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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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miR-424-5p disrupted the cell-cell adhesion complex by directly targeting ICAT.(a) HEK293 cells were transfected with 40 nM, 80 nM and 120 nM of miR-424-5p and cultured for 24 h. Cell lysates were extracted and subjected to immunoprecipitation with anti-β-catenin antibody followed by western blot analysis with anti-E-cadherin antibody. Proteins in whole-cell lysates were used as input control for analysis by western blot with anti-E-cadherin antibody and anti-ICAT antibody, respectively. (b) HEK293 cells were transfected with 0.5 μg, 1 μg and 2 μg of HA tagged ICAT recombinant and cultured for 24 h. Cell lysates were extracted and subjected to immunoprecipitation with anti-β-catenin antibody followed by western blot analysis with anti-E-cadherin antibody. Proteins in whole-cell lysates were used as input control. Similar results were got from three independent experiments. (c) HepG2 cells were transfected with 50 nM of 3 synthesized siRNAs targeting ICAT or nonsense control.48 h after transfection, the inhibitory effect of siRNA were analyzed by western blot. (d) The HepG2 cells were transfected with a mixture of Si-1 and Si-3 targeting ICAT, and cultured in anchorage deprived condition for 24 h before analysis of EMT related markers by real-time PCR. (e–f) HepG2 cells were transfected with miR-424-5p or HA-tagged ICAT recombinant and cultured in anchorage deprived condition for 24 h. Expression of E-cadherin, N-cadherin and ICAT was determined by western blot (e) and quantified by band intensity analysis of these proteins normalized to that of β-actin (f). (g–h) BEL7402, SMMC7721 and HepG2 HCC cells transfected with miR-424-5p, miR-424-5p inhibitor or Si-ICAT were cultured as anchorage deprived cells for 24 h before being harvested and analyzed for their invasion capability (g). The invaded cells were quantified and presented histogram were representative one from three independent experiments performed in duplicate (h). *P<0.05, **P < 0.01, ***P < 0.001.
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f6: miR-424-5p disrupted the cell-cell adhesion complex by directly targeting ICAT.(a) HEK293 cells were transfected with 40 nM, 80 nM and 120 nM of miR-424-5p and cultured for 24 h. Cell lysates were extracted and subjected to immunoprecipitation with anti-β-catenin antibody followed by western blot analysis with anti-E-cadherin antibody. Proteins in whole-cell lysates were used as input control for analysis by western blot with anti-E-cadherin antibody and anti-ICAT antibody, respectively. (b) HEK293 cells were transfected with 0.5 μg, 1 μg and 2 μg of HA tagged ICAT recombinant and cultured for 24 h. Cell lysates were extracted and subjected to immunoprecipitation with anti-β-catenin antibody followed by western blot analysis with anti-E-cadherin antibody. Proteins in whole-cell lysates were used as input control. Similar results were got from three independent experiments. (c) HepG2 cells were transfected with 50 nM of 3 synthesized siRNAs targeting ICAT or nonsense control.48 h after transfection, the inhibitory effect of siRNA were analyzed by western blot. (d) The HepG2 cells were transfected with a mixture of Si-1 and Si-3 targeting ICAT, and cultured in anchorage deprived condition for 24 h before analysis of EMT related markers by real-time PCR. (e–f) HepG2 cells were transfected with miR-424-5p or HA-tagged ICAT recombinant and cultured in anchorage deprived condition for 24 h. Expression of E-cadherin, N-cadherin and ICAT was determined by western blot (e) and quantified by band intensity analysis of these proteins normalized to that of β-actin (f). (g–h) BEL7402, SMMC7721 and HepG2 HCC cells transfected with miR-424-5p, miR-424-5p inhibitor or Si-ICAT were cultured as anchorage deprived cells for 24 h before being harvested and analyzed for their invasion capability (g). The invaded cells were quantified and presented histogram were representative one from three independent experiments performed in duplicate (h). *P<0.05, **P < 0.01, ***P < 0.001.

Mentions: We have shown that ectopic overexpression of miR-424-5p inhibited EMT process of these anchorage-deprived cells; then we further performed immunoprecipitation analysis to verify the direct binding between E-cadherin and β-catenin in the miR-424-5p or HA-ICAT transfected cells. Our data showed that ectopic overexpression of miR-424-5p enhanced the binding between E-cadherin and β-catenin, while ICAT overexpression inhibited their interactions (Fig. 6a–b). These data further verified that miR-424-5p-mediated downregulation of ICAT is responsible for this deregulation of E-cadherin/β-catenin complex on the cell membrane. In addition, three siRNAs specifically targeting ICAT were synthesized, and western blot verification showed that Si-1 and Si-3 had significant inhibitory effect on ICAT (Fig. 6c). Thus the following experiments were done with the mixture of these two SiRNAs. Our data showed that Si-ICAT transfection significantly inhibited EMT markers in the detached HCC cells (Fig. 6d), while overexpression of ICAT rescued the effect of miR-424-5p on EMT of these detached HCC cells (Fig. 6e–f). Thus these data confirmed that deregulation of miR-424-5p, through targeting ICAT, is involved in EMT process of the detached HCC cell. Moreover, we are interested in clarifying whether the malignancy linked with EMT process was also mediated by miR-424-5p-induced downregulation of ICAT. Our data showed that knockdown of ICAT could indeed photocopy the observed effect on invasion upon overexpression of miR-424-5p to a great extent (Fig. 6g–h). These data indicated that the involvement of miR-424-5p in EMT could take effect, at least to a great degree, via directly targeting ICAT.


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)

miR-424-5p disrupted the cell-cell adhesion complex by directly targeting ICAT.(a) HEK293 cells were transfected with 40 nM, 80 nM and 120 nM of miR-424-5p and cultured for 24 h. Cell lysates were extracted and subjected to immunoprecipitation with anti-β-catenin antibody followed by western blot analysis with anti-E-cadherin antibody. Proteins in whole-cell lysates were used as input control for analysis by western blot with anti-E-cadherin antibody and anti-ICAT antibody, respectively. (b) HEK293 cells were transfected with 0.5 μg, 1 μg and 2 μg of HA tagged ICAT recombinant and cultured for 24 h. Cell lysates were extracted and subjected to immunoprecipitation with anti-β-catenin antibody followed by western blot analysis with anti-E-cadherin antibody. Proteins in whole-cell lysates were used as input control. Similar results were got from three independent experiments. (c) HepG2 cells were transfected with 50 nM of 3 synthesized siRNAs targeting ICAT or nonsense control.48 h after transfection, the inhibitory effect of siRNA were analyzed by western blot. (d) The HepG2 cells were transfected with a mixture of Si-1 and Si-3 targeting ICAT, and cultured in anchorage deprived condition for 24 h before analysis of EMT related markers by real-time PCR. (e–f) HepG2 cells were transfected with miR-424-5p or HA-tagged ICAT recombinant and cultured in anchorage deprived condition for 24 h. Expression of E-cadherin, N-cadherin and ICAT was determined by western blot (e) and quantified by band intensity analysis of these proteins normalized to that of β-actin (f). (g–h) BEL7402, SMMC7721 and HepG2 HCC cells transfected with miR-424-5p, miR-424-5p inhibitor or Si-ICAT were cultured as anchorage deprived cells for 24 h before being harvested and analyzed for their invasion capability (g). The invaded cells were quantified and presented histogram were representative one from three independent experiments performed in duplicate (h). *P<0.05, **P < 0.01, ***P < 0.001.
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f6: miR-424-5p disrupted the cell-cell adhesion complex by directly targeting ICAT.(a) HEK293 cells were transfected with 40 nM, 80 nM and 120 nM of miR-424-5p and cultured for 24 h. Cell lysates were extracted and subjected to immunoprecipitation with anti-β-catenin antibody followed by western blot analysis with anti-E-cadherin antibody. Proteins in whole-cell lysates were used as input control for analysis by western blot with anti-E-cadherin antibody and anti-ICAT antibody, respectively. (b) HEK293 cells were transfected with 0.5 μg, 1 μg and 2 μg of HA tagged ICAT recombinant and cultured for 24 h. Cell lysates were extracted and subjected to immunoprecipitation with anti-β-catenin antibody followed by western blot analysis with anti-E-cadherin antibody. Proteins in whole-cell lysates were used as input control. Similar results were got from three independent experiments. (c) HepG2 cells were transfected with 50 nM of 3 synthesized siRNAs targeting ICAT or nonsense control.48 h after transfection, the inhibitory effect of siRNA were analyzed by western blot. (d) The HepG2 cells were transfected with a mixture of Si-1 and Si-3 targeting ICAT, and cultured in anchorage deprived condition for 24 h before analysis of EMT related markers by real-time PCR. (e–f) HepG2 cells were transfected with miR-424-5p or HA-tagged ICAT recombinant and cultured in anchorage deprived condition for 24 h. Expression of E-cadherin, N-cadherin and ICAT was determined by western blot (e) and quantified by band intensity analysis of these proteins normalized to that of β-actin (f). (g–h) BEL7402, SMMC7721 and HepG2 HCC cells transfected with miR-424-5p, miR-424-5p inhibitor or Si-ICAT were cultured as anchorage deprived cells for 24 h before being harvested and analyzed for their invasion capability (g). The invaded cells were quantified and presented histogram were representative one from three independent experiments performed in duplicate (h). *P<0.05, **P < 0.01, ***P < 0.001.
Mentions: We have shown that ectopic overexpression of miR-424-5p inhibited EMT process of these anchorage-deprived cells; then we further performed immunoprecipitation analysis to verify the direct binding between E-cadherin and β-catenin in the miR-424-5p or HA-ICAT transfected cells. Our data showed that ectopic overexpression of miR-424-5p enhanced the binding between E-cadherin and β-catenin, while ICAT overexpression inhibited their interactions (Fig. 6a–b). These data further verified that miR-424-5p-mediated downregulation of ICAT is responsible for this deregulation of E-cadherin/β-catenin complex on the cell membrane. In addition, three siRNAs specifically targeting ICAT were synthesized, and western blot verification showed that Si-1 and Si-3 had significant inhibitory effect on ICAT (Fig. 6c). Thus the following experiments were done with the mixture of these two SiRNAs. Our data showed that Si-ICAT transfection significantly inhibited EMT markers in the detached HCC cells (Fig. 6d), while overexpression of ICAT rescued the effect of miR-424-5p on EMT of these detached HCC cells (Fig. 6e–f). Thus these data confirmed that deregulation of miR-424-5p, through targeting ICAT, is involved in EMT process of the detached HCC cell. Moreover, we are interested in clarifying whether the malignancy linked with EMT process was also mediated by miR-424-5p-induced downregulation of ICAT. Our data showed that knockdown of ICAT could indeed photocopy the observed effect on invasion upon overexpression of miR-424-5p to a great extent (Fig. 6g–h). These data indicated that the involvement of miR-424-5p in EMT could take effect, at least to a great degree, via directly targeting ICAT.

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