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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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Expression of EMT related markers in attached and detached HCC cells.(a) BEL7402, SMMC7721, and HepG2 cells were seeded in 6-well plates without or with poly-HEMA coating as attached and detached HCC cells. After 24 h of culture, cells were collected, and the expression of EMT markers in BEL7402, SMMC7721 and HepG2 cells was detected by western blot. β-actin was used as an internal control. (b) Quantitation of band intensities from western blot analysis was analyzed densitometrically using Image J software and normalized to β-actin. (c) After the detached and attached cells were cultured for 24 h, expression of EMT related markers were detected by real-time PCR, and the histograms represented the relative mRNA expression levels normalized to β-actin. Presented data were representative one from at least three independent experiments. *P<0.05. **P<0.01.
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f2: Expression of EMT related markers in attached and detached HCC cells.(a) BEL7402, SMMC7721, and HepG2 cells were seeded in 6-well plates without or with poly-HEMA coating as attached and detached HCC cells. After 24 h of culture, cells were collected, and the expression of EMT markers in BEL7402, SMMC7721 and HepG2 cells was detected by western blot. β-actin was used as an internal control. (b) Quantitation of band intensities from western blot analysis was analyzed densitometrically using Image J software and normalized to β-actin. (c) After the detached and attached cells were cultured for 24 h, expression of EMT related markers were detected by real-time PCR, and the histograms represented the relative mRNA expression levels normalized to β-actin. Presented data were representative one from at least three independent experiments. *P<0.05. **P<0.01.

Mentions: Both anoikis-resistant process and EMT are regarded as important events in the metastatic process of cancer cells. Our previous study showed that acquisition of resistance to anoikis conferred HCC cells more malignant behaviors, including resistance to apoptosis induction, resistance to extracellular stimuli and increased capability of invasion10. These properties are also known as features of EMT-transformed mesenchymal cells. Therefore it is intriguing to see whether these anchorage independent HCC cells also undergo EMT process. Thus we detected EMT related markers in these detached HCC cells by western blot, with β-actin as an internal control. Our data showed that expression levels of epithelial markers, including E-cadherin and β-catenin were significantly down-regulated, accompanied with up-regulation of mesenchymal markers, including N-cadherin and vimentin (Fig. 2a–b). The expression changes of these markers were also verified by quantitative real-time PCR (Fig. 2c). Snail, an identified transcription factor repressing epithelial genes via E-boxes in the corresponding promoters, was also analyzed. Our data showed that snail was significantly upregulated in these detached HCC cells (Fig. 2a–c). These data suggested that EMT was dramatically induced by acquisition of resistance to anoikis in the detached HCC cells. Moreover, our previous data showed that anchorage deprival also resulted in the acquisition of other properties involved in cancer progression, such as an increased ability to migrate, a higher resistance to apoptosis and a higher capability of colony formation11. Thus, all of these properties are in consistence with a more invasive and malignant behavior of EMT-transformed cells in cancer progression.


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)

Expression of EMT related markers in attached and detached HCC cells.(a) BEL7402, SMMC7721, and HepG2 cells were seeded in 6-well plates without or with poly-HEMA coating as attached and detached HCC cells. After 24 h of culture, cells were collected, and the expression of EMT markers in BEL7402, SMMC7721 and HepG2 cells was detected by western blot. β-actin was used as an internal control. (b) Quantitation of band intensities from western blot analysis was analyzed densitometrically using Image J software and normalized to β-actin. (c) After the detached and attached cells were cultured for 24 h, expression of EMT related markers were detected by real-time PCR, and the histograms represented the relative mRNA expression levels normalized to β-actin. Presented data were representative one from at least three independent experiments. *P<0.05. **P<0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Expression of EMT related markers in attached and detached HCC cells.(a) BEL7402, SMMC7721, and HepG2 cells were seeded in 6-well plates without or with poly-HEMA coating as attached and detached HCC cells. After 24 h of culture, cells were collected, and the expression of EMT markers in BEL7402, SMMC7721 and HepG2 cells was detected by western blot. β-actin was used as an internal control. (b) Quantitation of band intensities from western blot analysis was analyzed densitometrically using Image J software and normalized to β-actin. (c) After the detached and attached cells were cultured for 24 h, expression of EMT related markers were detected by real-time PCR, and the histograms represented the relative mRNA expression levels normalized to β-actin. Presented data were representative one from at least three independent experiments. *P<0.05. **P<0.01.
Mentions: Both anoikis-resistant process and EMT are regarded as important events in the metastatic process of cancer cells. Our previous study showed that acquisition of resistance to anoikis conferred HCC cells more malignant behaviors, including resistance to apoptosis induction, resistance to extracellular stimuli and increased capability of invasion10. These properties are also known as features of EMT-transformed mesenchymal cells. Therefore it is intriguing to see whether these anchorage independent HCC cells also undergo EMT process. Thus we detected EMT related markers in these detached HCC cells by western blot, with β-actin as an internal control. Our data showed that expression levels of epithelial markers, including E-cadherin and β-catenin were significantly down-regulated, accompanied with up-regulation of mesenchymal markers, including N-cadherin and vimentin (Fig. 2a–b). The expression changes of these markers were also verified by quantitative real-time PCR (Fig. 2c). Snail, an identified transcription factor repressing epithelial genes via E-boxes in the corresponding promoters, was also analyzed. Our data showed that snail was significantly upregulated in these detached HCC cells (Fig. 2a–c). These data suggested that EMT was dramatically induced by acquisition of resistance to anoikis in the detached HCC cells. Moreover, our previous data showed that anchorage deprival also resulted in the acquisition of other properties involved in cancer progression, such as an increased ability to migrate, a higher resistance to apoptosis and a higher capability of colony formation11. Thus, all of these properties are in consistence with a more invasive and malignant behavior of EMT-transformed cells in cancer progression.

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