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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 inhibited the tumorigenicity of HCC cells in vivo in BALB/c nude mice.(a) The 5-week BALB/c nude mice were subcutaneously injected with SMMC7721 cells. Images presented were the isolated tumors transfected with miR-424-5p plasmid or mock control on day 12 after transfection. (b–c) The volume (b) and weight(c) of the miR-424-5p plasmid or mock control transfected tumors were assayed on day 18 after transplantation of HCC cells. (d) Expression of miR-424-5p in the miR-424-5p plasmid or mock control transfected HCC tumors was analyzed by real-time PCR and normalized to U6. (e) Expression of ICAT in HCC tumors transfected with the miR-424-5p or mock plasmid was analyzed by real-time PCR and normalized to β-actin. (f) The correlation analysis between the expression levels of miR-424-5p and ICAT. (g–i) The expression of E-cadherin(g), N-cadherin(h) and β-catenin(i) in the HCC tumors transfected with miR-424-5p plasmid or mock control. *P<0.05, **P<0.01.
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f7: miR-424-5p inhibited the tumorigenicity of HCC cells in vivo in BALB/c nude mice.(a) The 5-week BALB/c nude mice were subcutaneously injected with SMMC7721 cells. Images presented were the isolated tumors transfected with miR-424-5p plasmid or mock control on day 12 after transfection. (b–c) The volume (b) and weight(c) of the miR-424-5p plasmid or mock control transfected tumors were assayed on day 18 after transplantation of HCC cells. (d) Expression of miR-424-5p in the miR-424-5p plasmid or mock control transfected HCC tumors was analyzed by real-time PCR and normalized to U6. (e) Expression of ICAT in HCC tumors transfected with the miR-424-5p or mock plasmid was analyzed by real-time PCR and normalized to β-actin. (f) The correlation analysis between the expression levels of miR-424-5p and ICAT. (g–i) The expression of E-cadherin(g), N-cadherin(h) and β-catenin(i) in the HCC tumors transfected with miR-424-5p plasmid or mock control. *P<0.05, **P<0.01.

Mentions: To further assess the effect of miR-424-5p on HCC tumor growth in vivo, the xenografted tumor growth model was made by subcutaneous injection with SMMC7721 HCC cells in nude mice. The formed tumors were then transfected with pCMV-MIR-424-5p plasmid or mock control before the mice were sacrificed on day 18 after transplantation. Our data showed that the average size of tumors transfected with miR-424-5p plasmid was significantly smaller than those transfected with mock control on day 12 after transfection (Fig. 7a–b). The average weight of tumors in the miR-424-5p transfected group was significantly less than those in the mock control group (Fig. 7c). Quantitative real-time PCR showed that expression of miR-424-5p in tumors transfected with miR-424-5p plasmid was indeed significantly higher than those transfected with mock control (Fig. 7d); while expression of its target gene, ICAT was significantly lower in the miR-424-5p transfected tumors (Fig. 7e). Correlation analysis showed that the expression level of ICAT was negatively correlated with that of miR-424-5p (Fig. 7f), which further confirmed that ICAT was a target of miR-424-5p in HCC tissues. We further analyzed the expression of EMT related markers and our data showed that Epithelial marker E-cadherin and β-catenin was significantly upregulated while mesenchymal marker N-cadherin was significantly downregulated in miR-424-5p transfected group (Fig. 7g–i). These in vivo data was consistent with our in vitro data, showing that miR-424-5p inhibited HCC progression through its downregulation of ICAT, which further inhibited EMT through maintaining the E-cadherin/β-catenin complex on the cell membrane.


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 inhibited the tumorigenicity of HCC cells in vivo in BALB/c nude mice.(a) The 5-week BALB/c nude mice were subcutaneously injected with SMMC7721 cells. Images presented were the isolated tumors transfected with miR-424-5p plasmid or mock control on day 12 after transfection. (b–c) The volume (b) and weight(c) of the miR-424-5p plasmid or mock control transfected tumors were assayed on day 18 after transplantation of HCC cells. (d) Expression of miR-424-5p in the miR-424-5p plasmid or mock control transfected HCC tumors was analyzed by real-time PCR and normalized to U6. (e) Expression of ICAT in HCC tumors transfected with the miR-424-5p or mock plasmid was analyzed by real-time PCR and normalized to β-actin. (f) The correlation analysis between the expression levels of miR-424-5p and ICAT. (g–i) The expression of E-cadherin(g), N-cadherin(h) and β-catenin(i) in the HCC tumors transfected with miR-424-5p plasmid or mock control. *P<0.05, **P<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4150107&req=5

f7: miR-424-5p inhibited the tumorigenicity of HCC cells in vivo in BALB/c nude mice.(a) The 5-week BALB/c nude mice were subcutaneously injected with SMMC7721 cells. Images presented were the isolated tumors transfected with miR-424-5p plasmid or mock control on day 12 after transfection. (b–c) The volume (b) and weight(c) of the miR-424-5p plasmid or mock control transfected tumors were assayed on day 18 after transplantation of HCC cells. (d) Expression of miR-424-5p in the miR-424-5p plasmid or mock control transfected HCC tumors was analyzed by real-time PCR and normalized to U6. (e) Expression of ICAT in HCC tumors transfected with the miR-424-5p or mock plasmid was analyzed by real-time PCR and normalized to β-actin. (f) The correlation analysis between the expression levels of miR-424-5p and ICAT. (g–i) The expression of E-cadherin(g), N-cadherin(h) and β-catenin(i) in the HCC tumors transfected with miR-424-5p plasmid or mock control. *P<0.05, **P<0.01.
Mentions: To further assess the effect of miR-424-5p on HCC tumor growth in vivo, the xenografted tumor growth model was made by subcutaneous injection with SMMC7721 HCC cells in nude mice. The formed tumors were then transfected with pCMV-MIR-424-5p plasmid or mock control before the mice were sacrificed on day 18 after transplantation. Our data showed that the average size of tumors transfected with miR-424-5p plasmid was significantly smaller than those transfected with mock control on day 12 after transfection (Fig. 7a–b). The average weight of tumors in the miR-424-5p transfected group was significantly less than those in the mock control group (Fig. 7c). Quantitative real-time PCR showed that expression of miR-424-5p in tumors transfected with miR-424-5p plasmid was indeed significantly higher than those transfected with mock control (Fig. 7d); while expression of its target gene, ICAT was significantly lower in the miR-424-5p transfected tumors (Fig. 7e). Correlation analysis showed that the expression level of ICAT was negatively correlated with that of miR-424-5p (Fig. 7f), which further confirmed that ICAT was a target of miR-424-5p in HCC tissues. We further analyzed the expression of EMT related markers and our data showed that Epithelial marker E-cadherin and β-catenin was significantly upregulated while mesenchymal marker N-cadherin was significantly downregulated in miR-424-5p transfected group (Fig. 7g–i). These in vivo data was consistent with our in vitro data, showing that miR-424-5p inhibited HCC progression through its downregulation of ICAT, which further inhibited EMT through maintaining the E-cadherin/β-catenin complex on the cell membrane.

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