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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 regulates HDAC2 and CDK2 expression by binding 3′-UTR in hepatocellular carcinoma(A) Differential expression of HDAC2 and CDK2 mRNAs from the mRNA microarray data obtained from NCBI, GEO database (Accession No: GSE36376 and GSE45436). The comparative expression values were showed as scatter plots. The median expression is indicated by horizontal line. The microRNA expression levels are shown on the y axis (log2 intensity, ***P<0.001, Student's t test). (B) Western blot analysis. SNU-449 and SKHep-1 cells were transfected with miR-31 mimics after transfected with Dicer siRNA or negative control siRNA (N.C). The protein levels of HDAC2, CDK2 and Dicer were detected with their specific antibodies. GAPDH was used as the endogenous loading control. (C) The target sites of miR-31 in 3′-UTR of HDAC2 and CDK2 are shown as a schematic representation. The target sequence was predicted by miRNA target prediction program, miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/). (D) Luciferase reporter assay. Wild type or mutant 3′-UTR construct of HDAC2 and CDK2 were cloned into a psi-CHECK2 vector, respectively, and co-transfected with miR-31 mimics in SNU-449 and SKHep-1 cells. Renilla luciferase activities were normalized to firefly luciferase activities. All assays were performed in triplicates and repeated at least three times. (means ± SD; *P<0.05; **P<0.005, ***P<0.001, Student's t test). (E) The Biotin-labeled miR-31-mRNA pull down assay. SNU-449 and SKHep-1 cells were transfected with Biotin-labeled microRNA control (Bio-N.C) or Biotin-labeled miR-31 mimics for 48 hours. The expressions of HDAC2 and CDK2 were measured by qRT-PCR and normalized to GAPDH (means ± SD; **P<0.005; ***P<0.001).
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Figure 2: MiR-31 regulates HDAC2 and CDK2 expression by binding 3′-UTR in hepatocellular carcinoma(A) Differential expression of HDAC2 and CDK2 mRNAs from the mRNA microarray data obtained from NCBI, GEO database (Accession No: GSE36376 and GSE45436). The comparative expression values were showed as scatter plots. The median expression is indicated by horizontal line. The microRNA expression levels are shown on the y axis (log2 intensity, ***P<0.001, Student's t test). (B) Western blot analysis. SNU-449 and SKHep-1 cells were transfected with miR-31 mimics after transfected with Dicer siRNA or negative control siRNA (N.C). The protein levels of HDAC2, CDK2 and Dicer were detected with their specific antibodies. GAPDH was used as the endogenous loading control. (C) The target sites of miR-31 in 3′-UTR of HDAC2 and CDK2 are shown as a schematic representation. The target sequence was predicted by miRNA target prediction program, miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/). (D) Luciferase reporter assay. Wild type or mutant 3′-UTR construct of HDAC2 and CDK2 were cloned into a psi-CHECK2 vector, respectively, and co-transfected with miR-31 mimics in SNU-449 and SKHep-1 cells. Renilla luciferase activities were normalized to firefly luciferase activities. All assays were performed in triplicates and repeated at least three times. (means ± SD; *P<0.05; **P<0.005, ***P<0.001, Student's t test). (E) The Biotin-labeled miR-31-mRNA pull down assay. SNU-449 and SKHep-1 cells were transfected with Biotin-labeled microRNA control (Bio-N.C) or Biotin-labeled miR-31 mimics for 48 hours. The expressions of HDAC2 and CDK2 were measured by qRT-PCR and normalized to GAPDH (means ± SD; **P<0.005; ***P<0.001).

Mentions: It has been demonstrated that all the known processes of cancer biology, including apoptosis, proliferation, survival, and metastasis, are regulated by small regulatory non-coding RNAs consisting of approximately 19–25 nucleotides; e.g. miRNAs [5]. Therefore, we hypothesized that some cancer-driver genes targeted by miR-31 are up-regulated in HCC as miR-31 was down-regulated in HCC. Thus, to identify miR-31 target genes, we used the target prediction program, miRWALK (http://www.umm.uniheidelberg.de/apps/zmf/mirwalk/), a comprehensive database on miRNAs with eight established program (RNA22, miRanda, miRDB, TargetScan, RNAhybrid, PITA, PICTAR, and Diana-microT) [16]. From this database, at least in six out of eight different prediction programs, 399 genes were predicted to be targeted by miR-31 (data not shown). Of these 399 genes, we were able to identify 36 genes that were commonly up-regulated in three different HCC cohort data sets, GSE14520, GSE22058 and GSE16757, respectively (Supplementary Table S1). Among these, our previous study has demonstrated that histone deacetylase 2 (HDAC2) and cyclin-dependent kinase 2 (CDK2) were overexpressed in HCC [17]. We then recapitulated the expression of HDAC2 and CDK2 genes from two more cohorts of HCC patients to generalize our finding. Consistently, HDAC2 and CDK2 genes were significantly over-expressed in these two different HCC cohorts (Fig. 2A). The fact that HDAC2 and CDK2 are up-regulated in HCC led us to hypothesize that normal HDAC2 and CDK2 expressions are balanced by endogenous miR-31, which selectively controls HDAC2 and CDK2 mRNA translation in normal hepatic liver cells. Thus, to support our hypothesis that HDAC2 and CDK2 expressions are regulated by miR-31 in HCC cell lines, we introduced Dicer specific siRNAs to block miRNA biogenesis in HCC cells. As shown in Fig. 2B, Dicer knockdown augmented HDAC2 and CDK2 protein expressions in SNU-449 and SKHep-1 cells, whereas co-transfection of miR-31 mimics attenuated Dicer knockdown effect on the same cells. Then, to determine whether HDAC2 and CDK2 are selectively regulated by miR-31 via direct interaction with the 3′-UTR of these genes, we cloned the 3′-UTR of HDAC2 and CDK2 into a reporter vector linking the luciferase open reading frame downstream to generate psi-CHECK2-HDAC2_3′-UTR and psiCHECK-CDK2_3′-UTR plasmid, respectively (Fig. 2C and Supplementary Fig. S1). Next, to verify that miR-31 specifically binds to 3′UTRs of CDK2 and HDAC2 to interfere translation of those transcripts, mutant vectors harboring random mutation sequences of miR-31 biding sites of the 3′UTR of CDK2 and HDAC2 genes were generated, and then each vector was co-transfected with miR-31 into SNU-449 and SKHep-1 cells. It was found that miR-31 was able to suppress reporter gene activity in these cells, whereas mutants plasmids showed no changes in the reporter gene activity in both SNU-449 and SKHep-1 cells indicating miR-31 selectively regulate both HDAC2 and CDK2 expressions in HCC cells in vitro (Fig. 2D). In addition, to clarify the direct interaction between miR-31 and 3′-UTRs of the two transcripts, we carried out biotin-labeled RNA pull-down assays. As expected, when Bio-miR-31 mimics were transfected to both SNU-449 and SKHep-1 cells, HDAC2 and CDK2 transcripts were enriched in these cells compared to that of Bio-microRNA control transfectants (Fig. 2E). These results demonstrate that miR-31 is a direct regulator of endogenous expression HDAC2 and CDK2 in liver cancer cells.


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 regulates HDAC2 and CDK2 expression by binding 3′-UTR in hepatocellular carcinoma(A) Differential expression of HDAC2 and CDK2 mRNAs from the mRNA microarray data obtained from NCBI, GEO database (Accession No: GSE36376 and GSE45436). The comparative expression values were showed as scatter plots. The median expression is indicated by horizontal line. The microRNA expression levels are shown on the y axis (log2 intensity, ***P<0.001, Student's t test). (B) Western blot analysis. SNU-449 and SKHep-1 cells were transfected with miR-31 mimics after transfected with Dicer siRNA or negative control siRNA (N.C). The protein levels of HDAC2, CDK2 and Dicer were detected with their specific antibodies. GAPDH was used as the endogenous loading control. (C) The target sites of miR-31 in 3′-UTR of HDAC2 and CDK2 are shown as a schematic representation. The target sequence was predicted by miRNA target prediction program, miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/). (D) Luciferase reporter assay. Wild type or mutant 3′-UTR construct of HDAC2 and CDK2 were cloned into a psi-CHECK2 vector, respectively, and co-transfected with miR-31 mimics in SNU-449 and SKHep-1 cells. Renilla luciferase activities were normalized to firefly luciferase activities. All assays were performed in triplicates and repeated at least three times. (means ± SD; *P<0.05; **P<0.005, ***P<0.001, Student's t test). (E) The Biotin-labeled miR-31-mRNA pull down assay. SNU-449 and SKHep-1 cells were transfected with Biotin-labeled microRNA control (Bio-N.C) or Biotin-labeled miR-31 mimics for 48 hours. The expressions of HDAC2 and CDK2 were measured by qRT-PCR and normalized to GAPDH (means ± SD; **P<0.005; ***P<0.001).
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Related In: Results  -  Collection

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Figure 2: MiR-31 regulates HDAC2 and CDK2 expression by binding 3′-UTR in hepatocellular carcinoma(A) Differential expression of HDAC2 and CDK2 mRNAs from the mRNA microarray data obtained from NCBI, GEO database (Accession No: GSE36376 and GSE45436). The comparative expression values were showed as scatter plots. The median expression is indicated by horizontal line. The microRNA expression levels are shown on the y axis (log2 intensity, ***P<0.001, Student's t test). (B) Western blot analysis. SNU-449 and SKHep-1 cells were transfected with miR-31 mimics after transfected with Dicer siRNA or negative control siRNA (N.C). The protein levels of HDAC2, CDK2 and Dicer were detected with their specific antibodies. GAPDH was used as the endogenous loading control. (C) The target sites of miR-31 in 3′-UTR of HDAC2 and CDK2 are shown as a schematic representation. The target sequence was predicted by miRNA target prediction program, miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/). (D) Luciferase reporter assay. Wild type or mutant 3′-UTR construct of HDAC2 and CDK2 were cloned into a psi-CHECK2 vector, respectively, and co-transfected with miR-31 mimics in SNU-449 and SKHep-1 cells. Renilla luciferase activities were normalized to firefly luciferase activities. All assays were performed in triplicates and repeated at least three times. (means ± SD; *P<0.05; **P<0.005, ***P<0.001, Student's t test). (E) The Biotin-labeled miR-31-mRNA pull down assay. SNU-449 and SKHep-1 cells were transfected with Biotin-labeled microRNA control (Bio-N.C) or Biotin-labeled miR-31 mimics for 48 hours. The expressions of HDAC2 and CDK2 were measured by qRT-PCR and normalized to GAPDH (means ± SD; **P<0.005; ***P<0.001).
Mentions: It has been demonstrated that all the known processes of cancer biology, including apoptosis, proliferation, survival, and metastasis, are regulated by small regulatory non-coding RNAs consisting of approximately 19–25 nucleotides; e.g. miRNAs [5]. Therefore, we hypothesized that some cancer-driver genes targeted by miR-31 are up-regulated in HCC as miR-31 was down-regulated in HCC. Thus, to identify miR-31 target genes, we used the target prediction program, miRWALK (http://www.umm.uniheidelberg.de/apps/zmf/mirwalk/), a comprehensive database on miRNAs with eight established program (RNA22, miRanda, miRDB, TargetScan, RNAhybrid, PITA, PICTAR, and Diana-microT) [16]. From this database, at least in six out of eight different prediction programs, 399 genes were predicted to be targeted by miR-31 (data not shown). Of these 399 genes, we were able to identify 36 genes that were commonly up-regulated in three different HCC cohort data sets, GSE14520, GSE22058 and GSE16757, respectively (Supplementary Table S1). Among these, our previous study has demonstrated that histone deacetylase 2 (HDAC2) and cyclin-dependent kinase 2 (CDK2) were overexpressed in HCC [17]. We then recapitulated the expression of HDAC2 and CDK2 genes from two more cohorts of HCC patients to generalize our finding. Consistently, HDAC2 and CDK2 genes were significantly over-expressed in these two different HCC cohorts (Fig. 2A). The fact that HDAC2 and CDK2 are up-regulated in HCC led us to hypothesize that normal HDAC2 and CDK2 expressions are balanced by endogenous miR-31, which selectively controls HDAC2 and CDK2 mRNA translation in normal hepatic liver cells. Thus, to support our hypothesis that HDAC2 and CDK2 expressions are regulated by miR-31 in HCC cell lines, we introduced Dicer specific siRNAs to block miRNA biogenesis in HCC cells. As shown in Fig. 2B, Dicer knockdown augmented HDAC2 and CDK2 protein expressions in SNU-449 and SKHep-1 cells, whereas co-transfection of miR-31 mimics attenuated Dicer knockdown effect on the same cells. Then, to determine whether HDAC2 and CDK2 are selectively regulated by miR-31 via direct interaction with the 3′-UTR of these genes, we cloned the 3′-UTR of HDAC2 and CDK2 into a reporter vector linking the luciferase open reading frame downstream to generate psi-CHECK2-HDAC2_3′-UTR and psiCHECK-CDK2_3′-UTR plasmid, respectively (Fig. 2C and Supplementary Fig. S1). Next, to verify that miR-31 specifically binds to 3′UTRs of CDK2 and HDAC2 to interfere translation of those transcripts, mutant vectors harboring random mutation sequences of miR-31 biding sites of the 3′UTR of CDK2 and HDAC2 genes were generated, and then each vector was co-transfected with miR-31 into SNU-449 and SKHep-1 cells. It was found that miR-31 was able to suppress reporter gene activity in these cells, whereas mutants plasmids showed no changes in the reporter gene activity in both SNU-449 and SKHep-1 cells indicating miR-31 selectively regulate both HDAC2 and CDK2 expressions in HCC cells in vitro (Fig. 2D). In addition, to clarify the direct interaction between miR-31 and 3′-UTRs of the two transcripts, we carried out biotin-labeled RNA pull-down assays. As expected, when Bio-miR-31 mimics were transfected to both SNU-449 and SKHep-1 cells, HDAC2 and CDK2 transcripts were enriched in these cells compared to that of Bio-microRNA control transfectants (Fig. 2E). These results demonstrate that miR-31 is a direct regulator of endogenous expression HDAC2 and CDK2 in liver cancer cells.

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