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MicroRNA-490-3p inhibits colorectal cancer metastasis by targeting TGFβR1.

Xu X, Chen R, Li Z, Huang N, Wu X, Li S, Li Y, Wu S - BMC Cancer (2015)

Bottom Line: Artificially overexpression miR-490-3p in CRC cell lines inhibited cell migration and invasion abilities while knockdown miR-490-3p expression caused the reverse effects.Inhibition of TGFβR1 could partially recover the tumor suppression effect of miR-490-3p. miR-490-3p is downregulated during CRC malignant progression. miR-490-3p represses CRC cell migration and invasion abilities, partially by targeting to the TGF-β signaling pathway.Taken together, miR-490-3p is acting as a tumor suppressor in CRC.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. xxh@gzhmu.edu.cn.

ABSTRACT

Background: Colorectal cancer (CRC) is one of the most common malignances worldwide. Metastasis is responsible for the rapid recurrence and poor prognosis of CRC. However, the underlying molecular mechanism of CRC metastasis remains largely unclear. In this study we purposed to investigate the expression and biological functions of miR-490-3p in CRC metastasis, as well as to identify its downstream target genes and influenced pathway.

Methods: The expression level of miR-490-3p in CRC cell lines, CRC adjacent normal tissues, non-metastasis and metastasis tissues were assessed by quantitative real-time PCR. Patient survivals were follow-up up to 7 years. Gain-of-function and loss-of-function study on cell migration and invasion abilities were carried out by transfection of miR-490-3p mimics or inhibitors respectively. The molecular targets of miR-490-3p were computationally identified and experimentally verified by dual-luciferase reporter assay and western blot. Functional rescue was also conducted to confirm miR-490-3p inhibits CRC metastasis by targeting TGF-β signaling pathway.

Results: miR-490-3p expression was persistently downregulated during CRC malignant progression, as well as in CRC cell lines. Artificially overexpression miR-490-3p in CRC cell lines inhibited cell migration and invasion abilities while knockdown miR-490-3p expression caused the reverse effects. TGFβR1 and MMP2/9 were the downstream targets of miR-490-3p in CRC. Inhibition of TGFβR1 could partially recover the tumor suppression effect of miR-490-3p.

Conclusion: miR-490-3p is downregulated during CRC malignant progression. miR-490-3p represses CRC cell migration and invasion abilities, partially by targeting to the TGF-β signaling pathway. Taken together, miR-490-3p is acting as a tumor suppressor in CRC.

No MeSH data available.


Related in: MedlinePlus

miR-490-3p targets TGFβR1 and MMP2/9 in CRC cells. a TGFβR1 was predicted to be a theoretical miR-490-3p target. Complete base pairing in seed region of TGFβR1 3‘UTR was disrupted in constructed mutant plasmid to verify direct interaction. b Western blot results showed that overexpression of miR-490-3p in CRC cell lines decreased the protein level of TGFβR1, and knockdown expression of miR-490-3p increased the protein level of TGFβR1 inversely. c Dual-luciferase reporter assay indicated miR-490-3p bound to TGFβR1 3‘UTR directly. d Disruption of seed region base pairing blocked the interaction between miR-490-3p and TGFβR1. e Quantitative real-time PCR analysis suggested MMP2 and MMP9 were the downstream responses of miR-490-3p interference. f The relative miR-490-3p expression and TGFβR1 protein level in 8 CRC tissues were quantified by real-time PCR and western blot respectively. Linear regression analysis of them revealed a strong negative correlation. *: A two-tailed t-test P-value <0.05
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Fig5: miR-490-3p targets TGFβR1 and MMP2/9 in CRC cells. a TGFβR1 was predicted to be a theoretical miR-490-3p target. Complete base pairing in seed region of TGFβR1 3‘UTR was disrupted in constructed mutant plasmid to verify direct interaction. b Western blot results showed that overexpression of miR-490-3p in CRC cell lines decreased the protein level of TGFβR1, and knockdown expression of miR-490-3p increased the protein level of TGFβR1 inversely. c Dual-luciferase reporter assay indicated miR-490-3p bound to TGFβR1 3‘UTR directly. d Disruption of seed region base pairing blocked the interaction between miR-490-3p and TGFβR1. e Quantitative real-time PCR analysis suggested MMP2 and MMP9 were the downstream responses of miR-490-3p interference. f The relative miR-490-3p expression and TGFβR1 protein level in 8 CRC tissues were quantified by real-time PCR and western blot respectively. Linear regression analysis of them revealed a strong negative correlation. *: A two-tailed t-test P-value <0.05

Mentions: By using a popular miRNA targets prediction algorithm TargetScan, TGFβR1 was predicted to be a theoretical target of miR-490-3p (Fig. 5a), which miRNA recognition element (MRE) in 3′UTR was conserved among mammals. Western blot results showed that overexpression of miR-490-3p in CRC cell lines decreased the protein level of TGFβR1, and knockdown expression of miR-490-3p increased the protein level of TGFβR1 inversely (Fig. 5b). Dual-luciferase reporter assay indicated that miR-490-3p suppressed TGFβR1 expression by direct binding to the MRE (Fig. 5c), while mutants in the seed region disrupted their interaction (Fig. 5d). MMP2 and MMP9 have been previously reported to be direct target genes of miR-490-3p in ovarian cancer [8]. Here we found that MMP2 and MMP9 mRNA expression was downregulated after overexpression of miR-490-3p and upregulated after knockdown of miR-490-3p (Fig. 5e), confirming the interactions between them. Moreover, in eight CRC patient tissue samples, a significant reversed correlation between miR-490-3p expression and TGFβR1 protein level had been identified (Fig. 5f, r = −0.92, P < 0.05), which further proved the true inhibitory relationship.Fig. 5


MicroRNA-490-3p inhibits colorectal cancer metastasis by targeting TGFβR1.

Xu X, Chen R, Li Z, Huang N, Wu X, Li S, Li Y, Wu S - BMC Cancer (2015)

miR-490-3p targets TGFβR1 and MMP2/9 in CRC cells. a TGFβR1 was predicted to be a theoretical miR-490-3p target. Complete base pairing in seed region of TGFβR1 3‘UTR was disrupted in constructed mutant plasmid to verify direct interaction. b Western blot results showed that overexpression of miR-490-3p in CRC cell lines decreased the protein level of TGFβR1, and knockdown expression of miR-490-3p increased the protein level of TGFβR1 inversely. c Dual-luciferase reporter assay indicated miR-490-3p bound to TGFβR1 3‘UTR directly. d Disruption of seed region base pairing blocked the interaction between miR-490-3p and TGFβR1. e Quantitative real-time PCR analysis suggested MMP2 and MMP9 were the downstream responses of miR-490-3p interference. f The relative miR-490-3p expression and TGFβR1 protein level in 8 CRC tissues were quantified by real-time PCR and western blot respectively. Linear regression analysis of them revealed a strong negative correlation. *: A two-tailed t-test P-value <0.05
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Fig5: miR-490-3p targets TGFβR1 and MMP2/9 in CRC cells. a TGFβR1 was predicted to be a theoretical miR-490-3p target. Complete base pairing in seed region of TGFβR1 3‘UTR was disrupted in constructed mutant plasmid to verify direct interaction. b Western blot results showed that overexpression of miR-490-3p in CRC cell lines decreased the protein level of TGFβR1, and knockdown expression of miR-490-3p increased the protein level of TGFβR1 inversely. c Dual-luciferase reporter assay indicated miR-490-3p bound to TGFβR1 3‘UTR directly. d Disruption of seed region base pairing blocked the interaction between miR-490-3p and TGFβR1. e Quantitative real-time PCR analysis suggested MMP2 and MMP9 were the downstream responses of miR-490-3p interference. f The relative miR-490-3p expression and TGFβR1 protein level in 8 CRC tissues were quantified by real-time PCR and western blot respectively. Linear regression analysis of them revealed a strong negative correlation. *: A two-tailed t-test P-value <0.05
Mentions: By using a popular miRNA targets prediction algorithm TargetScan, TGFβR1 was predicted to be a theoretical target of miR-490-3p (Fig. 5a), which miRNA recognition element (MRE) in 3′UTR was conserved among mammals. Western blot results showed that overexpression of miR-490-3p in CRC cell lines decreased the protein level of TGFβR1, and knockdown expression of miR-490-3p increased the protein level of TGFβR1 inversely (Fig. 5b). Dual-luciferase reporter assay indicated that miR-490-3p suppressed TGFβR1 expression by direct binding to the MRE (Fig. 5c), while mutants in the seed region disrupted their interaction (Fig. 5d). MMP2 and MMP9 have been previously reported to be direct target genes of miR-490-3p in ovarian cancer [8]. Here we found that MMP2 and MMP9 mRNA expression was downregulated after overexpression of miR-490-3p and upregulated after knockdown of miR-490-3p (Fig. 5e), confirming the interactions between them. Moreover, in eight CRC patient tissue samples, a significant reversed correlation between miR-490-3p expression and TGFβR1 protein level had been identified (Fig. 5f, r = −0.92, P < 0.05), which further proved the true inhibitory relationship.Fig. 5

Bottom Line: Artificially overexpression miR-490-3p in CRC cell lines inhibited cell migration and invasion abilities while knockdown miR-490-3p expression caused the reverse effects.Inhibition of TGFβR1 could partially recover the tumor suppression effect of miR-490-3p. miR-490-3p is downregulated during CRC malignant progression. miR-490-3p represses CRC cell migration and invasion abilities, partially by targeting to the TGF-β signaling pathway.Taken together, miR-490-3p is acting as a tumor suppressor in CRC.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. xxh@gzhmu.edu.cn.

ABSTRACT

Background: Colorectal cancer (CRC) is one of the most common malignances worldwide. Metastasis is responsible for the rapid recurrence and poor prognosis of CRC. However, the underlying molecular mechanism of CRC metastasis remains largely unclear. In this study we purposed to investigate the expression and biological functions of miR-490-3p in CRC metastasis, as well as to identify its downstream target genes and influenced pathway.

Methods: The expression level of miR-490-3p in CRC cell lines, CRC adjacent normal tissues, non-metastasis and metastasis tissues were assessed by quantitative real-time PCR. Patient survivals were follow-up up to 7 years. Gain-of-function and loss-of-function study on cell migration and invasion abilities were carried out by transfection of miR-490-3p mimics or inhibitors respectively. The molecular targets of miR-490-3p were computationally identified and experimentally verified by dual-luciferase reporter assay and western blot. Functional rescue was also conducted to confirm miR-490-3p inhibits CRC metastasis by targeting TGF-β signaling pathway.

Results: miR-490-3p expression was persistently downregulated during CRC malignant progression, as well as in CRC cell lines. Artificially overexpression miR-490-3p in CRC cell lines inhibited cell migration and invasion abilities while knockdown miR-490-3p expression caused the reverse effects. TGFβR1 and MMP2/9 were the downstream targets of miR-490-3p in CRC. Inhibition of TGFβR1 could partially recover the tumor suppression effect of miR-490-3p.

Conclusion: miR-490-3p is downregulated during CRC malignant progression. miR-490-3p represses CRC cell migration and invasion abilities, partially by targeting to the TGF-β signaling pathway. Taken together, miR-490-3p is acting as a tumor suppressor in CRC.

No MeSH data available.


Related in: MedlinePlus