Figures & data
Figure 1 Increase of TRIM32 expression in colorectal cancer. (A) mRNA expression levels of TRIM32 in 25 pairs of colorectal cancer patients’ tumor tissues and paracancerous tissues were evaluated by qRT-PCR analysis. (B) mRNA expression levels of TRIM32 in FHC, SW480 and HT29 cells were detected by qRT-PCR analysis. (C) Protein levels of TRIM32 in colorectal cancer tissues, paracancerous tissues, FHC, SW480 and HT29 were detected by Western blot assays.
Notes: Data was expressed as mean±SD. #P<0.05 vs paracancerous tissues, *P<0.05 vs FHC group.
Abbreviations: TRIM32, tripartite motif 32; mRNA, messenger RNA; qRT-PCR, quantitative real-time polymerase chain reaction.
Figure 2 Knockdown of TRIM32 increases the sensitivity of colorectal cancer cells to cisplatin. (A) Effect of TRIM32 siRNA and plasmid on changing the TRIM32 expression was detected by Western blot assay. (B) SW480 and HT29 cells were transfected with TRIM32 siRNA or plasmid. 24 hrs later, these cells were treated with different concentrations of cisplatin (0–30 μM) for another 48 hrs. CCK-8 assays were performed to measure the cell viability of these cells. (C) Effect of TRIM32 siRNA and plasmid on changing the IC50 of cisplatin to SW480 and HT29.
Notes: Data was expressed as mean±SD. *P<0.05 vs NCO group.
Abbreviations: TRIM32, tripartite motif 32; CCK-8, Cell Counting Kit-8; IC50, 50% inhibiting concentration; siRNA, small interfering RNA.
Figure 3 TRIM32 is targeted by miR-519d in colorectal cancer. (A) The bioinformatics software TargetScan, miRanda and PicTar were used to identify the binding region of miR-519d in the TRIM32 3ʹ UTR. (B) Expression levels of miR-519d in 25 pairs of colorectal cancer patients’ tumor tissues and paracancerous tissues were evaluated by qRT-PCR analysis. (C) Expression levels of miR-519d in FHC, SW480 and HT29 were measured by qRT-PCR analysis. (D) Transfection with miR-519d mimics significantly increased the miR-519d levels in SW480 and HT29 cells. (E) Effect of miR-519d mimics on changing the protein expression of TRIM32 was evaluated by Western blot assays. (F) Effect of miR-519d mimics on changing the luciferase activities of pMIR-Reporters carrying wild type or mutant type of TRIM32 3ʹ UTR.
Notes: Data was expressed as mean±SD. #P<0.05 vs paracancerous tissues, *P<0.05 vs FHC group, &P<0.05 vs NCO group, $P<0.05.
Abbreviations: TRIM32, tripartite motif 32; 3ʹ UTR, 3ʹuntranslated region; qRT-PCR, quantitative real-time polymerase chain reaction.
Figure 4 Inhibition of TRIM32 induced by miR-519d increases the sensitivity of colorectal cancer cells to cisplatin. (A) Effect of miR-519d and TRIM32 plasmid on changing the TRIM32 expression was evaluated by Western blot assay. (B) SW480 and HT29 cells were transfected with miR-519d and TRIM32 plasmid. 24 hrs later, these cells were treated with cisplatin (1 μM) for another 48 hrs. CCK-8 assays were performed to measure the cell viability of these cells. (C) SW480 and HT29 cells were transfected with anti-miR-519d. 24 hrs later, these cells were treated with cisplatin (5 μM) for another 48 hrs. CCK-8 assays were performed to measure the cell viability of these cells.
Notes: Data was expressed as mean±SD. *P<0.05 vs NCO group, #P<0.05 vs cisplatin + NCO group, &P<0.05 vs cisplatin + miR-519d group.
Abbreviations: TRIM32, tripartite motif 32; CCK-8, Cell Counting Kit-8.
Figure 5 Inhibition of TRIM32 induced by miR-519d suppresses Bcl-2 expression and promotes the cisplatin-induced generation of ROS. (A) Effect of miR-519d and TRIM32 plasmid on changing the Bcl-2 expression was evaluated by Western blot assay. (B) SW480 and HT29 cells were transfected with miR-519d and TRIM32 plasmid. 24 hrs later, these cells were treated with cisplatin (1 μM) for another 48 hrs. Flow cytometry analysis was performed to detect the generation of ROS.
Notes: Data was expressed as mean±SD. *P<0.05 vs NCO group, #P<0.05 vs cisplatin + NCO group, &P<0.05 vs cisplatin + miR-519d group.
Abbreviations: TRIM32, tripartite motif 32; ROS, reactive oxygen species.
Figure 6 Inhibition of TRIM32 induced by miR-519d promotes mitochondria-mediated intrinsic apoptosis induced by cisplatin. (A) SW480 and HT29 cells were transfected with miR-519d and TRIM32 plasmid. 24 hrs later, these cells were treated with cisplatin (1 μM) for another 48 hrs. After staining with JC-1, flow cytometry analysis was performed to detect the MMP (Δφ) of cells. (B) Activation of caspase-9 and caspase-3 in SW480 and HT29 cells was detected by Western blot assay. (C) Apoptosis of SW480 and HT29 cells was detected by flow cytometry analysis.
Notes: Data was expressed as mean±SD. *P<0.05 vs cisplatin + NCO group, #P<0.05 vs cisplatin + miR-519d group.
Abbreviations: TRIM32, tripartite motif 32; MMP, mitochondrial membrane potential.
Figure 7 Overexpression of miR-519d sensitizes colorectal cancer to cisplatin treatment in vivo. (A) Tumor growth of miR-519d-overexpressed or control HT29 tumors which were treated with cisplatin (2 mg/kg) twice a week. (B) Expression of miR-519d in resected tumor tissues. (C) Expression of TRIM32 and Bcl-2 in resected tumor tissues.
Notes: Data was expressed as mean±SD. *P<0.05 vs HT29-control group, #P<0.05 vs HT29-control + cisplatin group.
Abbreviations: TRIM32, tripartite motif 32; Bcl-2, B-cell lymphoma-2.
Figure 8 Inhibition of TRIM32 induced by miR-519d increases the sensitivity of colorectal cancer cells to oxaliplatin and carboplatin. (A) SW480 and HT29 cells were transfected with miR-519d and TRIM32 plasmid. 24 hrs later, these cells were treated with oxaliplatin (1 μM) for another 48 hrs. CCK-8 assays were performed to measure the cell viability of these cells. (B) SW480 and HT29 cells were transfected with miR-519d and TRIM32 plasmid. 24 hrs later, these cells were treated with carboplatin (1 μM) for another 48 hrs. CCK-8 assays were performed to measure the cell viability of these cells.
Notes: Data was expressed as mean±SD. *P<0.05 vs oxaliplatin + NCO group, #P<0.05 vs oxaliplatin + miR-519d group, &P<0.05 vs carboplatin + NCO group, $P<0.05 vs carboplatin + miR-519d group.
Abbreviations: TRIM32, tripartite motif 32; CCK-8, Cell Counting Kit-8.
Figure 9 Schema of the predicted mechanisms implicated in SW480 and HT29 cells response to cisplatin and miR-519d. MiR-519d inhibits TRIM32 expression and thus suppresses the expression of Bcl-2 and promotes the cisplatin-caused generation of ROS. Because of the high level of ROS and inhibition of Bcl-2, apoptosis signals produced by cisplatin were expanded to trigger the mitochondria-mediated intrinsic apoptosis pathway. As a result, MMP (Δφ) was decreased and the effector caspases (caspase-9 and caspase-3) are triggered to cause the apoptotic cell death of SW480 and HT29 cells.
Abbreviations: TRIM32, tripartite motif 32; Bcl-2, B-cell lymphoma-2; MMP, mitochondrial membrane potential, ROS, reactive oxygen species.
