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

Inactivation mechanism of tumor suppressor miR-31 in liver cancer(A) Cross-cancer summary of homozygous mutations and copy number variations in all cancers available on cBioPortal (http://www.cbioportal.org). The arrows represents hepatocellular carcinoma. (B) Western blot analysis. SNU-449 and SKHep-1 cells were treated with DZNep (0.1% DMSO or 5 μM of 5-zaz-dC). The expressions of EZH2, H3 Lys-27 hyper-methylation (H3K27me3), HDAC2 and CDK2 were analyzed with immunoblotting. (C) A qRT-PCR analysis of miR-31 expression in DZNep treated liver cancer cell lines (means ± SD, *P<0.05, Student's t test). (D) Western blot analysis. The liver cancer cell lines, SNU-449 and SKHep-1, were treated with indicated drug (01% DMSO or 10 μM of 5-aza-dC), or transfected with siRNA (negative control siRNA, si-DNMT1), and analyzed protein expressions of miR-31 target genes, HDAC2 and CDK2. (E) A qRT-PCR analysis of miR-31 in 5-aza-dC treated, or siRNA (negative control siRNA, si-DNMT1) transfected SNU-449 and SKHep-1 cells (means ± SD; **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 6: Inactivation mechanism of tumor suppressor miR-31 in liver cancer(A) Cross-cancer summary of homozygous mutations and copy number variations in all cancers available on cBioPortal (http://www.cbioportal.org). The arrows represents hepatocellular carcinoma. (B) Western blot analysis. SNU-449 and SKHep-1 cells were treated with DZNep (0.1% DMSO or 5 μM of 5-zaz-dC). The expressions of EZH2, H3 Lys-27 hyper-methylation (H3K27me3), HDAC2 and CDK2 were analyzed with immunoblotting. (C) A qRT-PCR analysis of miR-31 expression in DZNep treated liver cancer cell lines (means ± SD, *P<0.05, Student's t test). (D) Western blot analysis. The liver cancer cell lines, SNU-449 and SKHep-1, were treated with indicated drug (01% DMSO or 10 μM of 5-aza-dC), or transfected with siRNA (negative control siRNA, si-DNMT1), and analyzed protein expressions of miR-31 target genes, HDAC2 and CDK2. (E) A qRT-PCR analysis of miR-31 in 5-aza-dC treated, or siRNA (negative control siRNA, si-DNMT1) transfected SNU-449 and SKHep-1 cells (means ± SD; **P<0.005; ***P<0.001, Student's t test).

Mentions: Lastly, to gain further insight into the inactivation mechanism of miR-31 in liver cancer, we investigated whether miR-31 locus is deleted in liver cancer using TCGA data available on cBioPortal (www.cbioportal.org). From this, we found that the incidence of homozygous deletion of miR-31 gene locus was very low (1.3 ~ 1.6%) in liver cancer compared to that of bladder or pancreatic cancer (Fig. 6A). In addition to genomic deletion, epigenetic gene silencing is another tumor suppressor inactivation mechanism. Since EZH2, a core component of polycomb repressive complex2 (PRC2), was reported to be over-expressed in HCC, we assumed that hyper-methylation of H3 Lys-27 residue may be related with the suppression of miR-31 [20]. To clarify that expression of miR-31 is regulated by EZH2, cells were treated with DZNep (3-Deazaneplanocin A, an inhibitor of S-adenosylmethionine-dependent methyltransferase, and stimulates degradation of EZH2). Notably, treatment of DZNep elicited remarkable suppression of EZH2, HDAC2 and CDK2 proteins with concomitant increase of miR-31 expression in SNU-449 and SKHep-1 cells (Fig. 6B and C). Promoter hyper-methylation is also another efficient way of gene deregulation. In prostate cancer, hyper-methylation of miR-31 promoter was responsible for its low expression and contributed tumorigenesis [21]. Therefore, cells were treated with 5-aza-dC (Azacitidine, 5-aza-2′deoxycytidine), a chemical analogue of the cytosine nucleoside causing hypomethylation of DNA, or DNMT1 (DNA-methyltrasnferase 1)-siRNA, and performed western blot analysis. Disruption of DNA methylation by either 5-aza-dC treatment or DNMT1 knockdown caused the induction of miR-31 expression, and thereby suppressed HDAC2 and CDK2 expression in both SNU-449 and SKHep-1cells (Fig. 6D and E). These results provide the underlying mechanisms leading to the suppression of endogenous miR-31 in HCC.


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)

Inactivation mechanism of tumor suppressor miR-31 in liver cancer(A) Cross-cancer summary of homozygous mutations and copy number variations in all cancers available on cBioPortal (http://www.cbioportal.org). The arrows represents hepatocellular carcinoma. (B) Western blot analysis. SNU-449 and SKHep-1 cells were treated with DZNep (0.1% DMSO or 5 μM of 5-zaz-dC). The expressions of EZH2, H3 Lys-27 hyper-methylation (H3K27me3), HDAC2 and CDK2 were analyzed with immunoblotting. (C) A qRT-PCR analysis of miR-31 expression in DZNep treated liver cancer cell lines (means ± SD, *P<0.05, Student's t test). (D) Western blot analysis. The liver cancer cell lines, SNU-449 and SKHep-1, were treated with indicated drug (01% DMSO or 10 μM of 5-aza-dC), or transfected with siRNA (negative control siRNA, si-DNMT1), and analyzed protein expressions of miR-31 target genes, HDAC2 and CDK2. (E) A qRT-PCR analysis of miR-31 in 5-aza-dC treated, or siRNA (negative control siRNA, si-DNMT1) transfected SNU-449 and SKHep-1 cells (means ± SD; **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 6: Inactivation mechanism of tumor suppressor miR-31 in liver cancer(A) Cross-cancer summary of homozygous mutations and copy number variations in all cancers available on cBioPortal (http://www.cbioportal.org). The arrows represents hepatocellular carcinoma. (B) Western blot analysis. SNU-449 and SKHep-1 cells were treated with DZNep (0.1% DMSO or 5 μM of 5-zaz-dC). The expressions of EZH2, H3 Lys-27 hyper-methylation (H3K27me3), HDAC2 and CDK2 were analyzed with immunoblotting. (C) A qRT-PCR analysis of miR-31 expression in DZNep treated liver cancer cell lines (means ± SD, *P<0.05, Student's t test). (D) Western blot analysis. The liver cancer cell lines, SNU-449 and SKHep-1, were treated with indicated drug (01% DMSO or 10 μM of 5-aza-dC), or transfected with siRNA (negative control siRNA, si-DNMT1), and analyzed protein expressions of miR-31 target genes, HDAC2 and CDK2. (E) A qRT-PCR analysis of miR-31 in 5-aza-dC treated, or siRNA (negative control siRNA, si-DNMT1) transfected SNU-449 and SKHep-1 cells (means ± SD; **P<0.005; ***P<0.001, Student's t test).
Mentions: Lastly, to gain further insight into the inactivation mechanism of miR-31 in liver cancer, we investigated whether miR-31 locus is deleted in liver cancer using TCGA data available on cBioPortal (www.cbioportal.org). From this, we found that the incidence of homozygous deletion of miR-31 gene locus was very low (1.3 ~ 1.6%) in liver cancer compared to that of bladder or pancreatic cancer (Fig. 6A). In addition to genomic deletion, epigenetic gene silencing is another tumor suppressor inactivation mechanism. Since EZH2, a core component of polycomb repressive complex2 (PRC2), was reported to be over-expressed in HCC, we assumed that hyper-methylation of H3 Lys-27 residue may be related with the suppression of miR-31 [20]. To clarify that expression of miR-31 is regulated by EZH2, cells were treated with DZNep (3-Deazaneplanocin A, an inhibitor of S-adenosylmethionine-dependent methyltransferase, and stimulates degradation of EZH2). Notably, treatment of DZNep elicited remarkable suppression of EZH2, HDAC2 and CDK2 proteins with concomitant increase of miR-31 expression in SNU-449 and SKHep-1 cells (Fig. 6B and C). Promoter hyper-methylation is also another efficient way of gene deregulation. In prostate cancer, hyper-methylation of miR-31 promoter was responsible for its low expression and contributed tumorigenesis [21]. Therefore, cells were treated with 5-aza-dC (Azacitidine, 5-aza-2′deoxycytidine), a chemical analogue of the cytosine nucleoside causing hypomethylation of DNA, or DNMT1 (DNA-methyltrasnferase 1)-siRNA, and performed western blot analysis. Disruption of DNA methylation by either 5-aza-dC treatment or DNMT1 knockdown caused the induction of miR-31 expression, and thereby suppressed HDAC2 and CDK2 expression in both SNU-449 and SKHep-1cells (Fig. 6D and E). These results provide the underlying mechanisms leading to the suppression of endogenous miR-31 in HCC.

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