F-box and leucine-rich repeat 6 promotes gastric cancer progression via the promotion of epithelial-mesenchymal transition

Figure 1 F-box and leucine-rich repeat 6 is highly expressed in gastric cancer samples and gastric cancer cell lines. A and B: Higher expression of F-box and leucine-rich repeat 6 (FBXL6) was found in gastric cancer (GC) samples than the matched normal tissues (based on GSE54129 and TCGA database); C: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of FBXL6 mRNA expression in 68 pairs of GC patient samples; D: Western blot analysis was performed using an antibody against FBXL6 in 4 pairs of GC patients’ samples; E: Outcomes of immunohistochemical staining was performed using an antibody against FBXL6 and representative photographs of FBXL6 in GC patients. Scale bar: 100 μm; F: Relationship between FBXL6 expression and clinical parameters in GC by logistic analysis; G: RT-qPCR and Western blot analysis show the expression of FBXL6 in different GC cell lines (MKN-45, MGC-803 and HGC-27) and GES-1; H and I: Immunofluorescence staining examine the expression and localization of FBXL6 were using in GC cells (H) and tissues (I); J: Kaplan-Meier plots of overall survival and progression-free survival for GC samples from the K-M Plotter database. ^bP < 0.01; ^cP < 0.001. FBXL6: F-box and leucine-rich repeat 6.

Figure 2 F-box and leucine-rich repeat 6 promotes gastric cancer cell proliferation. A and B: Reverse transcription-quantitative polymerase chain reaction and Western blot analysis of MKN45 and HGC-27 stably transfected with F-box and leucine-rich repeat 6 (FBXL6) knockdown and overexpression of FBXL6 in MGC-803 cells; C and D: CCK8 assay analyzed the proliferation of stable knockdown in MKN-45 and HGC-27 cell lines and overexpression in MGC-803 cell; E and F: Colony formation assays were used to detect cell growth of knockdown FBXL6 in MKN-45 and HGC-27 or overexpression FBXL6 in MGC-803 cells; G and H: 5-ethynyl-2’-deoxyuridine assays help in the analysis of the viability of knockdown of FBXL6 in MKN-45 and HGC-27 cell or overexpression of FBXL6 in MGC-803; ^bP < 0.01; ^cP < 0.001. FBXL6: F-box and leucine-rich repeat 6.

Figure 3 F-box and leucine-rich repeat 6 regulates gastric cancer cell migration. A and B: Wound healing analysis to assess the migration of F-box and leucine-rich repeat 6 (FBXL6) knockdown stable cell lines or overexpression of FBXL6 in MGC-803 cells at 0 h and 24 h; C and D: Cell migration and invasion of gastric cancer cells with (C) knockdown of FBXL6 or (D) overexpression of FBXL6 by transwell assay. E and F: The experimental results revealed that the knockdown of FBXL6 significantly reduced migration and invasion of MKN-45 cells (E), whereas the overexpression of FBXL6 increased migration in MGC-803 (F). ^bP < 0.01; ^cP < 0.001. FBXL6: F-box and leucine-rich repeat 6.

Figure 4 Knockdown of F-box and leucine-rich repeat 6 in gastric cancer cells suppressed tumorigenesis invivo. A: Images of tumor formation in nude BALB/c mice after injection of sh-NC and sh-F-box and leucine-rich repeat 6 (FBXL6) #1 of HGC-27 cells into their subcutaneous; B: Tumor shape of shRNA and sh-NC groups after dissection; C: The tumor growth curve in sh-FBXL6#1 and sh-NC groups; D: Hematoxylin and eosin staining detected tumor in sh-FBXL6#1 and sh-NC groups; E and F: Immunohistochemical staining was performed to determine the tumor's expression of FBXL6, E-cadherin, N-cadherin, Vimentin, and Ki-67. ^bP < 0.01; ^cP < 0.001. FBXL6: F-box and leucine-rich repeat 6.

Figure 5 F-box and leucine-rich repeat 6 promotes the progression of gastric cancer cells through the epithelial-mesenchymal transition pathway. A and B: The expression of epithelial-mesenchymal transition-associated markers (E-cadherin, N-cadherin and Vimentin) and Snail is shown using western blot analysis; C and D: The expression of MMP-2 and MMP-9 was detected in the transfected gastric cancer cells by western blot. ^bP < 0.01; ^cP < 0.001. FBXL6: F-box and leucine-rich repeat 6.