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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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Enforced miR-424-5p expression in HCC cells reversed anoikis resistance of HCC cells.(a) BEL7402, SMMC7721, and HepG2 cells were transiently transfected with 50 nM miR-424-5p or random mock control, respectively. MiR-424-5p level in the transfected HCC cells was detected by real-time PCR and normalized to internal control U6. (b–c) The miR-424-5p transftected HepG2 cells were cultured in anchorage-deprived condition for 24 h; and cell viabilities were determined by trypan blue staining(b) and CCK8 kit(c). (d) The miR-424-5p transfected HepG2 cells were transferred to the poly-HEMA coated wells and cultured as anchorage-deprived cells for another 24 h. Caspase 3 activation was determined by caspase 3 substrate assay kit. (e–g) The miR-424-5p transfected HepG2 and SMMC7721 cells were cultured in the poly-HEMA coated wells as detached cells for another 24 h, caspase 3 activity was detected by western blot (e). The band densities were analyzed by Image J software and normalized to β-actin(f–g). (h) Apoptotic rate of the miR-424-5p transfected detached HCC cells were determined by ANNEXIN V-PI apoptotic assay kit followed by flow cytometry analysis. Presented data were representative one from at least three independent experiments. * P < 0.05, **P < 0.01.
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f3: Enforced miR-424-5p expression in HCC cells reversed anoikis resistance of HCC cells.(a) BEL7402, SMMC7721, and HepG2 cells were transiently transfected with 50 nM miR-424-5p or random mock control, respectively. MiR-424-5p level in the transfected HCC cells was detected by real-time PCR and normalized to internal control U6. (b–c) The miR-424-5p transftected HepG2 cells were cultured in anchorage-deprived condition for 24 h; and cell viabilities were determined by trypan blue staining(b) and CCK8 kit(c). (d) The miR-424-5p transfected HepG2 cells were transferred to the poly-HEMA coated wells and cultured as anchorage-deprived cells for another 24 h. Caspase 3 activation was determined by caspase 3 substrate assay kit. (e–g) The miR-424-5p transfected HepG2 and SMMC7721 cells were cultured in the poly-HEMA coated wells as detached cells for another 24 h, caspase 3 activity was detected by western blot (e). The band densities were analyzed by Image J software and normalized to β-actin(f–g). (h) Apoptotic rate of the miR-424-5p transfected detached HCC cells were determined by ANNEXIN V-PI apoptotic assay kit followed by flow cytometry analysis. Presented data were representative one from at least three independent experiments. * P < 0.05, **P < 0.01.

Mentions: MiR-424-5p was among the top significantly down-regulated microRNAs in the detached HCC cells, and previous reports indicated its involvement in carcinogenesis121314. Thus we are interested in defining its role in resistance to anoikis and in EMT process of these anchorage-deprived HCC cells. To clarify this issue, we transfected miR-424-5p into the detached HCC cells, and its enforced expression efficiency was verified by real-time PCR (Fig. 3a). Our data showed that after anchorage deprival, cell viabilities of the miR-424-5p transfected cells were significantly decreased compared with the mock control (Fig. 3b–c). Further analysis showed a significant activation of caspase 3 in the miR-424-5p transfected detached HCC cells (Fig. 3d–g). Because cleavage of caspase 3 was a definite evidence of apoptosis, these data indicated that massive apoptosis was induced in the mir-424-5p transfected cells. Apoptosis occurrence in the miR-424-5p transfected detached HCC cells was further confirmed by ANNEXIN V-FITC apoptosis assay (Fig. 3h). Altogether, these data indicated that miR-424-5p transfection could reverse resistance to anoikis in the anchorage-deprived HCC cells.


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)

Enforced miR-424-5p expression in HCC cells reversed anoikis resistance of HCC cells.(a) BEL7402, SMMC7721, and HepG2 cells were transiently transfected with 50 nM miR-424-5p or random mock control, respectively. MiR-424-5p level in the transfected HCC cells was detected by real-time PCR and normalized to internal control U6. (b–c) The miR-424-5p transftected HepG2 cells were cultured in anchorage-deprived condition for 24 h; and cell viabilities were determined by trypan blue staining(b) and CCK8 kit(c). (d) The miR-424-5p transfected HepG2 cells were transferred to the poly-HEMA coated wells and cultured as anchorage-deprived cells for another 24 h. Caspase 3 activation was determined by caspase 3 substrate assay kit. (e–g) The miR-424-5p transfected HepG2 and SMMC7721 cells were cultured in the poly-HEMA coated wells as detached cells for another 24 h, caspase 3 activity was detected by western blot (e). The band densities were analyzed by Image J software and normalized to β-actin(f–g). (h) Apoptotic rate of the miR-424-5p transfected detached HCC cells were determined by ANNEXIN V-PI apoptotic assay kit followed by flow cytometry analysis. 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

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getmorefigures.php?uid=PMC4150107&req=5

f3: Enforced miR-424-5p expression in HCC cells reversed anoikis resistance of HCC cells.(a) BEL7402, SMMC7721, and HepG2 cells were transiently transfected with 50 nM miR-424-5p or random mock control, respectively. MiR-424-5p level in the transfected HCC cells was detected by real-time PCR and normalized to internal control U6. (b–c) The miR-424-5p transftected HepG2 cells were cultured in anchorage-deprived condition for 24 h; and cell viabilities were determined by trypan blue staining(b) and CCK8 kit(c). (d) The miR-424-5p transfected HepG2 cells were transferred to the poly-HEMA coated wells and cultured as anchorage-deprived cells for another 24 h. Caspase 3 activation was determined by caspase 3 substrate assay kit. (e–g) The miR-424-5p transfected HepG2 and SMMC7721 cells were cultured in the poly-HEMA coated wells as detached cells for another 24 h, caspase 3 activity was detected by western blot (e). The band densities were analyzed by Image J software and normalized to β-actin(f–g). (h) Apoptotic rate of the miR-424-5p transfected detached HCC cells were determined by ANNEXIN V-PI apoptotic assay kit followed by flow cytometry analysis. Presented data were representative one from at least three independent experiments. * P < 0.05, **P < 0.01.
Mentions: MiR-424-5p was among the top significantly down-regulated microRNAs in the detached HCC cells, and previous reports indicated its involvement in carcinogenesis121314. Thus we are interested in defining its role in resistance to anoikis and in EMT process of these anchorage-deprived HCC cells. To clarify this issue, we transfected miR-424-5p into the detached HCC cells, and its enforced expression efficiency was verified by real-time PCR (Fig. 3a). Our data showed that after anchorage deprival, cell viabilities of the miR-424-5p transfected cells were significantly decreased compared with the mock control (Fig. 3b–c). Further analysis showed a significant activation of caspase 3 in the miR-424-5p transfected detached HCC cells (Fig. 3d–g). Because cleavage of caspase 3 was a definite evidence of apoptosis, these data indicated that massive apoptosis was induced in the mir-424-5p transfected cells. Apoptosis occurrence in the miR-424-5p transfected detached HCC cells was further confirmed by ANNEXIN V-FITC apoptosis assay (Fig. 3h). Altogether, these data indicated that miR-424-5p transfection could reverse resistance to anoikis in the anchorage-deprived HCC cells.

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