Resveratrol restrains colorectal cancer metastasis by regulating miR-125b-5p/TRAF6 signaling axis

Abstract

Colorectal cancer is one of the most common malignancies with a high incidence, metastatic tendency and low 5-year survival rate. Resveratrol, a polyphenolic compound has been shown to inhibit colorectal cancer metastasis in recent studies. Its underlying molecular mechanism remains to be elucidated. Our findings demonstrated that miR-125b-5p, acting as a tumor suppressor, was conspicuously down-regulated in both colorectal cancer tissues and cell lines. The expression of miR-125b-5p negatively correlated with the expression of its direct target TNF receptor associated factor 6 (TRAF6). Both miR-125b-5p overexpression and TRAF6 knockdown inhibited metastasis of colorectal cancer cells. In addition, we uncovered that resveratrol up-regulated miR-125b-5p by increasing its stability and suppressed TRAF6-induced signal pathway in a dose/time-dependent manner. Resveratrol could significantly curtail the migration and invasion of colorectal cancer cells, which was counteracted by miR-125b-5p knockdown or TRAF6 overexpression. These results indicated that resveratrol could restrain colorectal cancer metastasis by promoting miR-125b-5p/TRAF6 signaling axis. Furthermore, lung metastasis models of colorectal cancer were constructed by tail vein injection. Down-regulation of miR-125b-5p could facilitate colorectal cancer metastasis in vivo, which could be impeded by resveratrol. In conclusion, our findings delineated the miR-125b-5p/TRAF6 signaling axis as a novel molecular mechanism underlying the metastatic process in colorectal cancer, as well as a prospective therapeutic target. Resveratrol disrupts colorectal cancer metastasis by activating miR-125b-5p/TRAF6 signal pathway and might improve the clinical outcome of colorectal cancer patients with low expression of miR-125b-5p.

Keywords: Resveratrol; TRAF6; colorectal cancer; metastasis; miR-125b-5p.

Figure 1

MiR-125b-5p suppressed metastasis and proliferation of colorectal cancer and was under-expressed in colorectal cancer. A. TCGA bioinformatics analysis showed that the expression of miR-125b-5p gene was relatively low in tumor tissues. B. The expression of miR-125b-5p in normal colorectal epithelial cell line (NCM460) and five colorectal cancer cell lines (HCT-8, HCT-116, SW480, SW620, and RKO) was analyzed by qRT-PCR. C. HCT-8 cells were transfected with 50 nM miR-125b-5p mimics or miR-125b-5p inhibitors. D. Colony formation assay revealed that miR-125b-5p inhibited colony formation ability. E, F. Scratch wound healing assay and trans-well assay showed that miR-125b-5p inhibited the metastasis of HCT-8 cell line. G, H. The expression of E-cadherin (E-cad), N-cadherin (N-cad) and SNAIL after overexpression of miR-125b-5p was detected by qRT-PCR and western blotting (WB). I, J. Scratch wound healing assay and trans-well assay were performed to analyzed cells migration and invasion. K, L. WB and qRT-PCR indicated that low expression of miR-125b-5p promoted EMT in HCT-8 cell line. All experiments were independently repeated at least three times, and the data were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant (*, P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 2

MiR-125b-5p targeted TRAF6 and reduced its expression in a time-dependent manner. A. Public database searches by TargetScan, miRWark, miRBD, and starBase predicted TRAF6 as a target of miR-125-5p. B, C. Dual luciferase reporter assay was performed to detect the binding of miR-125b-5p and TRAF6 mRNA 3’-UTR. D, E. WB and qRT-PCR indicated that miR-125b-5p suppressed the expression of TRAF6 in HCT-8 cell line. F, G. WB and qRT-PCR showed that miR-125b-5p mimics reduced the expression of TRAF6 in a time-dependent manner. H, I. WB showed that miR-125-5p inhibited AKT/GSK-3β pathway. All experiments were independently repeated at least three times, and the data were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant (*, P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 3

TRAF6 promoted colorectal cancer cell metastasis through activating AKT/GSK-3β/SNAIL pathway. A. WB and qRT-PCR showed that the expression of TRAF6 in five colorectal cancer cell lines was higher than that in normal intestinal epithelial cell lines. B. HCT-8 cell were transfected with OE-TRAF6 and HCT-116 cell were transfected with siRNAs. The TRAF6 level was analyzed by qRT-PCR and WB. C. The proliferation ability of the cells was detected by clonal formation assay. D-G. TRAF6 promoted cell migration and invasion in scratch wound healing assay and trans-well assay. H, I. SNAIL, E-cad and N-cad were detected by qRT-PCR. J, K. WB showed that TRAF6 promoted EMT through AKT/GSK-3β pathway. L. Trans-well migration assay indicated that co-transfection of miR-125b-5p inhibitor and OE-TRAF6 abrogated the migration ability of HCT-8 cell line. All experiments were independently repeated at least three times, and the data were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant (*, P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 4

Resveratrol could promote the stability of miR-125b-5p and inhibited the TRAF6/AKT/GSK-3β pathway. A. Cell Counting Kit-8 assay was performed to investigate the cytotoxic effects of resveratrol on HCT-8, HCT-116 and RKO cell lines. B. The expression of miR-125b-5p was detected by qRT-PCR after resveratrol treatment. C, D. qRT-PCR results showed that resveratrol increased the expression of miR-125b-5p in a dose- and time-dependent manner. E. Decay of miR-125-5p was monitored in HCT-8 cell co-treated with Actinomycin D and resveratrol. F. The expression of TRAF6 was detected after resveratrol treatment with different concentration. G. WB showed that resveratrol inhibited the TRAF6/AKT/GSK-3β pathway in a dose-dependent manner. All experiments were independently repeated at least three times, and the data were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant (*, P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 5

Low expressed miR-125b-5p could antagonize the inhibitory effect of resveratrol on colorectal cancer metastasis. A. The scratch wound healing assay revealed that the Res group inhibited the migration of colorectal cancer cells, and the inhibitory effect was impeded by co-administrating with miR-125b-5p inhibitor. B. The ability of migration and invasion of cells was detected by trans-well migration and invasion assay. C, D. HCT-8 cells were treated with miR-125-5p inhibitor, resveratrol or their combination. The expression of RNA or protein was analyzed by qRT-PCR or WB, respectively. All experiments were independently repeated at least three times, and the data were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant (*, P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 6

Overexpression of TRAF6 antagonized the inhibitory effect of resveratrol on colorectal cancer metastasis. A. The scratch wound healing assay revealed that the Res group inhibited the migration of colorectal cancer cells, and the inhibitory effect was impeded by co-administrating with OE-TRAF6. B. The ability of migration and invasion of cells was detected by trans-well migration and invasion assay. C, D. HCT-8 cells were treated with OE-TRAF6, resveratrol or their combination. The expression of RNA or protein was analyzed by qRT-PCR or WB, respectively. All experiments were independently repeated at least three times, and the data were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant (*, P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 7

Resveratrol antagonized the promotion effect of low expressed miR-125b-5p on colorectal cancer metastasis in vivo. A. Live fluorescence imaging revealing lung metastasis of colorectal cancer in mice administered different treatments. B. Total fluxes of mouse fluorescence signals were analyzed among different groups. C. H&E staining revealed histopathological changes of tumor foci in lung tissue. D. The relative proportion of tumors in lung tissue was calculated after H&E staining. E. Immunohistochemical staining assays were conducted to evaluate the expression of Ki-67 in mouse tumor tissues. F-H. Liver and kidney function indexes were detected in mouse serum, and there was no significant difference among all groups. I. H&E staining showed no significant changes in liver, kidney and spleen morphology of the four groups. P < 0.05 was considered statistically significant (*, P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 8

Resveratrol suppresses the metastasis of colorectal cancer by regulating miR-125b/TRAF6/GSK-3β/AKT/SNAIL pathway.