MiR-200b-3p is a post-transcriptional regulator of PRDX2
To identify miRNAs targeting PRDX2, bioinformatic algorithms, including TargetscanHuman.7.0, DIANA LAB and miRanda, were used to screen for miRNA candidates. As shown in Fig. 1a, miR-1228-3p, miR-200b-3p, miR-200c-3p and miR-429 were agreed by all three algorithms. To further narrow down miRNA candidates, we measured each miRNA expression level and PRDX2 protein level in six CRC cell lines with different metastatic potentials. Western-blot analysis showed that PRDX2 protein level was higher in the metastatic cell lines (SW620 and LoVo) than in the pre-invasive cell lines (SW480, Caco2, HT29 and HCT116) (Fig. 1b). Conversely, qPCR showed that miR-1228-3p and miR-200b-3p had lower expression levels in the metastatic cell lines than in the pre-invasive cell lines (Fig. 1c). Pearson correlation analysis showed that only miR-200b-3p expression level was significantly inverse correlation with PRDX2 protein level(r = − 0.977, p = 0.001, Fig. 1d), suggesting that miR-200b-3p might be a negative regulator of PRDX2.
To confirm whether miR-200b-3p regulates PRDX2 negatively, we constructed pmirGLO-3′UTRs of PRDX2 luciferase vectors (Fig. 1e). Reporter assays showed that ectopic miR-200b-3p expression dramatically suppressed the luciferase activity of wild-type (wt) PRDX2 3′UTR in 293T cells and LoVo cells, while it did not suppress the luciferase activity of mutant-type (mut) PRDX2 3′UTR (Fig. 1f). Consistent with results of reporter assays, we found ectopic miR-200b-3p reduced PRDX2 protein level (Fig. 1g). These results showed that miR-200b-3p targeted PRDX2 3′UTR and disrupted its protein expression.
MiR-200b-3p represses oncogenic properties of CRC cells by targeting PRDX2 in vitro
To investigate the effects of miR-200b-3p, we established LoVo/miR cells stably expressing miR-200b-3p, LoVo/miR + PRDX2 cells stably co-expressing miR-200b-3p and nontargetable PRDX2 and SW480/Zip-miR cells stably silencing miR-200b-3p (Additional file 1: Figure S1a). CCK8 proliferation assays showed that miR-200b-3p overexpression inhibited CRC cell proliferation, whereas miR-200b-3p silencing promoted CRC cell proliferation (Additional file 1: Figure S1b). Similarly, transwell invasive assays showed that miR-200b-3p overexpression dramatically inhibited invasive behavior of LoVo cells (Fig. 2a), while miR-200b-3p silencing showed the opposite effect in SW480 cells (Fig. 2b). Noticeably, miR-200b-3p overexpression reduced frequencies of cells with fibroblastic or spindle-like morphology and concomitantly increased frequencies of cobblestone-like cells (Fig. 2c). In contrast, miR-200b-3p silencing showed the opposite effect (Fig. 2d). This suggested that miR-200b-3p might inhibit CRC cell EMT. Further supporting this notion, miR-200b-3p overexpression increased the expression of the epithelial marker E-cadherin and decreased the expression of the mesenchymal markers N-cadherin and vimentin, and vice versa (Fig. 2e, f). Importantly, these suppressive effects of miR-200b-3p on malignant behaviors of LoVo cells were substantially weakened by the nontargetable PRDX2 (Fig. 2a, c, e), suggesting that PRDX2 is a functional target of miR-200b-3p in regulating biological behaviors of CRC cells in vitro.
MiR-200b-3p suppresses growth, invasion and metastasis of CRC cells by targeting PRDX2 in vivo
Based on our in vitro results, we speculated that miR-200b-3p might be involved in regulating biological behaviors of CRC cells in vivo. To test it, we injected LoVo/miR, LoVo/miR + PRDX2 and SW480/Zip-miR cells subcutaneously in mice. We found that mice with LoVo/miR cells injection displayed inhibited tumor growth (Additional file 1: Figure S1c, d), while mice with SW480/Zip-miR cells injection showed the opposite effect (Additional file 1: Figure S1e). Intriguingly, the subcutaneous tumors derived from LoVo/miR cells were well encapsulated in false fibrous membrane (Fig. 2g), suggesting that miR-200b-3p could inhibit invasive behavior of CRC cell in vivo. To model metastatic process of CRC, we transplanted the subcutaneous tumor in the mesentery at the distal end of caecum of nude mice. We found that mice with miR-200b-3p overexpressing tumors developed fewer metastatic nodules in intestine and liver than control mice with tumors of similar size (Fig. 3a–c). In contrast, mice with miR-200b-3p silencing tumors showed the more metastatic nodules than control mice (Fig. 3d–f). Unexpectedly, a lung metastasis was observed under microscope in a mouse with miR-200b-3p silencing tumor (Additional file 1: Figure S1f). Importantly, the nontargetable PRDX2 rescued the miR-200b-3p-induced repression of CRC cell growth, invasion and metastasis in vivo (Figs. 2g, 3a–c and Additional file 1: Figure S1c, d), suggesting that PRDX2 is sufficient to mediate the effects of miR-200b-3p on regulating biological behaviors of CRC cells in vivo.
MiR-200b-3p is transcriptionally regulated by c-Myc
Growing studies have shown the regulation of miRNAs’ expression by transcription factors [23, 24]. As described in the previous study [24], we chose 2-kb region directly upstream of miR-200b-3p stem loop as the putative promoter. Possible binding motifs of c-Myc were found in the putative promoter of miR-200b-3p using Consite (http://consite.genereg.net/) on line database. An inverse correlation between c-Myc protein and miR-200b-3p was observed in six CRC cells (r = − 0.908, p = 0.012, Fig. 1c and Additional file 2: Figure S2a). Luciferase report assay showed that c-Myc strongly repressed the activity of the miR-200b-3p promoter in 293T and SW480 cells (Fig. 4a and Additional file 2: Figure S2b). ChIP assay showed that c-Myc bound to the region 1 (R1, − 371 to − 210 bp) and region 2 (R2, − 993 to − 830 bp) of the miR-200b-3p promoter (Fig. 4b). Further supporting the role of c-Myc in regulating miR-200b-3p expression, we found that overexpression of c-Myc inhibited miR-200b-3p expression level (Additional file 2: Figure S2c) in CRC cells. These results indicated that c-Myc bound to the promoter of miR-200b-3p and suppressed its expression.
c-Myc strengthens malignant capacities of CRC cells by repressing miR-200b-3p in vitro and in vivo
To determine whether miR-200b-3p was responsible for the effects of c-Myc on CRC progression, we established SW480/c-Myc cells stably expressing c-Myc and SW480/c-Myc + miR cells stably co-expressing c-Myc and miR-200b-3p (Additional file 2: Figure S2d). We found that overexpression of c-Myc in SW480 cells enhanced cell invasiveness and EMT in vitro, which was dampened by re-expression of miR-200b-3p (Fig. 4c, d and Additional file 3: Figure S3a, b). In accord with in vitro results, in vivo metastatic assays demonstrated that overexpression of c-Myc strongly promoted proliferation and metastasis of SW480 cells, while the promoting effects could be rescued by re-expression of miR-200b-3p, at least partly (Fig. 4e–g and Additional file 3: Figure S3c, d). These results showed c-Myc/miR-200b-3p signal pathway was involved in regulating aggressive behaviors of CRC cells in vitro and in vivo.
MiR-200b-3p counteracts c-Myc and disrupts its protein stability by inhibiting AKT2/GSK3β pathway
Our findings reveal that the c-Myc/miR-200b-3p/PRDX2 regulatory axis plays an important role in regulating CRC progression. Interestingly, we noticed that c-Myc protein level in SW480/c-Myc cells was decreased after re-expression of miR-200b-3p (Additional file 2: Figure S2d). The same phenomenon was also observed in Caco2 cells (Fig. 5a), suggesting that miR-200b-3p was involved in regulation of c-Myc protein expression. Based on bioinformatic analysis, the possibility that direct effect of miR-200b-3p on c-Myc by binding to its 3′UTR was ruled out (data not shown). Recent studies show that altered phosphorylation status of c-Myc protein at S62 and T58 residues affects the protein stability [25, 26]. The phosphorylation at T58 residue initiates c-Myc ubiquitylation, which contributes to the protein degradation. GSK3β has been reported to play an important role in c-Myc protein phosphorylation modification [27]. Therefore, we hypothesized GSK3β could mediate the effect of miR-200b-3p on regulation of c-Myc protein stability. As expected, our results demonstrated that miR-200b-3p silencing increased Ser9 phosphorylation of GSK3β without altering total GSK3β protein level (Fig. 5b). MiR-200b-3p silencing also decreased T58 phosphorylation but increased S62 phosphorylation of c-Myc protein (Fig. 5b). Conversely, miR-200b-3p overexpression showed the opposite changes of phosphorylation in GSK-3β and c-Myc proteins (Fig. 5b). Noticeably, following treating for 72 h with Tws119, a selective GSK-3β inhibitor, we found that the increased Ser9 phosphorylation of GSK3β and S62 phosphorylation of c-Myc, and the decreased T58 phosphorylation of c-Myc induced by miR-200b-3p silencing were well abolished (Fig. 5b).
Unexpectedly, miR-200b-3p overexpression decreased total Protein Kinase B 1/2 (AKT1/2) protein levels, particularly total AKT2 protein level, and vice versa (Fig. 5c). AKT2 represses GSK3β activity by increasing the phosphorylation level of GSK3β protein at Ser9 residues [28], and has been reported to be upregulated in CRC tissues compared to normal colon mucosa and promote tumor progression [29, 30]. The opposite effects between AKT2 and miR-200b-3p on CRC progression made us investigate the potential interaction between them. Indeed, bioinformatic analysis showed a conserved complementary sequence of miR-200b-3p present in AKT2 mRNA 3′UTR, suggesting AKT2 was a potential target of miR-200b-3p (Fig. 5d). Luciferase reporter assay showed ectopic miR-200b-3p expression inhibited the activity of wt 3′UTR of AKT2, but failed to inhibit the activity of mut 3′UTR of AKT2 in 293T cells (Fig. 5e). Moreover, we found that miR-200b-3p overexpression decreased total AKT2 protein and AKT2 Ser474 phosphorylation levels, which led to deceased Ser9 phosphorylation of GSK3β, increased T58 phosphorylation and deceased S62 phosphorylation of c-Myc, and a decrease in c-Myc protein level (Fig. 5f). Our results demonstrated that miR-200b-3p disrupted c-Myc protein stability through inhibiting AKT2/GSK3β pathway, suggesting that a regulatory loop consisting of the c-Myc/miR-200b-3p/PRDX2 axis and AKT2/GSK3β pathway was involved in regulating CRC progression.
The expression of c-Myc, miR-200b-3p and PRDX2 is disrupted in human CRC tissues and their expression levels are associated with clinicopathological features and survival of CRC patients
Having uncovered the important involvement of the c-Myc/miR-200b-3p/PRDX2 regulatory loop in CRC progression, we speculated that their expression profiles might be perturbed in human CRC tissues. To test it, we assessed c-Myc, miR-200b-3p and PRDX2 expression profiles in 97 cases of paired CRC samples. IHC results showed c-Myc and PRDX2 protein levels were frequently higher in CRC tissues than in adjacent normal mucosa tissues (Fig. 6a, b and Additional file 4: Figure S4b). These samples were then scored based on c-Myc and PRDX2 staining extent (Additional file 4: Figure S4a) for survival analysis. As shown in Fig. 6c, d, the overall survival of CRC patients with low c-Myc or PRDX2 score (0 or 1) was significantly longer than that of CRC patients with high c-Myc or PRDX2 score (2 or 3). qPCR analysis showed that miR-200b-3p expression was downregulated in most CRC tissues compared to in PNCM tissues (Fig. 6e) and its expression level was negatively correlated with c-Myc and PRDX2 protein levels (Additional file 4: Figure S4c, d). To evaluate the effect of miR-200b-3p on overall survival of CRC patients, we stratified all cases into two groups (low miR-200b-3p expression: less than 60% expression of the PNCM tissue, high miR-200b-3p expression: more than and equal to 60% expression of the PNCM tissue) according to their miR-200b-3p level (Additional file 4: Figure S4e). As shown in Fig. 6f, CRC patients with high miR-200b-3p expression had a longer overall survival than CRC patients with low miR-200b-3p expression. Moreover, we found that c-Myc, miR-200b-3p and PRDX2 expression levels were well associated with tumor differentiation, size and pathological Tumor-Node-Metastasis (pTNM) stage (Additional file 5: Table S1).
Disruption of the c-Myc/miR-200b-3p/PRDX2 regulatory loop enhances chemotherapeutic resistance of CRC cells
Chemotherapeutic resistance is associated with metastasis [21]. We proposed that disruption of the c-Myc/miR-200b-3p/PRDX2 loop might enhance resistance of CRC cells to chemotherapy. As expected, We found that the half inhibitory concentration (IC50) of SW480/c-Myc cells to oxaliplatin (OXL), the most commonly used drug in CRC chemotherapy, was dramatically higher than that of the control SW480 cells (10.39 μM vs. 6.365 μM), while re-expression of miR-200b-3p partly abolished the increased resistance to oxaliplatin (7.054 μM vs. 10.39 μM), as shown in Fig. 7a. We also found that LoVo/miR cells had decreased resistance to oxaliplatin compared to the control LoVo cells (4.859 μM vs. 8.521 μM), while the decreased resistance was restored by nontargetable PRDX2 (4.859 μM vs. 7.325 μM, Fig. 7b). Moreover, we investigated the effect of the loop on drug-induced cytotoxicity. After CRC cells were treated with 5 μM of oxaliplatin for 72 h, we found that overexpression of c-Myc reduced apoptosis ratios compared to the control, while re-expression of miR-200b-3p abrogated the reduced effects (Fig. 7c, e). We also found that miR-200b-3p overexpression increased apoptosis ratios, while the increased effect was rescued by nontargetable PRDX2 (Fig. 7d, f). These findings indicated that perturbation of the c-Myc/miR-200b/PRDX2 regulatory loop increased chemotherapeutic resistance.