Limits...
MicroRNA-31 functions as a tumor suppressor by regulating cell cycle and epithelial-mesenchymal transition regulatory proteins in liver cancer.

Kim HS, Lee KS, Bae HJ, Eun JW, Shen Q, Park SJ, Shin WC, Yang HD, Park M, Park WS, Kang YK, Nam SW - Oncotarget (2015)

Bottom Line: MiR-31 expression was down-regulated in a large cohort of hepatocellular carcinoma (HCC) patients, and low expression of miR-31 was significantly associated with poor prognosis of HCC patients.We also found that ectopic expression of miR-31 mimics reduced metastatic potential of HCC cells by selectively regulating epithelial-mesenchymal transition (EMT) regulatory proteins such as N-cadherin, E-cadherin, vimentin and fibronectin.HCC tissues derived from chemical-induced rat liver cancer models validated that miR-31 expression is significantly down-regulated, and that those cell cycle- and EMT-regulatory proteins are deregulated in rat liver cancer.

View Article: PubMed Central - PubMed

Affiliation: Lab of Oncogenomics, Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.

ABSTRACT
MicroRNA-31 (miR-31) is among the most frequently altered microRNAs in human cancers and altered expression of miR-31 has been detected in a large variety of tumor types, but the functional role of miR-31 still hold both tumor suppressive and oncogenic roles in different tumor types. MiR-31 expression was down-regulated in a large cohort of hepatocellular carcinoma (HCC) patients, and low expression of miR-31 was significantly associated with poor prognosis of HCC patients. Ectopic expression of miR-31 mimics suppressed HCC cell growth by transcriptional deregulation of cell cycle proteins. Additional study evidenced miR-31 directly to suppress HDAC2 and CDK2 expression by inhibiting mRNA translation in HCC cells. We also found that ectopic expression of miR-31 mimics reduced metastatic potential of HCC cells by selectively regulating epithelial-mesenchymal transition (EMT) regulatory proteins such as N-cadherin, E-cadherin, vimentin and fibronectin. HCC tissues derived from chemical-induced rat liver cancer models validated that miR-31 expression is significantly down-regulated, and that those cell cycle- and EMT-regulatory proteins are deregulated in rat liver cancer. Overall, we suggest that miR-31 functions as a tumor suppressor by selectively regulating cell cycle and EMT regulatory proteins in human hepatocarcinogenesis providing a novel target for the molecular treatment of liver malignancies.

No MeSH data available.


Related in: MedlinePlus

MiR-31 suppressed motility and invasion of HCC cells(A) Motility and invasion assay of liver cancer cells transfected with miR-31 or co-transfected miR-31 with AS-miR-31. Invasion assay was performed with transwell-inserts coated with Matrigel. Images were taken with invert microscope (magnification, x100). (B) The cell number of migrated cells were counted in randomly selected fields and presented in bar graph (means ± SD; *P<0.05; **P<0.005; ***P<0.001, Student's t test). (C and D) Western blot analysis of E-cadherin, N-cadherin, vimentin and fibronectin of SNU-449 (C) and SKHep-1 (D) cells. GAPDH was used as the endogenous loading control. Quantitative analysis of western blot data. Densitometry was used to quantify western blot data for E-cadherin, N-cadherin, vimentin and fibronectin, respectively (means ± SD; *P<0.05; **P<0.005; ***P<0.001, Student's t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480737&req=5

Figure 4: MiR-31 suppressed motility and invasion of HCC cells(A) Motility and invasion assay of liver cancer cells transfected with miR-31 or co-transfected miR-31 with AS-miR-31. Invasion assay was performed with transwell-inserts coated with Matrigel. Images were taken with invert microscope (magnification, x100). (B) The cell number of migrated cells were counted in randomly selected fields and presented in bar graph (means ± SD; *P<0.05; **P<0.005; ***P<0.001, Student's t test). (C and D) Western blot analysis of E-cadherin, N-cadherin, vimentin and fibronectin of SNU-449 (C) and SKHep-1 (D) cells. GAPDH was used as the endogenous loading control. Quantitative analysis of western blot data. Densitometry was used to quantify western blot data for E-cadherin, N-cadherin, vimentin and fibronectin, respectively (means ± SD; *P<0.05; **P<0.005; ***P<0.001, Student's t test).

Mentions: EMT has been proposed as a key process in cancer progression. Tumor cells that acquired EMT and metastatic ability show mesenchymal, fibroblast-like phenotypes with cell-cell contact, polarity loss, enabling cells migration and invasion [19]. MiR-31 is well known metastatic suppressor by direct targeting integrin family, RhoA and RDX in various cancers but unknown in liver cancer [14]. Thus, to elucidate the role of miR-31 in the malignant behavior of liver cancer cells, we performed in vitro motility and invasion assays. A modified Boyden chamber assays revealed that ectopic expression of miR-31 mimics significantly suppressed chemoattractant (5% fetal bovine serum)-stimulated migratory and invasive responses of both SNU-449 and SKHep-1 cells, whereas AS-miR-31 co-transfection significantly rescued anti-migratory and invasion effects in the same cells (Fig. 4A and B). In similar, when ras-transformed NIH-3T3 cells were transfected with miR-31 mimics to generalize the effect of miR-31 in the regulation of metastatic potential, we obtained consistent results in both motility and invasion assays (Supplementary Fig. S3). To gain further insight into the regulatory effect of miR-31 on EMT, western blot analysis was performed for the EMT regulatory proteins in in liver cancer cells. Notably, N-cadherin, vimentin and fibronectin, hallmarks of EMT, were dramatically decreased in miR-31 mimics transfectants, whereas E-cadherin, an epithelial markers, was increased in both SNU-449 and SKHep-1 cells (Fig. 4C and D). In contrast, the suppressive effect of miR-31 on EMT molecules in liver cancer cells was significantly rescued by the con-transfection of AS-miR-31. These results suggest that anti-metastatic potential of miR-31 could be attributed to the selective regulation of EMT proteins in liver cancer cells. Next, to validate our observations in in vivo model, we prepared diethylnitrosamine (DEN)–induced liver cancer rat models and examined cell cycle molecules and HDAC2 expressions in hepatocellular carcinoma tissues (Fig. 5A). DEN-induced rat HCC tissues showed the upregulated expression levels of HDAC2, CDK2, cyclin D1, cyclin A, N-cadherin, fibronectin and suppressed expression levels of p21WAF1/Cip1 and E-cadherin accompanied by reduced-miR31 expression compared to normal rat hepatic tissues (Fig. 5B and C).


MicroRNA-31 functions as a tumor suppressor by regulating cell cycle and epithelial-mesenchymal transition regulatory proteins in liver cancer.

Kim HS, Lee KS, Bae HJ, Eun JW, Shen Q, Park SJ, Shin WC, Yang HD, Park M, Park WS, Kang YK, Nam SW - Oncotarget (2015)

MiR-31 suppressed motility and invasion of HCC cells(A) Motility and invasion assay of liver cancer cells transfected with miR-31 or co-transfected miR-31 with AS-miR-31. Invasion assay was performed with transwell-inserts coated with Matrigel. Images were taken with invert microscope (magnification, x100). (B) The cell number of migrated cells were counted in randomly selected fields and presented in bar graph (means ± SD; *P<0.05; **P<0.005; ***P<0.001, Student's t test). (C and D) Western blot analysis of E-cadherin, N-cadherin, vimentin and fibronectin of SNU-449 (C) and SKHep-1 (D) cells. GAPDH was used as the endogenous loading control. Quantitative analysis of western blot data. Densitometry was used to quantify western blot data for E-cadherin, N-cadherin, vimentin and fibronectin, respectively (means ± SD; *P<0.05; **P<0.005; ***P<0.001, Student's t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480737&req=5

Figure 4: MiR-31 suppressed motility and invasion of HCC cells(A) Motility and invasion assay of liver cancer cells transfected with miR-31 or co-transfected miR-31 with AS-miR-31. Invasion assay was performed with transwell-inserts coated with Matrigel. Images were taken with invert microscope (magnification, x100). (B) The cell number of migrated cells were counted in randomly selected fields and presented in bar graph (means ± SD; *P<0.05; **P<0.005; ***P<0.001, Student's t test). (C and D) Western blot analysis of E-cadherin, N-cadherin, vimentin and fibronectin of SNU-449 (C) and SKHep-1 (D) cells. GAPDH was used as the endogenous loading control. Quantitative analysis of western blot data. Densitometry was used to quantify western blot data for E-cadherin, N-cadherin, vimentin and fibronectin, respectively (means ± SD; *P<0.05; **P<0.005; ***P<0.001, Student's t test).
Mentions: EMT has been proposed as a key process in cancer progression. Tumor cells that acquired EMT and metastatic ability show mesenchymal, fibroblast-like phenotypes with cell-cell contact, polarity loss, enabling cells migration and invasion [19]. MiR-31 is well known metastatic suppressor by direct targeting integrin family, RhoA and RDX in various cancers but unknown in liver cancer [14]. Thus, to elucidate the role of miR-31 in the malignant behavior of liver cancer cells, we performed in vitro motility and invasion assays. A modified Boyden chamber assays revealed that ectopic expression of miR-31 mimics significantly suppressed chemoattractant (5% fetal bovine serum)-stimulated migratory and invasive responses of both SNU-449 and SKHep-1 cells, whereas AS-miR-31 co-transfection significantly rescued anti-migratory and invasion effects in the same cells (Fig. 4A and B). In similar, when ras-transformed NIH-3T3 cells were transfected with miR-31 mimics to generalize the effect of miR-31 in the regulation of metastatic potential, we obtained consistent results in both motility and invasion assays (Supplementary Fig. S3). To gain further insight into the regulatory effect of miR-31 on EMT, western blot analysis was performed for the EMT regulatory proteins in in liver cancer cells. Notably, N-cadherin, vimentin and fibronectin, hallmarks of EMT, were dramatically decreased in miR-31 mimics transfectants, whereas E-cadherin, an epithelial markers, was increased in both SNU-449 and SKHep-1 cells (Fig. 4C and D). In contrast, the suppressive effect of miR-31 on EMT molecules in liver cancer cells was significantly rescued by the con-transfection of AS-miR-31. These results suggest that anti-metastatic potential of miR-31 could be attributed to the selective regulation of EMT proteins in liver cancer cells. Next, to validate our observations in in vivo model, we prepared diethylnitrosamine (DEN)–induced liver cancer rat models and examined cell cycle molecules and HDAC2 expressions in hepatocellular carcinoma tissues (Fig. 5A). DEN-induced rat HCC tissues showed the upregulated expression levels of HDAC2, CDK2, cyclin D1, cyclin A, N-cadherin, fibronectin and suppressed expression levels of p21WAF1/Cip1 and E-cadherin accompanied by reduced-miR31 expression compared to normal rat hepatic tissues (Fig. 5B and C).

Bottom Line: MiR-31 expression was down-regulated in a large cohort of hepatocellular carcinoma (HCC) patients, and low expression of miR-31 was significantly associated with poor prognosis of HCC patients.We also found that ectopic expression of miR-31 mimics reduced metastatic potential of HCC cells by selectively regulating epithelial-mesenchymal transition (EMT) regulatory proteins such as N-cadherin, E-cadherin, vimentin and fibronectin.HCC tissues derived from chemical-induced rat liver cancer models validated that miR-31 expression is significantly down-regulated, and that those cell cycle- and EMT-regulatory proteins are deregulated in rat liver cancer.

View Article: PubMed Central - PubMed

Affiliation: Lab of Oncogenomics, Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.

ABSTRACT
MicroRNA-31 (miR-31) is among the most frequently altered microRNAs in human cancers and altered expression of miR-31 has been detected in a large variety of tumor types, but the functional role of miR-31 still hold both tumor suppressive and oncogenic roles in different tumor types. MiR-31 expression was down-regulated in a large cohort of hepatocellular carcinoma (HCC) patients, and low expression of miR-31 was significantly associated with poor prognosis of HCC patients. Ectopic expression of miR-31 mimics suppressed HCC cell growth by transcriptional deregulation of cell cycle proteins. Additional study evidenced miR-31 directly to suppress HDAC2 and CDK2 expression by inhibiting mRNA translation in HCC cells. We also found that ectopic expression of miR-31 mimics reduced metastatic potential of HCC cells by selectively regulating epithelial-mesenchymal transition (EMT) regulatory proteins such as N-cadherin, E-cadherin, vimentin and fibronectin. HCC tissues derived from chemical-induced rat liver cancer models validated that miR-31 expression is significantly down-regulated, and that those cell cycle- and EMT-regulatory proteins are deregulated in rat liver cancer. Overall, we suggest that miR-31 functions as a tumor suppressor by selectively regulating cell cycle and EMT regulatory proteins in human hepatocarcinogenesis providing a novel target for the molecular treatment of liver malignancies.

No MeSH data available.


Related in: MedlinePlus