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MicroRNA-431 inhibits migration and invasion of hepatocellular carcinoma cells by targeting the ZEB1-mediated epithelial-mensenchymal transition.

Sun K, Zeng T, Huang D, Liu Z, Huang S, Liu J, Qu Z - FEBS Open Bio (2015)

Bottom Line: Herein, we found that miR-431 expression was reduced in HCC tissues compared to noncancerous tissues.We found that up-regulation of miR-431 expression decreased zinc finger E-box binding homeobox 1 (ZEB1) expression and inhibited the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and decreased vimentin expression in HCCLM3 cells.In conclusion, miR-431 inhibits migration and invasion of HCC cells by suppressing ZEB1-mediated EMT.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China.

ABSTRACT
MicroRNA-431 (miR-431) has been recognized as an oncogenic miRNA, being implicated in the initiation and development of human cancers. Recently, deregulation of miR-431 has been reported in several tumors. However, the clinical significance of miR-431 and its underlying role in human hepatocellular carcinoma (HCC) are poorly explored. Herein, we found that miR-431 expression was reduced in HCC tissues compared to noncancerous tissues. Otherwise, down-regulation of miR-431 was observed in aggressive tumor tissues. The levels of miR-431 expression in HCC cell lines were significantly lower than that in a nontransformed hepatic cell line. Clinical association analyses disclosed that a low level of miR-431 was prominently associated with poor prognostic features of HCC including venous infiltration, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) tumor stage. Our in vitro studies showed that up-regulation of miR-431 expression reduced cell invasion and migration in HCCLM3 cells. In contrast, down-regulation of miR-431 expression promoted SMMC-7721 cell invasion and migration. We found that up-regulation of miR-431 expression decreased zinc finger E-box binding homeobox 1 (ZEB1) expression and inhibited the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and decreased vimentin expression in HCCLM3 cells. Otherwise, down-regulation of miR-431 expression increased ZEB1 expression and promoted EMT in SMMC-7721 cells. Significantly, ZEB1 was identified as a target of miR-431 in HCC. ZEB1 knockdown abrogated the effect of miR-431 silencing on EMT and cell mobility in SMMC-7721 cells. In conclusion, miR-431 inhibits migration and invasion of HCC cells by suppressing ZEB1-mediated EMT.

No MeSH data available.


Related in: MedlinePlus

MiR-431 inhibits EMT by targeting ZEB1 in HCC cells. MiR-431 silenced SMMC-7721 cells and control cells that were transfected with scrambled siRNA or ZEB1 siRNA were subjected to Western blot and Transwell assays. (A) ZEB1 knockdown led to up-regulation of E-cadherin and down-regulation of vimentin in negative control (NC) vectors transfected SMMC-7721 cells. Otherwise, ZEB1 deletion abolished the effect of miR-431 down-regulation on EMT with elevated expression of E-cadherin and reduced expression of vimentin in SMMC-7721 cells. n = 6; ∗P < 0.05. (B) ZEB1 knockdown reduced cell migration and invasion in control SMMC-7721 cells. Furthermore, ZEB1 knockdown abrogated the effect of miR-431 silencing on SMMC-7721 cell mobility with reduced abilities of cell migration and invasion. n = 3; ∗P < 0.05.
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f0025: MiR-431 inhibits EMT by targeting ZEB1 in HCC cells. MiR-431 silenced SMMC-7721 cells and control cells that were transfected with scrambled siRNA or ZEB1 siRNA were subjected to Western blot and Transwell assays. (A) ZEB1 knockdown led to up-regulation of E-cadherin and down-regulation of vimentin in negative control (NC) vectors transfected SMMC-7721 cells. Otherwise, ZEB1 deletion abolished the effect of miR-431 down-regulation on EMT with elevated expression of E-cadherin and reduced expression of vimentin in SMMC-7721 cells. n = 6; ∗P < 0.05. (B) ZEB1 knockdown reduced cell migration and invasion in control SMMC-7721 cells. Furthermore, ZEB1 knockdown abrogated the effect of miR-431 silencing on SMMC-7721 cell mobility with reduced abilities of cell migration and invasion. n = 3; ∗P < 0.05.

Mentions: To uncover the underlying mechanisms by which miR-431 exert its functional role in HCC cells, we searched two publicly available databases (TargetScan 6.2 and miRanDa) for the predicted target of miR-431. ZEB1, an important regulator of EMT in HCC, was found to be one of the predicted targets of miR-431. HCCLM3 cells that were transduced with miR-control or miR-431 were subjected to WB for ZEB1 and EMT markers. As shown in Fig. 3, overexpression of miR-431 significantly reduced the expression of ZEB1 protein and resulted in increase of E-cadherin level and decrease of vimentin level in HCCLM3 cells (P < 0.05, respectively, Fig. 3). In contrast, knockdown of miR-431 increased the level of ZEB1 protein and led to decrease of E-cadherin expression and increase of vimentin expression (P < 0.05, respectively). Herein, a dual-luciferase reporter assay was performed to disclose whether miR-431 directly binds to the 3′-UTR of ZEB1 mRNA. As expected, miR-431 obviously reduced the luciferase activity of ZEB1 containing a wt 3′-UTR (P < 0.05, Fig. 4A and B). While an increase in the luciferase activity of wt ZEB1 3′-UTR was observed after anti-miR-431 tranfection (P < 0.05, Fig. 4A and B). However, there were no significant changes of the luciferase activity of ZEB1 with an mt 3′-UTR after corresponding miRNA vectors transfection (Fig. 4A and B). Notably, ZEB1 knockdown by a specific siRNA abolished the effect of miR-431 down-regulation on EMT with elevated expression of E-cadherin and reduced expression of vimentin in SMMC-7721 cells (P < 0.05, respectively, Fig. 5A). And ZEB1 knockdown abrogated the effect of miR-431 silencing on SMMC-7721 cell mobility with reduced abilities of cell migration and invasion (P < 0.05, respectively, Fig. 5B). Thus, our results indicate that ZEB1 is a functional target of miR-431 in HCC.


MicroRNA-431 inhibits migration and invasion of hepatocellular carcinoma cells by targeting the ZEB1-mediated epithelial-mensenchymal transition.

Sun K, Zeng T, Huang D, Liu Z, Huang S, Liu J, Qu Z - FEBS Open Bio (2015)

MiR-431 inhibits EMT by targeting ZEB1 in HCC cells. MiR-431 silenced SMMC-7721 cells and control cells that were transfected with scrambled siRNA or ZEB1 siRNA were subjected to Western blot and Transwell assays. (A) ZEB1 knockdown led to up-regulation of E-cadherin and down-regulation of vimentin in negative control (NC) vectors transfected SMMC-7721 cells. Otherwise, ZEB1 deletion abolished the effect of miR-431 down-regulation on EMT with elevated expression of E-cadherin and reduced expression of vimentin in SMMC-7721 cells. n = 6; ∗P < 0.05. (B) ZEB1 knockdown reduced cell migration and invasion in control SMMC-7721 cells. Furthermore, ZEB1 knockdown abrogated the effect of miR-431 silencing on SMMC-7721 cell mobility with reduced abilities of cell migration and invasion. n = 3; ∗P < 0.05.
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f0025: MiR-431 inhibits EMT by targeting ZEB1 in HCC cells. MiR-431 silenced SMMC-7721 cells and control cells that were transfected with scrambled siRNA or ZEB1 siRNA were subjected to Western blot and Transwell assays. (A) ZEB1 knockdown led to up-regulation of E-cadherin and down-regulation of vimentin in negative control (NC) vectors transfected SMMC-7721 cells. Otherwise, ZEB1 deletion abolished the effect of miR-431 down-regulation on EMT with elevated expression of E-cadherin and reduced expression of vimentin in SMMC-7721 cells. n = 6; ∗P < 0.05. (B) ZEB1 knockdown reduced cell migration and invasion in control SMMC-7721 cells. Furthermore, ZEB1 knockdown abrogated the effect of miR-431 silencing on SMMC-7721 cell mobility with reduced abilities of cell migration and invasion. n = 3; ∗P < 0.05.
Mentions: To uncover the underlying mechanisms by which miR-431 exert its functional role in HCC cells, we searched two publicly available databases (TargetScan 6.2 and miRanDa) for the predicted target of miR-431. ZEB1, an important regulator of EMT in HCC, was found to be one of the predicted targets of miR-431. HCCLM3 cells that were transduced with miR-control or miR-431 were subjected to WB for ZEB1 and EMT markers. As shown in Fig. 3, overexpression of miR-431 significantly reduced the expression of ZEB1 protein and resulted in increase of E-cadherin level and decrease of vimentin level in HCCLM3 cells (P < 0.05, respectively, Fig. 3). In contrast, knockdown of miR-431 increased the level of ZEB1 protein and led to decrease of E-cadherin expression and increase of vimentin expression (P < 0.05, respectively). Herein, a dual-luciferase reporter assay was performed to disclose whether miR-431 directly binds to the 3′-UTR of ZEB1 mRNA. As expected, miR-431 obviously reduced the luciferase activity of ZEB1 containing a wt 3′-UTR (P < 0.05, Fig. 4A and B). While an increase in the luciferase activity of wt ZEB1 3′-UTR was observed after anti-miR-431 tranfection (P < 0.05, Fig. 4A and B). However, there were no significant changes of the luciferase activity of ZEB1 with an mt 3′-UTR after corresponding miRNA vectors transfection (Fig. 4A and B). Notably, ZEB1 knockdown by a specific siRNA abolished the effect of miR-431 down-regulation on EMT with elevated expression of E-cadherin and reduced expression of vimentin in SMMC-7721 cells (P < 0.05, respectively, Fig. 5A). And ZEB1 knockdown abrogated the effect of miR-431 silencing on SMMC-7721 cell mobility with reduced abilities of cell migration and invasion (P < 0.05, respectively, Fig. 5B). Thus, our results indicate that ZEB1 is a functional target of miR-431 in HCC.

Bottom Line: Herein, we found that miR-431 expression was reduced in HCC tissues compared to noncancerous tissues.We found that up-regulation of miR-431 expression decreased zinc finger E-box binding homeobox 1 (ZEB1) expression and inhibited the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and decreased vimentin expression in HCCLM3 cells.In conclusion, miR-431 inhibits migration and invasion of HCC cells by suppressing ZEB1-mediated EMT.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China.

ABSTRACT
MicroRNA-431 (miR-431) has been recognized as an oncogenic miRNA, being implicated in the initiation and development of human cancers. Recently, deregulation of miR-431 has been reported in several tumors. However, the clinical significance of miR-431 and its underlying role in human hepatocellular carcinoma (HCC) are poorly explored. Herein, we found that miR-431 expression was reduced in HCC tissues compared to noncancerous tissues. Otherwise, down-regulation of miR-431 was observed in aggressive tumor tissues. The levels of miR-431 expression in HCC cell lines were significantly lower than that in a nontransformed hepatic cell line. Clinical association analyses disclosed that a low level of miR-431 was prominently associated with poor prognostic features of HCC including venous infiltration, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) tumor stage. Our in vitro studies showed that up-regulation of miR-431 expression reduced cell invasion and migration in HCCLM3 cells. In contrast, down-regulation of miR-431 expression promoted SMMC-7721 cell invasion and migration. We found that up-regulation of miR-431 expression decreased zinc finger E-box binding homeobox 1 (ZEB1) expression and inhibited the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and decreased vimentin expression in HCCLM3 cells. Otherwise, down-regulation of miR-431 expression increased ZEB1 expression and promoted EMT in SMMC-7721 cells. Significantly, ZEB1 was identified as a target of miR-431 in HCC. ZEB1 knockdown abrogated the effect of miR-431 silencing on EMT and cell mobility in SMMC-7721 cells. In conclusion, miR-431 inhibits migration and invasion of HCC cells by suppressing ZEB1-mediated EMT.

No MeSH data available.


Related in: MedlinePlus