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. 2014 May;390(1-2):19-30.
doi: 10.1007/s11010-013-1950-x. Epub 2014 Jan 10.

MiR-429 inhibits cells growth and invasion and regulates EMT-related marker genes by targeting Onecut2 in colorectal carcinoma

Yingnan Sun  1 , Shourong Shen, Xiaoping Liu, Hailin Tang, Zeyou Wang, Zhibin Yu, Xiayu Li, Minghua Wu
Affiliations

MiR-429 inhibits cells growth and invasion and regulates EMT-related marker genes by targeting Onecut2 in colorectal carcinoma

Yingnan Sun et al. Mol Cell Biochem. 2014 May.

Erratum in

Abstract

The 5-year survival rate for colorectal cancer is approximately 55 % because of its invasion and metastasis. The epithelial-mesenchymal transition (EMT) is one of the well-defined processes during the invasion and distant metastasis of primary epithelial tumors. miR-429, a member of the miR-200 family of microRNAs, was previously shown to inhibit the expression of transcriptional repressors ZEB1/delta EF1 and SIP1/ZEB2, and regulate EMT. In this study, we showed that miR-429 was significantly downregulated in colorectal carcinoma (CRC) tissues and cell lines. We found that miR-429 inhibited the proliferation and growth of CRC cells in vitro and in vivo, suggesting that miR-429 could play a role in CRC tumorigenesis. We also showed that downregulation of miR-429 may contribute to carcinogenesis and the initiation of EMT of CRC by targeting Onecut2. Further researches indicated that miR-429 inhibited the cells migration and invasion and reversed TGF-β-induced EMT changes in SW620 and SW480 cells. miR-429 could reverse the change of EMT-related markers genes induced by TGF-β1, such as E-cadherin, CTNNA1, CTNNB1, TFN, CD44, MMP2, Vimentin, Slug, Snail, and ZEB2 by targeting Onecut2. Taken together, our data showed that transcript factor Onecut2 is involved in the EMT, migration and invasion of CRC cells; miR-429 inhibits the initiation of EMT and regulated expression of EMT-related markers by targeting Onecut2; and miR-429 or Onecut2 is the important therapy target for CRC.

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Figures

Fig. 1
Fig. 1
miR-429 inhibits proliferation and tumorigenesis in CRC cells. a miR-429 downregulated expression in the CRC tissues (10 cases II and III stage CRC tissues and 10 cases corresponding to the adjacent normal tissue) and multiple cell lines (HT-29, SW620, and SW480 cell). **P < 0.01 compared to the expression of CRC tissues. b miR-429 inhibited the cell proliferation of CRC cells for different times in vitro. c miR-429 attenuated CRC growth in mouse xenograft models when aliquots of 40 μl of PBS containing 1 μg of mature miR-429 or scramble were directly injected into the tumor. *P < 0.05; **P < 0.01
Fig. 2
Fig. 2
miR-429 targets transcript factor Onecut2 and is inversely correlated with the mRNA or protein levels of Onecut2 in CRC tissues. a Luciferase assay on SW620 cells, which were cotransfected with miR-429 and a luciferase reporter containing the full length of Onecut2 3′-UTR (Luc-wt) or a mutant (Luc-mut) in which the four nucleotides of the miR-429-binding site were mutated. Luciferase activities were measured 48-h post transfection. miR-429 markedly suppressed luciferase activity in Luc-wt reporter constructs. The data are mean ± SD for separate transfections (n = 6). b Onecut2 mRNA level was analyzed upon miR-429 or scramble transfection in HT-29, SW620, and SW480 cells by real-time PCR. All data are shown as mean ± SD. **P < 0.01. c miR-429 transfection affects Onecut2 protein levels by Western blot analysis. HT-29, SW620, and SW480 cells were transfected with miR-429 or scramble control. d Representative expression levels of miR429 and Onecut2 mRNA by real-time PCR in clinical specimens (n = 50), and the correlation between miR-429 and Onecut2 was analyzed by Spearman’s correlation test. e ISH detects the position and expression of miR-429, and IHC detects the position and expression of Onecut2 in CRC (103 cases) relative to paracancerous normal tissues (103 cases). Scale bars 100 μm
Fig. 2
Fig. 2
miR-429 targets transcript factor Onecut2 and is inversely correlated with the mRNA or protein levels of Onecut2 in CRC tissues. a Luciferase assay on SW620 cells, which were cotransfected with miR-429 and a luciferase reporter containing the full length of Onecut2 3′-UTR (Luc-wt) or a mutant (Luc-mut) in which the four nucleotides of the miR-429-binding site were mutated. Luciferase activities were measured 48-h post transfection. miR-429 markedly suppressed luciferase activity in Luc-wt reporter constructs. The data are mean ± SD for separate transfections (n = 6). b Onecut2 mRNA level was analyzed upon miR-429 or scramble transfection in HT-29, SW620, and SW480 cells by real-time PCR. All data are shown as mean ± SD. **P < 0.01. c miR-429 transfection affects Onecut2 protein levels by Western blot analysis. HT-29, SW620, and SW480 cells were transfected with miR-429 or scramble control. d Representative expression levels of miR429 and Onecut2 mRNA by real-time PCR in clinical specimens (n = 50), and the correlation between miR-429 and Onecut2 was analyzed by Spearman’s correlation test. e ISH detects the position and expression of miR-429, and IHC detects the position and expression of Onecut2 in CRC (103 cases) relative to paracancerous normal tissues (103 cases). Scale bars 100 μm
Fig. 3
Fig. 3
Effect of miR-429 on cells mobility and invasion by target Onecut2 in CRC cells. a Wound-healing assay analyzes the effect of miR-429 or si-Onecut2 on SW620 cells migration. *P < 0.05. b Wound-healing assay analyzes the rescued effect of Onecut2 overexpression on inhibitory healing induced by miR-429. *P < 0.05. c Matrigel invasion assay analyzes the rescued effect of Onecut2 overexpression on invasion inhibition induced by miR-429. **P < 0.01
Fig. 4
Fig. 4
The morphology observation that miR-429 or siOnecut2 inhibits EMT induced by TGF-β in SW620 cells (a) and SW480 cells (b)
Fig. 5
Fig. 5
miR-429 regulates the expression of EMT-related marker genes by targeting Onecut2 in CRC cells. a miR-429 rescued the expression alteration of EMT-related marker genes induced by TGF-β1 in SW620 cells. *P < 0.05. b si-Onecut2 rescued the expression alteration of EMT-related marker genes induced by TGF-β1 in SW620 cells. *P < 0.05
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