Deficiency of LIMA1 accelerates bladder cancer progression by disrupting PINK1/Parkin-mediated mitophagy

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  Fig. 1. LIMA1 is downregulated in bladder cancer patients. A The concentration of LIMA1 was compared by ELISA analysis in serum extracted from healthy controls and BLCA patients. BLCA patients: n = 51, healthy controls: n = 35. The concentration of LIMA1 by ELISA in 51 BLCA patients, stratified by clinical stage B, tumor stage C, node stage D, pathologic stage E, and recurrence F from our cohort. Clinical stage 0a-Ⅱ: n = 28, Ⅲ-Ⅳ: n = 23; Tumor stage Ta-T2: n = 28, T3-T4: n = 23; Node stage N0: n = 32, Nx: n = 19; Pathologic stage Ⅰ-Ⅱ: n = 14, Ⅲ: n = 37. G Representative IHC showed that LIMA1 were detected in different T stages from the tissue samples of BLCA patients, scale bar: 200 μm, 50 μm (enlarged). H The staining positive intensity of LIMA1 was compared by IHC analysis in bladder cancer sample and corresponding non-tumor normal tissues. Bladder cancer sample, n = 63; corresponding non-tumor normal tissues, n = 32. I The staining score of LIMA1 was compared by IHC analysis in BLCA sample and corresponding non-tumor normal tissues. J Kaplan-Meier survival plot depicting cumulative overall survival of 63 BLCA patients, stratified by LIMA1 protein levels using IHC staining scores. The…

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  Fig. 2. Overexpression of LIMA1 inhibits tumorigenesis of T24 and 5637 cells. A, B Expression of LIMA1 was assessed by Western blot analysis in protein extracted from bladder cancer and normal cells. C The relative expression of LIMA1 was compared by qRT-PCR analysis in mRNA extracted from bladder cancer and normal cells. D–G The overexpression efficiency of LIMA1 in 5637 D, F and T24 E, G cells were detected by WB and qRT-PCR. H–K The overexpression of LIMA1 markedly inhibits proliferation of 5637 H, I and T24 J, K cells. L–O The overexpression of LIMA1 significantly reduces the migration capacity of 5637 L, M and T24 N, O cells by wound healing assay. P–R Transwell assays displayed the migration and invasion of 5637 cells after overexpression of LIMA1. S–U Transwell assays displayed the migration and invasion of T24 cells after overexpression of LIMA1. All data were expressed as the means ± SD of at least 3 independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Fig. 3. Knockdown of LIMA1 promotes tumorigenesis of T24 and 5637 cells. A–D The knockdown efficiency of LIMA1 in 5637 A, B and T24 C, D cells were detected by WB and qRT-PCR. The knockdown of LIMA1 markedly promotes proliferation of 5637 E, F and T24 G, H cells. The knockdown of LIMA1 significantly increases the migration capacity of 5637 I, K and T24 J, L cells by wound healing assay. M–O Transwell assays displayed the migration and invasion of 5637 cells after knockdown of LIMA1. P–R Transwell assays displayed the migration and invasion of T24 cells after knockdown of LIMA1. All data were expressed as the means ± SD of at least 3 independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Fig. 4. Effects of overexpression of LIMA1 on the mitophagy of T24 and 5637 cells. A, B. JC-1 showing the mitochondrial membrane potential in 5637 A and T24 B cells treated with overexpression of LIMA1. Scale bar = 25 μm. C, D Levels of ATP in 5637 C and T24 D cells after overexpression of LIMA1 by an ATP determination kit. E, F Immunofluorescence staining showing LC3B (green) and TOM20 (red) in 5637 E and T24 F cells treated with overexpression of LIMA1. Scale bar = 10 µm. G, H The overexpression of LIMA1 treated 5637 G and T24 H cells were transfected with mitochondrially targeted mKeima and excitation at 550 nm (red) and 438 nm (green) by microscopy. Scale bar = 10 µm. All data were expressed as the means ± SD of at least 3 independent experiments, **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Fig. 5. Effects of knockdown LIMA1 on the mitophagy of T24 and 5637 cells. A, B JC-1 showing the mitochondrial membrane potential in 5637 A and T24 B cells treated with knockdown of LIMA1. Scale bar = 25 μm. C, D Levels of ATP in 5637 C and T24 D cells after knockdown of LIMA1 by an ATP determination kit. E, F Immunofluorescence staining showing LC3B (green) and TOM20 (red) in 5637 E and T24 F cells treated with knockdown of LIMA1. Scale bar = 10 µm. G, H The knockdown of LIMA1 treated 5637 G and T24 H cells were transfected with mitochondrially targeted mKeima and excitation at 550 nm (red) and 438 nm (green) by microscopy. Scale bar = 10 µm. All data were expressed as the means ± SD of at least 3 independent experiments, **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Fig. 6. The mitophagy promoter reversed the effect of knockdown of LIMA1 on T24 cell tumorigenesis. A JC-1 in siLIMA1-T24 cells treated with Urolithin A. B Urolithin A treated siLIMA1-T24 cells were transfected with mitochondrially targeted mKeima. C Levels of ATP in siLIMA1-T24 cells treated with Urolithin A. D, E The proliferation of siLIMA1-T24 cells treated with Urolithin A. F The migration of siLIMA1-T24 cells treated with Urolithin A. G The invasion of siLIMA1-T24 cells treated with Urolithin A. All data were expressed as the means ± SD of at least 3 independent experiments, **P < 0.01; ***P < 0.001.

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  Fig. 7. LIMA1 regulated mitophagy through the PINK1/Parkin pathway. A Graphical representation of the 3D structure of the LIMA1 and PINK1 interaction model. B Co-IP analysis of the endogenous interactions between LIMA1 and PINK1 in 5637 and T24 cells. C The co-localization between LIMA1 (green) with PINK1 (red) was analyzed by confocal microscopy in 5637 and T24 cells. Scale bar = 10 μm. D–G The expression of PINK1 and Parkin in 5637 D, E and T24 F, G cells with LIMA1 overexpression by Western blot and qRT-PCR. H–K The expression of PINK1 and Parkin in 5637 H, I and T24 J, K cells with LIMA1 knockdown by qRT-PCR. All data were expressed as the means ± SD of at least 3 independent experiments, **P < 0.01; ***P < 0.001.

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  Fig. 8. Knockdown of LIMA1 suppressed tumor growth in mice. A, B The highly immunodeficient mice grown normally to 6 weeks were injected with shCtrl-T24 or shLIMA1-T24 cells and sacrificed after 7 weeks. C The subcutaneous tumors were surgically excised and photographed in two experimental groups (n = 6 mice per group). D The tumor volume was measured at different time points (n = 6 mice per group). E The tumor weights of the different groups (n = 6 mice per group). F H&E staining revealed the tumor tissues in the shLIMA1 group had lesser necrotic areas than that in the shCtrl group. Scale bar:100 μm. G–I Representative visual field images of IHC (LIMA1, PINK1, Ki-67) of tumor tissue from two groups. Scale bar: 100 μm. J Immunofluorescence staining showing LC3B (green) and TOM20 (red) of tumor tissue from two groups. Scale bar = 25 µm. All data were expressed as the means ± SD of at least 3 independent experiments, **P < 0.01; ***P < 0.001.