; IgG: Immunoglobulin G; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LMO7: LIM domain only protein 7; NS: Not significant; shL1: Cells transfected with sh-LMO7-1; shL2: Cells transfected with sh-LMO7-2; ZEBI: Zinc finger E-box binding homeobox 1.

Figure 7 Transforming growth factor-β regulates the expression of zinc finger E-box binding homeobox 1 and LIM domain only protein 7. A: The expression of LIM domain only protein 7 mRNA in SW480 and LoVo cells treated with transforming growth factor-β; B: Zinc finger E-box binding homeobox 1 and LIM domain only protein 7 protein expression in SW480 and LoVo cells treated with transforming growth factor-β. Using the independent samples t-test. ^aP < 0.05. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LMO7: LIM domain only protein 7; TGF-β: Transforming growth factor-β; ZEBI: Zinc finger E-box binding homeobox 1.