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Knockdown of CD44 inhibits the invasion and metastasis of hepatocellular carcinoma both in vitro and in vivo by reversing epithelial-mesenchymal transition.

Gao Y, Ruan B, Liu W, Wang J, Yang X, Zhang Z, Li X, Duan J, Zhang F, Ding R, Tao K, Dou K - Oncotarget (2015)

Bottom Line: First, we noticed that CD44 expression was associated with the mesenchymal phenotype in HCC cell lines, and knocking down CD44 with lentivirus-mediated shRNA in HCC cell lines resulted in the mesenchymal-epithelial-transition (MET) and the subsequent impaired migration and invasion in vitro.Moreover, in a metastatic mice model established by tail vein injection of luciferase labelled MHCC97-H cells, we confirmed that CD44 knockdown resulted in the decreased metastasis of HCC cells.Furthermore, we found that the induction of MET by CD44 inhibition might be achieved, at least in part, by repressing the ERK/Snail pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepato-Biliary and Pancreto-Splenic Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an, China.

ABSTRACT
Mounting evidence has shown that induction of epithelial-mesenchymal transition (EMT) contributes to the the expression of CSC (cancer stem cell) markers. However, whether and how CSC markers could be involved in regulating EMT has rarely been reported. CD44, being one of the most commonly used CSC markers in hepatocellular carcinoma (HCC), has been demonstrated to act as a multidomain, transmembrane platform that serves to integrate a wide variety of extracellular signals. Therefore, we determined to seek whether CD44 is necessary for the EMT process in HCC. First, we noticed that CD44 expression was associated with the mesenchymal phenotype in HCC cell lines, and knocking down CD44 with lentivirus-mediated shRNA in HCC cell lines resulted in the mesenchymal-epithelial-transition (MET) and the subsequent impaired migration and invasion in vitro. Moreover, in a metastatic mice model established by tail vein injection of luciferase labelled MHCC97-H cells, we confirmed that CD44 knockdown resulted in the decreased metastasis of HCC cells. Furthermore, we found that the induction of MET by CD44 inhibition might be achieved, at least in part, by repressing the ERK/Snail pathway.

No MeSH data available.


Related in: MedlinePlus

Knockdown of CD44 induced the MET in SMMC-7721 and MHCC97-H cellsqRT-PCR (A) and Western-blot (B) analysis of CD44 expression in SMMC-7721 and MHCC97-H cells that were transfected with shRNA of CD44 (KD) and normal control (NC). The expression levels of proteins (C) and mRNA (D) of the EMT markers are shown. (E) The protein expression levels of E-cadherin and N-cadherin in SMMC-7721 cells transfected with shRNA of CD44 and NC are shown by immunofluorescences under a 400× field. (* means p<0.05, ** means p<0.01, *** means p<0.001 by T-test).
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Figure 2: Knockdown of CD44 induced the MET in SMMC-7721 and MHCC97-H cellsqRT-PCR (A) and Western-blot (B) analysis of CD44 expression in SMMC-7721 and MHCC97-H cells that were transfected with shRNA of CD44 (KD) and normal control (NC). The expression levels of proteins (C) and mRNA (D) of the EMT markers are shown. (E) The protein expression levels of E-cadherin and N-cadherin in SMMC-7721 cells transfected with shRNA of CD44 and NC are shown by immunofluorescences under a 400× field. (* means p<0.05, ** means p<0.01, *** means p<0.001 by T-test).

Mentions: To investigate the influence of CD44 knockdown on the EMT in HCC cells, we transfected lentivirus with shRNA of CD44 or NC into SMMC-7721 and MHCC97-H cells respectively, and got the stably passaged SMMC-7721-KD, SMMC-7721-NC, MHCC97-H-KD and MHCC97-H-NC. qRT-PCR showed a dramatic CD44 mRNA decrease of 75.01% in SMMC-7721 and 82.08% in MHCC97-H with lentivirus transfection (Fig. 2A). Western blot confirmed the inhibition of CD44 protein in both SMMC-7721 and MHCC97-H cells (Fig. 2B). Because of the CD44 knockdown, the enhanced expression of epithelial marker (E-cadherin) and the reduced expression of mesenchymal markers (N-cadherin and Vimentin) in MHCC97-H-KD and SMMC-7721-KD cells were detected by Western blot (Fig. 2C), qRT-PCR (Fig. 2D) and immunofluorescence (Fig. 2E). These results demonstrated that the knockdown of CD44 in SMMC-7721 and MHCC97-H cells induced a transition to the epithelial phenotype.


Knockdown of CD44 inhibits the invasion and metastasis of hepatocellular carcinoma both in vitro and in vivo by reversing epithelial-mesenchymal transition.

Gao Y, Ruan B, Liu W, Wang J, Yang X, Zhang Z, Li X, Duan J, Zhang F, Ding R, Tao K, Dou K - Oncotarget (2015)

Knockdown of CD44 induced the MET in SMMC-7721 and MHCC97-H cellsqRT-PCR (A) and Western-blot (B) analysis of CD44 expression in SMMC-7721 and MHCC97-H cells that were transfected with shRNA of CD44 (KD) and normal control (NC). The expression levels of proteins (C) and mRNA (D) of the EMT markers are shown. (E) The protein expression levels of E-cadherin and N-cadherin in SMMC-7721 cells transfected with shRNA of CD44 and NC are shown by immunofluorescences under a 400× field. (* means p<0.05, ** means p<0.01, *** means p<0.001 by T-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480719&req=5

Figure 2: Knockdown of CD44 induced the MET in SMMC-7721 and MHCC97-H cellsqRT-PCR (A) and Western-blot (B) analysis of CD44 expression in SMMC-7721 and MHCC97-H cells that were transfected with shRNA of CD44 (KD) and normal control (NC). The expression levels of proteins (C) and mRNA (D) of the EMT markers are shown. (E) The protein expression levels of E-cadherin and N-cadherin in SMMC-7721 cells transfected with shRNA of CD44 and NC are shown by immunofluorescences under a 400× field. (* means p<0.05, ** means p<0.01, *** means p<0.001 by T-test).
Mentions: To investigate the influence of CD44 knockdown on the EMT in HCC cells, we transfected lentivirus with shRNA of CD44 or NC into SMMC-7721 and MHCC97-H cells respectively, and got the stably passaged SMMC-7721-KD, SMMC-7721-NC, MHCC97-H-KD and MHCC97-H-NC. qRT-PCR showed a dramatic CD44 mRNA decrease of 75.01% in SMMC-7721 and 82.08% in MHCC97-H with lentivirus transfection (Fig. 2A). Western blot confirmed the inhibition of CD44 protein in both SMMC-7721 and MHCC97-H cells (Fig. 2B). Because of the CD44 knockdown, the enhanced expression of epithelial marker (E-cadherin) and the reduced expression of mesenchymal markers (N-cadherin and Vimentin) in MHCC97-H-KD and SMMC-7721-KD cells were detected by Western blot (Fig. 2C), qRT-PCR (Fig. 2D) and immunofluorescence (Fig. 2E). These results demonstrated that the knockdown of CD44 in SMMC-7721 and MHCC97-H cells induced a transition to the epithelial phenotype.

Bottom Line: First, we noticed that CD44 expression was associated with the mesenchymal phenotype in HCC cell lines, and knocking down CD44 with lentivirus-mediated shRNA in HCC cell lines resulted in the mesenchymal-epithelial-transition (MET) and the subsequent impaired migration and invasion in vitro.Moreover, in a metastatic mice model established by tail vein injection of luciferase labelled MHCC97-H cells, we confirmed that CD44 knockdown resulted in the decreased metastasis of HCC cells.Furthermore, we found that the induction of MET by CD44 inhibition might be achieved, at least in part, by repressing the ERK/Snail pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepato-Biliary and Pancreto-Splenic Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an, China.

ABSTRACT
Mounting evidence has shown that induction of epithelial-mesenchymal transition (EMT) contributes to the the expression of CSC (cancer stem cell) markers. However, whether and how CSC markers could be involved in regulating EMT has rarely been reported. CD44, being one of the most commonly used CSC markers in hepatocellular carcinoma (HCC), has been demonstrated to act as a multidomain, transmembrane platform that serves to integrate a wide variety of extracellular signals. Therefore, we determined to seek whether CD44 is necessary for the EMT process in HCC. First, we noticed that CD44 expression was associated with the mesenchymal phenotype in HCC cell lines, and knocking down CD44 with lentivirus-mediated shRNA in HCC cell lines resulted in the mesenchymal-epithelial-transition (MET) and the subsequent impaired migration and invasion in vitro. Moreover, in a metastatic mice model established by tail vein injection of luciferase labelled MHCC97-H cells, we confirmed that CD44 knockdown resulted in the decreased metastasis of HCC cells. Furthermore, we found that the induction of MET by CD44 inhibition might be achieved, at least in part, by repressing the ERK/Snail pathway.

No MeSH data available.


Related in: MedlinePlus