LASP2 suppresses colorectal cancer progression through JNK/p38 MAPK pathway meditated epithelial-mesenchymal transition

Figures

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  Fig. 1. LASP2 is downregulated in colorectal cancer tissues. (a) LASP2 mRNA expression in CRC cell lines by real-time PCR. Expression levels were determined by the normalization of GAPDH. Error bars represent mean SD averaged from 3 independent experiments. (b) LASP2 expression in CRC cell lines by western blot. Immunosignal represent the relative amount of protein expression and was quantified by greyscale scanning software, and relative protein abundance was normalized by β-tubulin. (c) LASP2 expression in normal and CRC tissues by IHC from paraffin blocks. Representatives of LASP1 staining intensity: (a) negative in adjacent tissue, (b) weak in adjacent tissue, (c) moderate in adjacent tissue, (d) strong in adjacent tissue, (e) negative in tumor tissue, (f) weak in tumor tissue, (g) moderate in tumor tissue and (h) strong in tumor tissue

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  Fig. 2. LASP2 was negatively associated with progression and poor survival in CRC. Kaplan-Meier survival analysis in patients with CRC. a Overall survival of all patients. (n = 89, log rank = 5.490, P = 0.019). b Overall survival of patients in T3 and T4 stage (n = 80, log rank = 4.384, P = 0.036)

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  Fig. 3. LASP2 suppresses human CRC cell growth and migration. Transfection of LASP2 vector and siRNA was detected by western blot (a) and real time RT-PCR (b). Function of exogenous LASP2 overexpression and knockdown was investigated by CCK-8 assay (c) and transwell assay (d). Representative figures were shown. Error bars represent mean SD averaged from 3 independent experiments. (*) indicates P < 0.05. (**) indicates P < 0.01

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  Fig. 4. JNK/p38 MAPK inactivation is required for LASP2-medatied cell behaviors in CRC cells. a Marks of MAPK pathway and EMT were detected by western blot in LASP2 knockdown cell line SW620. Immunosignals of protein expression level were quantified by greyscale scanning software and relative protein abundance was normalized by β-tubulin. b Relative expression of phosphorylation of p38 and SAPK/JNK compared with original one. Error bars represent mean SD averaged from 3 independent experiments. (**) indicates P < 0.01

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  Fig. 5. JNK/p38 MAPK inactivation is required for LASP2-medatied cell behaviors in CRC cells. a Western blot analysis of marks of MAPK pathway and EMT after treatment with JNK inhibitor JNK inhibitor II and p38 inhibitor SB203580 in LASP2 down-regulated CRC cell line SW620. Immunosignals of protein expression level were quantified by greyscale scanning software and relative protein abundance was normalized by β-tubulin. b Representative figures of transwell assay after treatment of JNK inhibitor JNK inhibitor II and p38 inhibitor SB203580. Error bars represent mean SD averaged from 3 independent experiments. (#) indicates not statistic significant while (*) indicate P < 0.05 and (**) P < 0.01

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  Fig. 6. LASP2 is negatively regulated by LASP1 in colorectal cancer cells and tissues. a Relative mRNA expression of LASP1 and LASP2 in CRC cell lines tested by real time RT-PCR. Error bars represent mean SD averaged from 3 independent experiments. b LASP2 and LASP1 expression level was examined by western blot after transfected with LASP1 vector in SW620 and siRNA in SW480. Immunosignals of protein expression level were quantified by greyscale scanning software and relative protein abundance was normalized by β-tubulin. c Paraffin-embedded CRC samples were stained with anti-LASP1 or anti-LASP2 antibodies in the same position. Graphical illustration was shown in the right of LASP2 expression in the existence of LASP1 in 89 CRC samples