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MiR-424-5p reversed epithelial-mesenchymal transition of anchorage-independent HCC cells by directly targeting ICAT and suppressed HCC progression.

Zhang Y, Li T, Guo P, Kang J, Wei Q, Jia X, Zhao W, Huai W, Qiu Y, Sun L, Han L - Sci Rep (2014)

Bottom Line: Microarray expression profiling revealed that expression of miR-424-5p was significantly decreased in anoikis-resistant HCC cells.Clinical investigation demonstrated that miR-424-5p was significantly downregulated in HCC tissues compared with that of the non-cancerous liver tissues, and this decreased expression of miR-424-5p was significantly correlated with higher pathological grades and more advanced TNM stages.Therefore, aberrant expression of miR-424-5p is critically involved in resistance to anoikis and EMT during the metastatic process of HCC, and its downregulation significantly contributes to liver cancer progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Shandong University School of Medicine, Jinan 250012, China.

ABSTRACT
Resistance to anoikis and Epithelial-mesenchymal transition (EMT) are two processes critically involved in cancer metastasis. In this study, we demonstrated that after anchorage deprival, hepatocellular carcinoma (HCC) cells not only resisted anoikis, but also exhibited EMT process. Microarray expression profiling revealed that expression of miR-424-5p was significantly decreased in anoikis-resistant HCC cells. Ectopic overexpression of miR-424-5p was sufficient to reverse resistance to anoikis, block EMT process and inhibit malignant behaviors of HCC cells. Target analysis showed that a potent β-catenin inhibitor, ICAT/CTNNBIP1 was a direct target of miR-424-5p. Further study demonstrated that miR-424-5p reversed resistance to anoikis and EMT of HCCs by directly targeting ICAT and further maintaining the E-cadherin/β-catanin complex on the cellular membrance. In vivo study further demonstrated that miR-424-5p significantly inhibited the tumorigenicity of HCC cells in nude mice. Clinical investigation demonstrated that miR-424-5p was significantly downregulated in HCC tissues compared with that of the non-cancerous liver tissues, and this decreased expression of miR-424-5p was significantly correlated with higher pathological grades and more advanced TNM stages. Therefore, aberrant expression of miR-424-5p is critically involved in resistance to anoikis and EMT during the metastatic process of HCC, and its downregulation significantly contributes to liver cancer progression.

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ICAT is a direct target of miR-424-5p.(a–b) Expression of ICAT mRNA and protein in detached HepG2 cells was analyzed by real-time PCR (a) and western blot (b), respectively. (c) The HepG2 cells were transfected with miR-424-5p or its inhibitor; and ICAT mRNA expression was detected by real-time PCR and quantified by normalization to β-actin. (d–e) The BEL7402, SMMC7721 and HepG2 HCC cells were transfected with 50 nM of miR-424-5p or nonsense control, the endogenous protein levels of ICAT in HCC cells were detected by western blot (d) and quantified by the band intensity of ICAT normalized to β-actin (e). (f) miR-424-5p binding site is located in the ICAT/CTNNBIP1-3′-UTR and mutation was generated in the ICAT/CTNNBIP1-3′-UTR sequence by mutating 3 nucleotides recognized by miR-424-5p. Either wild-type (WT) or mutant CTNNBIP1-3′-UTR was subcloned into the dual-luciferase reporter vector. (g) The luciferase reporter vector containing WT ICAT/CTNNBIP1-3′-UTR or mutant 3′-UTR was cotransfected with miR-424-5p or miR-NC into HEK293 cells. Firefly luciferase activities were determined 48 h post-transfection and normalized to Renilla luciferase. Presented data were representative one from at least three independent experiments. *P < 0.05, **P < 0.01,***P < 0.001.
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f5: ICAT is a direct target of miR-424-5p.(a–b) Expression of ICAT mRNA and protein in detached HepG2 cells was analyzed by real-time PCR (a) and western blot (b), respectively. (c) The HepG2 cells were transfected with miR-424-5p or its inhibitor; and ICAT mRNA expression was detected by real-time PCR and quantified by normalization to β-actin. (d–e) The BEL7402, SMMC7721 and HepG2 HCC cells were transfected with 50 nM of miR-424-5p or nonsense control, the endogenous protein levels of ICAT in HCC cells were detected by western blot (d) and quantified by the band intensity of ICAT normalized to β-actin (e). (f) miR-424-5p binding site is located in the ICAT/CTNNBIP1-3′-UTR and mutation was generated in the ICAT/CTNNBIP1-3′-UTR sequence by mutating 3 nucleotides recognized by miR-424-5p. Either wild-type (WT) or mutant CTNNBIP1-3′-UTR was subcloned into the dual-luciferase reporter vector. (g) The luciferase reporter vector containing WT ICAT/CTNNBIP1-3′-UTR or mutant 3′-UTR was cotransfected with miR-424-5p or miR-NC into HEK293 cells. Firefly luciferase activities were determined 48 h post-transfection and normalized to Renilla luciferase. Presented data were representative one from at least three independent experiments. *P < 0.05, **P < 0.01,***P < 0.001.

Mentions: Having identified miR-424-5p as a regulator of EMT, we were then interested in identifying the target gene of miR-424-5p responsible for these effects. We evaluated the potential targets of miR-424-5p using available public algorithms15, and the target gene annotations revealed that ICAT/CTNNBIP1, a novel β-catenin-interacting protein1617 was the predicted target of miR-424-5p. Through sequestering β-catenin, ICAT inhibits E-cadherin based cell adhesion and Wnt responsive gene expression, thus leads to an avenue for ICAT to play a role in tumorigenesis. Intriguingly, ICAT is overexpressed in some tumors, though its exact role in cancer progression has not been clarified18. Because ICAT is a regulator of E-cadherin/β-catenin complex of the cell adhesion, we hypothesized that up-regulation of ICAT in response to reduced level of miR-424-5p might be involved in EMT process of these detached HCC cells. To test this hypothesis, we firstly verified that ICAT was significantly up-regulated in detached HCC cells at both the mRNA and protein level (Fig. 5a–b). We further verified that ICAT expression was strongly inhibited by ectopic overexpression of miR-424-5p, while its expression was significantly enhanced by miR-424-5p inhibitor (Fig. 5c–e). Then we cloned a reporter construct containing the 3′-UTR of ICAT downstream of a luciferase open reading frame (Fig. 5f). This dual-luciferase reporter assay showed that co-transfection of ICAT-3′-UTR recombinant and miR-424-5p significantly reduced the relative luciferase activity in HEK293 cells, while transfection of mutant recombinant abrogated inhibition by miR-424-5p (Fig. 5g). These data suggested that ICAT was a direct target of miR-424-5p.


MiR-424-5p reversed epithelial-mesenchymal transition of anchorage-independent HCC cells by directly targeting ICAT and suppressed HCC progression.

Zhang Y, Li T, Guo P, Kang J, Wei Q, Jia X, Zhao W, Huai W, Qiu Y, Sun L, Han L - Sci Rep (2014)

ICAT is a direct target of miR-424-5p.(a–b) Expression of ICAT mRNA and protein in detached HepG2 cells was analyzed by real-time PCR (a) and western blot (b), respectively. (c) The HepG2 cells were transfected with miR-424-5p or its inhibitor; and ICAT mRNA expression was detected by real-time PCR and quantified by normalization to β-actin. (d–e) The BEL7402, SMMC7721 and HepG2 HCC cells were transfected with 50 nM of miR-424-5p or nonsense control, the endogenous protein levels of ICAT in HCC cells were detected by western blot (d) and quantified by the band intensity of ICAT normalized to β-actin (e). (f) miR-424-5p binding site is located in the ICAT/CTNNBIP1-3′-UTR and mutation was generated in the ICAT/CTNNBIP1-3′-UTR sequence by mutating 3 nucleotides recognized by miR-424-5p. Either wild-type (WT) or mutant CTNNBIP1-3′-UTR was subcloned into the dual-luciferase reporter vector. (g) The luciferase reporter vector containing WT ICAT/CTNNBIP1-3′-UTR or mutant 3′-UTR was cotransfected with miR-424-5p or miR-NC into HEK293 cells. Firefly luciferase activities were determined 48 h post-transfection and normalized to Renilla luciferase. Presented data were representative one from at least three independent experiments. *P < 0.05, **P < 0.01,***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150107&req=5

f5: ICAT is a direct target of miR-424-5p.(a–b) Expression of ICAT mRNA and protein in detached HepG2 cells was analyzed by real-time PCR (a) and western blot (b), respectively. (c) The HepG2 cells were transfected with miR-424-5p or its inhibitor; and ICAT mRNA expression was detected by real-time PCR and quantified by normalization to β-actin. (d–e) The BEL7402, SMMC7721 and HepG2 HCC cells were transfected with 50 nM of miR-424-5p or nonsense control, the endogenous protein levels of ICAT in HCC cells were detected by western blot (d) and quantified by the band intensity of ICAT normalized to β-actin (e). (f) miR-424-5p binding site is located in the ICAT/CTNNBIP1-3′-UTR and mutation was generated in the ICAT/CTNNBIP1-3′-UTR sequence by mutating 3 nucleotides recognized by miR-424-5p. Either wild-type (WT) or mutant CTNNBIP1-3′-UTR was subcloned into the dual-luciferase reporter vector. (g) The luciferase reporter vector containing WT ICAT/CTNNBIP1-3′-UTR or mutant 3′-UTR was cotransfected with miR-424-5p or miR-NC into HEK293 cells. Firefly luciferase activities were determined 48 h post-transfection and normalized to Renilla luciferase. Presented data were representative one from at least three independent experiments. *P < 0.05, **P < 0.01,***P < 0.001.
Mentions: Having identified miR-424-5p as a regulator of EMT, we were then interested in identifying the target gene of miR-424-5p responsible for these effects. We evaluated the potential targets of miR-424-5p using available public algorithms15, and the target gene annotations revealed that ICAT/CTNNBIP1, a novel β-catenin-interacting protein1617 was the predicted target of miR-424-5p. Through sequestering β-catenin, ICAT inhibits E-cadherin based cell adhesion and Wnt responsive gene expression, thus leads to an avenue for ICAT to play a role in tumorigenesis. Intriguingly, ICAT is overexpressed in some tumors, though its exact role in cancer progression has not been clarified18. Because ICAT is a regulator of E-cadherin/β-catenin complex of the cell adhesion, we hypothesized that up-regulation of ICAT in response to reduced level of miR-424-5p might be involved in EMT process of these detached HCC cells. To test this hypothesis, we firstly verified that ICAT was significantly up-regulated in detached HCC cells at both the mRNA and protein level (Fig. 5a–b). We further verified that ICAT expression was strongly inhibited by ectopic overexpression of miR-424-5p, while its expression was significantly enhanced by miR-424-5p inhibitor (Fig. 5c–e). Then we cloned a reporter construct containing the 3′-UTR of ICAT downstream of a luciferase open reading frame (Fig. 5f). This dual-luciferase reporter assay showed that co-transfection of ICAT-3′-UTR recombinant and miR-424-5p significantly reduced the relative luciferase activity in HEK293 cells, while transfection of mutant recombinant abrogated inhibition by miR-424-5p (Fig. 5g). These data suggested that ICAT was a direct target of miR-424-5p.

Bottom Line: Microarray expression profiling revealed that expression of miR-424-5p was significantly decreased in anoikis-resistant HCC cells.Clinical investigation demonstrated that miR-424-5p was significantly downregulated in HCC tissues compared with that of the non-cancerous liver tissues, and this decreased expression of miR-424-5p was significantly correlated with higher pathological grades and more advanced TNM stages.Therefore, aberrant expression of miR-424-5p is critically involved in resistance to anoikis and EMT during the metastatic process of HCC, and its downregulation significantly contributes to liver cancer progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Shandong University School of Medicine, Jinan 250012, China.

ABSTRACT
Resistance to anoikis and Epithelial-mesenchymal transition (EMT) are two processes critically involved in cancer metastasis. In this study, we demonstrated that after anchorage deprival, hepatocellular carcinoma (HCC) cells not only resisted anoikis, but also exhibited EMT process. Microarray expression profiling revealed that expression of miR-424-5p was significantly decreased in anoikis-resistant HCC cells. Ectopic overexpression of miR-424-5p was sufficient to reverse resistance to anoikis, block EMT process and inhibit malignant behaviors of HCC cells. Target analysis showed that a potent β-catenin inhibitor, ICAT/CTNNBIP1 was a direct target of miR-424-5p. Further study demonstrated that miR-424-5p reversed resistance to anoikis and EMT of HCCs by directly targeting ICAT and further maintaining the E-cadherin/β-catanin complex on the cellular membrance. In vivo study further demonstrated that miR-424-5p significantly inhibited the tumorigenicity of HCC cells in nude mice. Clinical investigation demonstrated that miR-424-5p was significantly downregulated in HCC tissues compared with that of the non-cancerous liver tissues, and this decreased expression of miR-424-5p was significantly correlated with higher pathological grades and more advanced TNM stages. Therefore, aberrant expression of miR-424-5p is critically involved in resistance to anoikis and EMT during the metastatic process of HCC, and its downregulation significantly contributes to liver cancer progression.

Show MeSH
Related in: MedlinePlus