miRNAs are a group of endogenous, small (18–25-nucleotide long), and non-coding RNA molecules that regulate the expression of specific mRNAs by either translational inhibition or mRNA degradation5. More than 50% of miRNAs are reported to be in cancer-associated genomic break points and function as tumor suppressors or oncogenic molecules6. Among the cancers, most CRCs form from the normal mucosa through the adenoma stage, which is accompanied by numerous gene mutations. Our study has screened circulating mmu-miR-762 expression in mice with CRC CT26 cell implantation. Further study demonstrate that CT26 cells transfected with mmu-miR-762 mimic promoted the cell viability and invasion/EMT of CRC cells. A similar result was also seen in human subjects. Expression of mmu-miR-762 in circulation was significantly higher in clinical CRC patients than in control donors. We subsequently confirmed that mmu-miR-762 significantly increased in distant metastasis of CRC, resulting in poor prognosis. Our study of the biological roles of miR-762 in CRC development revealed Wnt/β-catenin as a downstream signaling pathway. These findings suggest that miR-762 plays a fundamental role in colorectal cancer tumorigenesis by affecting cancer cell proliferation and invasion/EMT.
miR-762 is upregulated in radiation-induced tumors in mice and may affect the pathways involved in apoptosis in this context15. Previous studies reported abnormal miRNA expression in numerous cancers, and miR-762 overexpression has been reported in breast cancer and ovarian cancer, where it promoted cancer cell growth and metastasis16,17. A previous study also demonstrated an association between miR-762 expression and oral carcinogenesis18. However, no studies have examined the expression and function of circulating miR-762 in CRC. In this study, CRC cell line with mmu-miR-762 transfection was frequently up-regulated the cell viability and EMT, as well as has-miR-762 detected in serum extracted from CRC patients. Overexpression of mmu-miR-762 enhanced CRC cell proliferation and invasion. The ability of miR-762 to promote cell proliferation and invasion was confirmed by both overexpression and down-regulation experiments in vitro. Therefore, our results suggest that miR-762 is a novel oncogenic RNA in CRC.
To explore the molecular mechanisms by which miR-762 enhances CRC cell growth and invasion, Wnt/β-catenin was identified as a direct target of mmu-miR-762 in CRC cells. First, the Wnt/β-catenin signaling pathway was predicted to be regulated by miR-76219,20. Second, western blot analysis showed that the miR-762 mimic increased the expression of Wnt-1 and β-catenin. Third, the cytosolic and nuclear levels of transcription factor β-catenin were both up-regulated in miR-762 mimic-transfected cells. However, CT26 cells transfected with the mimic and inhibitor of miR-762 did not show altered sub-G1 levels in CT26 cells. The cell cycle was arrested at G0/G1 phase in cells treated with high concentration (150 nM) of miR-762 inhibitor. Additionally, MMP-9 was not changed in either mimic- and inhibitor-transfected cells. These data suggest that mmu-miR-762 regulates CRC CT26 cell viability and invasion/EMT might through the Wnt/β-catenin pathway, but does not regulate cell cycle and apoptosis.
Although miR-762 is overexpressed in several cancers, no studies have investigated miR-762 in CRC. In this study, real-time PCR analysis of human subjects confirmed that the expression of serum has-miR-762 in CRC patients was much higher than that in control donors. There was also an association between serum has-miR-762 expression and CRC patients with distant metastasis. miR-762 exerted its regulatory effects on cell proliferation and invasion/EMT by affecting the Wnt/β-catenin signaling pathway. β-Catenin can influence metastasis through its effects on the EMT21. An article reported that FOXP3 interacts with β-catenin could induce transcription of Wnt target genes to promote cell proliferation, invasion and EMT induction22. The present study demonstrated a similar result in CRC that miR-762 promoted CRC cell metastasis by promoting the Wnt/β-catenin pathway according to the results of the Transwell invasion assay and EMT molecules (vimentin and E-cadherin) by Western blotting.
To confirm that human miR-762 can regulate Wnt pathway as well as mouse miR-762, we used the TargetScan microRNA target prediction which algorithm is considered the conservation among species. The target prediction results were listed of both human hsa-miR-762 target (supplementary table S1) and murine mmu-miR-762 target prediction (supplementary table S2), demonstrating that the Wnt ligands were regulated by miR-762 in both human and murine. In human, has-miR-762 has predicted to suppress Wnt family such as 7B, 2B, 3A, 9A, 5A, 10B, 3, 7A, and 11. In mmu-miR-762, it has predicted to suppress Wnt ligands 9B, 7B, 6, 10B, 10A, 4, 3A, 7A, and 8. Among these, Wnt-7B, -3A, and − 10B have the genetic conservation of miR-762 binding site between human and murine 3’-UTR. Although the miR-762 has certain different target specify among human and mice, they still share some intersection in Wnt signaling. In the future, further analysis of the regulation of circulating miR-762 on cancer promotion and progression in CRC patients will be advanced elucidated.
Hou et al. (2017) reported that miR-762 can downregulate the expression of a tumor suppressor protein menin through a binding site in its 3'-UTR and consequently upregulate the Wnt cell signaling pathway to promote the development of ovarian cancer17. Our present study first revealed that circulating miR-762 expression may be useful as a biomarker for diagnosing CRC and higher serum miR-762 expression-caused distant metastasis of patients with CRC. While patients with high expression of miR-762 may have shorter survival times than those with low expression of miR-762, survival analysis could not be performed because of the relatively small sample size and short time. Furthermore, the associations between serum miR-762 and tumor size and lymph node invasion were not thoroughly investigated. Therefore, further large-scale studies are necessary to investigate whether serum miR-762 levels can be used as a predictive and a prognostic factor of patients with CRC.