SEPT2 crotonylation promotes metastasis and recurrence in hepatocellular carcinoma and is associated with poor survival

The crotonylome was highly expressed in highly metastasis potential HCC tissues and cells

To explore the role of crotonylation in HCC metastasis, we detected the pan-crotonylation in 100 HCC tissues. We found higher pan-crotonylation in primary HCC tissues of patients with metastasis within one year after surgery (N = 51), compared to HCC patients without metastasis (N = 49) (p < 0.01) (Fig. 1A, B). Patients with higher crotonylation had shorter disease-free survival period (p < 0.05) (Fig. 1C). Besides, we observed that the crotonylation level were similar in HBV positive and negative HCC (Additional file 1: Fig. S1A). EMT is a key process of cancer cell metastasis. During this process, epithelial cells acquire the characteristics of mesenchymal cells to enhance cell mobility and their migration ability [17]. We therefore examined the correlation between crotonylation and EMT makers by immunohistochemistry (IHC) staining. Our results showed that pan-crotonylation was negatively correlated with E-cadherin (p < 0.0001) and positively correlated with N-cadherin (p < 0.0001), suggesting that crotonylation level was associated with metastasis in HCC (Fig. 1D, E).

Fig. 1figure 1

The crotonylome in HCC tissues and cells. A, B Immunohistochemistry (IHC) assays showing that pan-crotonylation was higher in HCC patients with metastasis. The data are presented as the means ± SD. (Student’s t test). C Patients with high pan-crotonylation levels had a shorter disease-free survival period than those with low pan-crotonylation levels. D, E IHC assays of 100 HCC tissues showing that pan-crotonylation was correlated with E-Cadherin and N-Cadherin. F Flowchart showing the experimental procedure followed for identifying crotonylated proteins through stable isotope labeling by amino acids in cell culture (SILAC) and liquid chromatography with tandem mass spectrometry (LC–MS/MS). G Subcellular localization of the identified crotonylated proteins. H Gene set enrichment analysis (GSEA) showing the five most enriched pathways (p < 0.05). I The difference in protein expression and crotonylation between MHCC-97H and MHCC-97L cells. The level of protein expression and degree of crotonylation were increased in MHCC-97H cells (-lg false discovery rate [FDR] > 0). J Scatter diagram showing the differentially crotonylated sites in the MHCC-97H and MHCC-97L cell lines (p < 0.05). K Western blotting (WB) verified that the total crotonylation level was higher in the MHCC-97H cell line

To identify crotonylated proteins associated with tumor metastasis, two HCC cell lines MHCC-97L (minimally invasive) and MHCC-97H (highly invasive), which were isolated from one patient and showed differential invasive potential (Additional file 1: Fig. S1B) [18], were used to perform SILAC. Differentially crotonylated proteins (Additional file 2: Data S1) were identified by LC–MS/MS (Fig. 1F), including 1085 crotonylated proteins and 3540 crotonylation sites. The analysis on subcellular distribution of crotonylated proteins showed that 34.2% of crotonylated proteins were located in the cytosol, 24.2% in the nucleus, 8.7% in the cytoskeleton, and 7.6% in the mitochondrion (Fig. 1G). Crotonylated proteins were enriched in several metabolic pathways, including adipogenesis and fatty acid metabolism, oxidative phosphorylation, peroxisome, and xenobiotic metabolism pathways, compared to non-crotonylated proteins (Fig. 1H).

We next analyzed all protein sequences in modified 21-mers constituted with amino acids in specific positions (10 amino acids upstream and downstream of one site) (Additional file 1: Fig. S1C). The motif-X analysis showed that EKE, KE, and EKxE were overrepresented Kcr hotspot sites (Additional file 1: Fig. S1D). Further analysis on the differences between minimally and highly invasive cell proteomes showed that the levels of protein expression and crotonylation were generally increased in highly invasive cells, and the increase in crotonylation was more obvious than that in protein expression (Fig. 1I). When compared with minimally invasive cells, we identified 334 upregulated and 205 downregulated proteins (fold change [FC] > 1.5, p < 0.05) (Additional file 1: Fig. S1E), and 199 hypercrotonylated and 46 hypocrotonylated sites in highly invasive MHCC-97H cells (FC > 1.5, p < 0.05) (Fig. 1J). In addition, the total crotonylation level was significantly higher in highly invasive cells than in minimally invasive cells (p < 0.05) (Additional file 1: Fig. S1F), which were confirmed by Western blot (Fig. 1K). Collectively, our findings revealed the increase of crotonylation in HCC tissues with metastasis as well as highly invasive HCC cells.

Crotonylation was positively correlated with HCC cell migration and invasion

To investigate the association of crotonylation with metastasis, we analyzed differentially crotonylated proteins between the two HCC cell lines. As shown in Fig. 2A, highly invasive MHCC-97H cells had more hypercrotonylated proteins. Gene set enrichment analysis showed that in highly invasive cells, more crotonylated proteins were enriched in biological oxidation and metabolism of xenobiotics by cytochrome P450 processes, with fewer enriched in cell junction organization and focal adhesion processes, compared with crotonylated proteins in minimally invasive cells (Additional file 1: Fig. S2A, B). Gene Ontology (GO) annotation revealed that hypercrotonylated proteins in highly invasive cells were involved in diverse biological processes, particularly the extracellular matrix and cell adhesion molecule binding (Fig. 2B). In addition, we found higher levels of crotonylation on known metastasis-related proteins in highly invasive cells (Additional file 1: Fig. S2C).

Fig. 2figure 2

Crotonylation was positively correlated with HCC cell migration and invasion A Heatmap showing differentially crotonylated proteins in the MHCC-97H and MHCC-97L cell lines. B Gene ontology (GO)-based enrichment analysis of differentially crotonylated proteins. C Morphological changes in SNU449 and Huh7 cells after 25 mM sodium crotonate (NaCr) treatment. D Migration and invasion assays showing that SNU449 and Huh7 cells had higher invasive potential after 25 mM NaCr treatment. The data are presented as the means ± SD. **p < 0.001, ****p < 0.0001 (Student’s t test). E Wound healing assays showing that SNU449 and Huh7 cells exhibited greater migration capacity after 25 mM NaCr treatment. The data are presented as the means ± SD. **p < 0.01, ***p < 0.001 (Student’s t test). F Western blot analysis showing the changes in the expression of epithelial-mesenchymal transition (EMT)-related proteins after 25 mM NaCr treatment

For verification, we increased the overall level of crotonylation in SNU449, Huh7 and SMMC7721 cell lines by adding crotonylation substrate (NaCr, sodium crotonate). An anti-pan-crotonyl-lysine (pan-Kcr) antibody was used to detect the total crotonylation level in cells after NaCr treatment. As shown in Additional file 1: Fig. S3, the total crotonylation reached the maximum after NaCr (25 mM) treatment. SNU449, Huh7 and SMMC7721 cells treated with NaCr exhibited an elongated spindle shape (Fig. 2C, Additional file 1: Fig. S4A). Increasing crotonylation by NaCr treatment promoted migration and invasion of SNU449, Huh7 and SMMC7721 cells (Fig. 2D, E, Additional file 1: Fig. S4B, C). Furthermore, increasing NaCr concentration decreased the expression of epithelial markers E-cadherin, while enhancing the expression of mesenchymal markers N-cadherin and Snail in HCC cells (Fig. 2F, Additional file 1: Fig. S4D). These results indicated that increasing crotonylation exerted a promoting effect on HCC cell migration and invasion.

SEPT2 was identified in the crotonylome

To explore the mechanism by which crotonylation facilitates cell migration and invasion in HCC, the proteomes of highly and minimally invasive cells were analyzed. After normalization, the 3 most significantly differential crotonylated proteins- SEPT2, RAB35, and GRB2, were selected (Fig. 3A, Additional file 1: Fig. S5A). After NaCr treatment, the results showed that these three candidates were hypercrotonylated in MHCC-97L cells (Fig. 3B). Furthermore, analysis on endogenous crotonylation revealed that the crotonylation level of these three proteins was greater in highly invasive cells compared to minimally invasive cells, and these results were consistent with SILAC analysis (Fig. 3C).

Fig. 3figure 3

Lysine (K) 74 crotonylation of SEPT2 was identified. A Scatter diagram based on the fold change (FC) of protein expression (FC < 2) and crotonylation (FC > 1.5). B Validation of endogenous crotonylation of SEPT2, GRB2 and RAB35. MHCC-97L cells were treated with 25 mM sodium crotonate (NaCr) for 24 h. Cell lysates were immunoprecipitated with corresponding primary antibodies, followed by western blotting (WB) with an anti-pan-Kcr antibody. C Validation of endogenous differences in SEPT2, GRB2 and RAB35 crotonylation in MHCC-97H and MHCC-97L cell lines. SEPT2 crotonylation was highest crotonylation in the MHCC-97H cells. D The location of differentially crotonylated sites in the SEPT2 domain. E SEPT2-K74 is evolutionarily conserved in seven species. K74 in SEPT2 is highlighted in red. F The SEPT2-K74 and SEPT2-K318 mutants were crotonylated to a lesser extent than wild-type (WT) SEPT2. The degree of crotonylation in cells with ectopically expressed Flag-tagged SEPT2 WT, K74R and K318R was analyzed. G GTPase activity was impaired after SEPT2-K74R mutation. Flag-tagged SEPT2 WT and K74R proteins were purified, and an in vitro GTP hydrolysis assay was performed. Panels 1, 2, 4, and 5 show the negative controls, and panel 3 shows the positive control. H Western blot analysis of overexpressed Flag-tagged SEPT2 WT and K74R, pan-crotonylation and SEPT2 K74-crotonylation (K74-cr) in cells and crotonylation on SEPT2 with or without NaCr treatment

Among the 3 protein candidates, SEPT2 was eventually selected as it had the greatest level of crotonylation in highly invasive cells. SEPT2 is crucial in spindle formation and sister chromatid separation, thereby regulating G2/M phase transition and cell mitosis [19]. Whereas several studies have shown that SEPT2 can facilitate tumor growth and metastasis [20, 21]. We identified that the two crotonylation sites in SEPT2, K318 (H/L = 1.3, p < 0.05) and K74 (H/L = 2.19, p < 0.05), were significantly differentially crotonylated, as determined by LS-MS/MS. Analysis on the protein domains in SEPT2 showed that K74 site is in the GTPase domain, and K318 site is in the variable domain (Fig. 3D). Through analyzing the protein sequence alignment of SEPT2 homologs in different species, our results revealed that K74 was more evolutionarily conserved than K318 (Fig. 3E, Additional file 1: Fig. S5B). In addition, when the lysine residue at 74 site was replaced with an alanine, SEPT2 crotonylation was significantly reduced, largely abrogating SEPT2 crotonylation, whereas crotonylation of the K318R mutant was slightly reduced (Fig. 3F). These results indicated that K74cr may play a functional role in SEPT2. Therefore, we purified ectopically expressed SEPT2 wildtype (WT) and SEPT2-K74R in HEK293FT cells (Additional file 1: Fig. S5C) and performed GTPase assay. The results showed that SEPT2-K74R significantly impaired GTP degradation activity of SEPT2 (p < 0.001) (Fig. 3G). To better examine the role of SEPT2-K74cr, we generated a site-specific anti-SEPT2-K74 antibody that could target K74cr in SEPT2. The specificity of this antibody was confirmed, as the results of dot blot, and Western blot showed that K74cr preferentially detected SEPT-K74cr but not unmodified SEPT2 (Additional file 1: Fig. S5D, Fig. 3H).

SEPT2-K74R mutant inhibited HCC cell migration and invasion in vitro and in vivo

To investigate the functional role of SEPT2-K74 crotonylation, we stably overexpressed SEPT2-WT and SEPT2-K74R in SNU449, Huh7 and SMMC7721 cells. The forced expression of SEPT2-WT and SEPT2-K74R as well as whole-cell crotonylation and crotonylation of SEPT2 with or without NaCr treatment were verified by Western blot (Fig. 3, Additional file 1: Fig. S5E). Additional file 1: Fig. S5F showed that K74R could decrease the acetylation and succinylation of SEPT2, but NaCr treatment had no effect on SEPT2 acetylation and succinylation. We observed an increased metastatic potential of SEPT2-WT cells treated with NaCr compared to control cells. In contrast, migration and invasion of K74R-overexpressing cells with and without NaCr treatment were both inhibited, compared to SEPT2-WT and control cells (Fig. 4A–C, Additional file 1: Fig. S6A, B). These results thus implicated the anti-metastasis potential of SEPT2-K74R. Besides, we examined the impact of overexpressing SEPT2-WT and SEPT2-K74R on HCC cell growth and cell cycle with or without NaCr treatment. We found SEPT2-WT overexpressing has a positive effect on HCC cell growth, but K74R and NaCr treatment could hamper HCC cell growth (Additional file 1: Fig. S7A, B). Flow cytometry analysis of SEPT2-WT overexpressing cells, but not K74R, showed an increase in S phase and a reduction in G1 phase, and cells treated with NaCr showed a reduction of cells in S phase and an increase of cells in G1 phase (Additional file 1: Fig. S7C). It means that K74 decrotonylation could inhibit cell growth and cell cycle, but the role of NaCr inhibited cell growth and cycle may not due to SEPT2 crotonylation.

Fig. 4figure 4

SEPT2-K74R inhibited cell migration and invasion in vitro. A Overexpression of SEPT2-K74R inhibited migration of Huh7 cells (left panel) and SNU449 cells (right panel) with and without 25 mM sodium crotonate (NaCr) treatment. B Overexpression of SEPT2-K74R inhibited the invasion of Huh7 cells (left panel) and SNU449 cells (right panel) with and without 25 mM NaCr treatment. C Overexpression of SEPT2-K74R inhibited the migration of Huh7 cells (upper panel) and SNU449 cells (lower panel) with and without 25 mM NaCr treatment. The data are presented as the means ± SD. Ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA). NC, negative control; WT, wild type; 74R, SEPT2-K74R; NC-Cr, SEPT2-WT-Cr and K74R-Cr indicate NC, SEPT2-WT and SEPT2-K74R cells treated with 25 mM NaCr, respectively

To validate our in vitro findings, we established multiple mouse models with tail vein injection, orthotopic liver tumor implantation, and splenic vein injection. For mice with tail vein injection, Huh7 cells carrying a stable overexpression vector, SEPT2-WT, or SEPT2-K74R, were injected into the tail vein of NCG mice (N = 8 per group). Mice were sacrificed 8 weeks later, and HCC metastasis in the lungs was assessed (Additional file 1: Figure S8A). The results showed that mice in the SEPT2-K74R group were more nutrified (Additional file 1: Fig. S8B) with significantly higher body weight than the other two groups (both p < 0.05) (Fig. 5A). Mice in SEPT2-K74R (4/8) group also had fewer lung metastases than the SEPT2-WT (8/8) or vector (8/8) groups (Fig. 5B–D). In addition, we observed liver metastases in the vector (2/8) and SEPT2-WT (3/8) groups but not in the SEPT2-K74R group (Fig. 5E, Additional file 1: Fig. S8C).

Fig. 5figure 5

SEPT2-K74R inhibited cell migration and invasion in vivo A Mice in the SEPT2-K74R group gained the most weight. Huh7 stable cells were injected into the tail vein of NCG mice (8 mice each group). The mice were weighed every week (W) and were sacrificed 8 weeks after the injection. B Quantitative comparison of mice with lung metastases in each group. Fewer mice had lung metastasis in the SEPT2-K74R group. The red column indicates the mice had metastases. C Representative images of lung metastases in the tail vein injection model. After the mice were sacrificed, the lungs were dissected and photographed (upper panel). Blue arrows indicate lung metastases. Lung tissues were stained by H&E (bottom panel). D Quantitative comparison of the number of lung metastases in each group. E Quantitative comparison of mice with liver metastases in each group. The red column indicates the transferred mice. F Flowchart showing the orthotopic liver tumor implantation model. Huh7 stable cells were injected into the left lobe of the liver in NCG mice (8 mice per group). G Images of liver metastases in the orthotopic liver tumor implantation model. After the mice were sacrificed, the livers were dissected, photographed and weighed. Blue arrows indicate liver metastases. H Quantitative comparison of the number of liver metastases in each group. Mice in the SEPT2-K74R group had fewer liver metastases. I Representative images showing liver metastases in the orthotopic liver tumor implantation model. Tissues were stained by H&E. J, K Quantitative comparison of the number of mice with metastases in other lobes of the liver (J) and the number of mice with lung metastases (K). The red column indicates the transferred mice.The data are presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA, two-way ANOVA). NC, negative control; WT, wild-type SEPT2; K74R, SEPT2-K74R mutant

We then established an orthotopic mouse model with liver tumor implantation by injecting Huh7 cells into the left lobe of liver, and mice were sacrificed 10 weeks later (Fig. 5F). The relative liver weight in mice with K74R was significantly lower than the vector group (p < 0.05) (Additional file 1: Fig. S9A). We observed significantly fewer metastases in the left lobe of liver (Additional file 1: Fig. S9B) and the total liver (Fig. 5G, I) in mice with K74R than in the other two groups. Fewer metastases in other liver lobes (1/8) and lung (0/8) in the K74R group were also observed, compared to the vector group (6/8 with liver and 2/8 with lung metastases) and SEPT2-WT group (5/8 with liver and 3/8 with lung metastases) (Fig. 5J, K, Additional file 1: Fig. S9C). In addition, a further mouse model was established by injecting Huh7 cells into the spleen, and mice were sacrificed 8 weeks later (Additional file 1: Fig. S10A). Consistent with the findings of other mouse models, the K74 group had lower incidence of intrahepatic metastases, compared to the vector and SEPT2-WT groups (Additional file 1: Fig. S10B–E). Altogether, both in vitro and in vivo results illustrated that SEPT2-K74R mutant could markedly inhibit HCC metastasis.

SIRT2 decrotonylated SEPT2

We co-expressed SEPT2-WT with four decrotonylases, SIRT1, SIRT2, SIRT3 and HDAC3, which were verified in a previous study [8] in 293 T cells (Additional file 1: Fig. S11A). Notably, only SIRT2 had the ability to decrotonylate SEPT2, while the other three decrotonylases exerted no effect on SEPT2 Kcr. For verification, we performed co-IP assay (Additional file 1: Fig. S11B) and GST pull-down assay (Additional file 1: Fig. S11C), and both results confirmed that SIRT2 could interact with SEPT2. Furthermore, SIRT2 decronylated SEPT2 in a dose-dependent manner (Additional file 1: Fig. S11D), and knockdown of SIRT2 expression and SIRT2 inhibitor, AGK2, treatment led to a significant increase in SEPT2 crotonylation (Additional file 1: Fig. S11E, F). Therefore, our results indicated that SIRT2 could decrotonylate SEPT2. Moreover, we co-expressed SEPT2-WT with four crotonylases [10], CBP, P300, HMOF and PCAF in 293 T cells (Additional file 1: Fig. S11G). The result showed that CBP and P300 could interact with SEPT2 and had the ability to crotonylate SEPT2, while others exerted no effect on SEPT2 crotonylation.

Crotonylation promoted cell invasive capability mediated through the SEPT2-K74cr-P85α-Akt pathway

SEPT2 plays an important role in mitosis as it is crucially involved in spindle formation and sister chromatid separation. A previous study showed that P85α, a subunit of PI3K which is important for EMT [22, 23], interacted with SEPT2 to regulate mitosis and that SEPT2 GTPase activity is crucial for mitosis [24]. Therefore, we hypothesized that P85α may be related to SEPT2-K74cr, and P85α. By co-IP and GST pull-down assays, our results found that K74R mutation impaired the interaction of SEPT2 with P85α (Fig. 6A, B). A reduction of P85α expression in SEPT2-K74R-overexpressing cells was observed (Fig. 6C). While the mRNA expression of P85α did not change in SNU449 cells and even increased in Huh7 cells (Additional file 1: Fig. S12A), indicating that SEPT2-K74R regulated P85α protein level mainly at posttranscriptional level. For verification, SEPT2-74R was transiently overexpressed in SNU449 cells, and these cells were treated with the protein synthetase inhibitor CHX in FBS-free medium. The results showed that P85α protein level was decreased in SEPT2-K74R-overexpressing cells treated with CHX (p < 0.001) (Fig. 6D). In addition, we observed a decrease in the stability of P85α in SIRT2-overexpressing cells (Additional file 1: Fig. S12B).

Fig. 6figure 6

Crotonylation enhanced cell invasive capability by mediating SEPT2-K74Cr-P85α-Akt pathway activity (A, B) P85α interacts with SEPT2. Whole-cell lysates were immunoprecipitated with an anti-SEPT2 antibody (A) and GST-tagged SEPT2-WT or GST-tagged SEPT2-K74R were purified with anti-GST antibody-conjugated beads B. C SEPT2-K74R overexpression decreased the expression of downstream P85α. D SEPT2-K74R overexpression decreased P85α stability. Transient overexpression vectors carrying SEPT2 WT and SEPT2-K74R in SNU449 cells were treated with cycloheximide (CHX), and the P85α protein level was determined by WB (upper panel). The lower panel shows the relative protein levels in the different groups. Error bars represent ± SD on the basis of triplicate experiments. E, F P85α rescued SEPT2 function after sodium crotonate (NaCr) treatment. Knocking down P85α expression inhibited cell migration (E) and invasion (F) after NaCr treatment; overexpression of P85α in SEPT2-K74R-expressing cells rescued the cell migration (E) and invasion (F) abilities after NaCr treatment. G P85α rescued SEPT2 function after NaCr treatment. Quantitative comparison of wound-healing assays performed with Huh7 cells (upper panel) and SNU449 cells (lower panel). H Western blot analysis of proteins expressed in the P85α rescue assays.The data are presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA, two-way ANOVA). NC, negative control; WT, wild-type SEPT2; K74R, SEPT2-K74R mutant; Cr, NaCr treatment at25 mM; sh, short interfering RNA downregulation of p85α expression; oe, p85α overexpression

To identify which the protein degradation pathway K74R mutant regulates P85α level, SNU449 cells with transient SEPT2-74R overexpression were treated with the proteasome inhibitor MG132 and autophagy inhibitor Baf1A. The results showed that MG132 exerted no effect on P85α degradation, while Baf1A slowed P85α degradation in CHX-treated cells, indicating that SEPT2-K74R could enhance P85α stability by influencing its autophagic degradation (Additional file 1: Fig. S12C). Since P85α is a key protein in the PI3K-Akt pathway, we examined the interplay between this pathway and SEPT2-K74R. The results showed that PI3K-Akt pathway activities in SEPT2-K74R cells and SEPT2-K74R mouse tumor tissues were significantly decreased, and we observed a reduced expression of N-cadherin and an increased expression of E-cadherin (Fig. 6C, Additional file 1: Fig. S12D). Furthermore, knocking down P85α in conjunction with NaCr treatment inhibited the invasive capability of SEPT2-WT cells, while this trend was reversed by overexpressing P85α in SEPT2-K74R cells (Fig. 6E, F, Additional file 1: Fig. S13A, B). We also observed that P85α was overexpressed in SEPT2-WT cells after NaCr treatment but not in P85α-knockdown or SEPT2-K74R-overexpressing cells (Fig. 6G). These results collectively indicated that crotonylation could promote EMT through the SEPT2-K74Cr-P85α-AKT pathway.

Crotonylation of SEPT2 was associated with poor prognosis and recurrence in HCC patients

To examine the clinical importance of SEPT2-K74cr in HCC patients, we use the site-specific anti-SEPT2-K74 antibody to do IHC assays in HCC samples. The specificity of this antibody was confirmed by IHC in adherent cells (Additional file 1: Fig. S14A). A total of 126 HCC samples were collected for IHC analysis. Based on the percentage of positively stained cells and color intensity, each sample was given a score of 1 to 4 for SEPT2-K74cr level (Fig. 7A), and the score was then correlated with different clinical parameters. We found that HCC patients with low SEPT2-K74cr level had better prognosis (p < 0.05) (Fig. 7B) and lower recurrence rate (p < 0.05) (Fig. 7C). When correlating with cancer stages, SEPT2-K74cr protein level was found to be higher in patients with early HCC (stage I/II) than in patients with advanced HCC (stage II/III) (p < 0.05) (Fig. 7D, E). Moreover, we observed a significant increase (p < 0.05) of the SEPT2-K74cr level (Fig. 7F) in metastatic HCC tumors compared with primary HCC, but not pan-Kcr (Additional file 1: Fig. S14B). Figure 7G showed a higher SEPT2-K74cr level in bone metastasis tissue compared to its corresponding primary HCC tumor.

Fig. 7figure 7

SEPT2-K74cr may be a potential biomarker for predicting prognosis and HCC recurrence in patients (A) Scores indicate crotonylation of SEPT2-K74cr (K74-Cr) in representative tumor tissues as determined by immunohistochemistry (IHC) staining. Scores were calculated on the basis of the staining intensity and percentage of stained cells. B Patients with high K74-Cr levels had lower overall survival than those with low K74-Cr levels. C Patients with high K74-Cr levels had a higher recurrence rate than those with low K74-Cr levels. D Representative images of tissues with low and high stage pathology as determined by hemoxylin and eosin (H, E) and IHC staining. E Quantitative comparison of the IHC score in the tissues with low and high stage pathology. The data are presented as the means ± SD. *p < 0.05. F Quantitative comparison of the K74-Cr IHC scores in metastasis tissue and primary HCCs in Tissue microarray (HLivH060CD03) (G) Images showing bone metastasis tissue and the corresponding primary HCC tumor after H&E and IHC staining. H Graphic model showing the effect of SEPT2-K74 crotonylation in HCC. K74 crotonylation was negatively regulated by SIRT2, and K74 decrotonylation decreased P85α stability and PI3K pathway activity, which inhibited HCC metastasis. SEPT2-K74 crotonylation can serve as an independent predictor of low patient survival and HCC recurrence

We further assessed the association of SEPT2-K74cr with HCC prognosis. The distribution of patients’ characteristics based on survival and recurrence status were showed in Additional file 1: Table S1 and S2, respectively. By univariate Cox regression analysis (Table 1), BCLC stage and SEPT2-K74cr level were found to be significant prognostic factors of HCC (p < 0.05), whereas low SEPT2-K74cr level was associated with reduced risk of cancer-related death (relative risk [RR], 0.536; 95% confidence interval [CI] 0.293–0.982; p < 0.05). Moreover, by multivariate Cox regression analysis, the SEPT2-K74cr level was found to be an independent predictor for low survival (RR, 0.521; 95% CI 0.285–0.955; p < 0.05) and recurrence in HCC patients (RR, 0.575; 95% CI 0.330–0.999; p = 0.05) (Table 1 and Additional file 1: Tables S1–S3). Altogether, our findings demonstrated that SEPT2-K74cr could be a potential prognostic factor for HCC patients.

Table 1 Cox Regression Analysis of Potential Poor Prognostic Factors for HCC Patients

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