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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 inhibits liver cancer cell growth by targeting G1/S transition regulatory molecules(A) Ectopic expression of miR-31 suppressed SNU-449 and SKHep-1 cell proliferation. Transfection of antisense miR-31 (AS-miR-31) attenuated anti-growth effect of miR-31. The cell viability was determined by measuring MTT absorbance at A570. Cell growth was measured at every 24 hours. N.C represents negative control microRNA (means ± SD; **P<0.005, ***P<0.001 compared to control, Student's t test). (B) After transfection of miR-31 mimics or co-transfection with AS-miR-31 to SNU-449 and SKHep-1, the DNA content of PI-stained cells was analyzed by flow-cytometry (means ± SD; **P<0.005, ***P<0.001 compared to control, Student's t test). The stained cell number ratios are presented in bar graph (means ± SD; *P<0.05, **P<0.005, Student's t test). (C) Evaluation of apoptosis by annexin V-PITC (AV) and propidium iodide (PI) staining and analysis by flow-cytometry in SNU-449 and SKHep-1 cells after transfection of miR-31 mimics or negative control miRNA (N.C). (D) SNU-449 and SKHep-1 cells were transfected with miR-31 mimics or co-transfected with AS-miR-31. si-HDAC2 and si-CDK2 were used for knockdown of miR-31 target genes, respectively. The protein expression levels of G1/S regulatory molecules were analyzed by immunoblotting. N.C represents negative control miRNA. (E) Co-transfection of miR-31 with 3′UTR-deleted HDAC2 plasmid (pME18s-HDAC2-FLAG) rescued the expressions of G1/S regulatory molecules. The expressions were analyzed by immunoblotting.
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Figure 3: MiR-31 inhibits liver cancer cell growth by targeting G1/S transition regulatory molecules(A) Ectopic expression of miR-31 suppressed SNU-449 and SKHep-1 cell proliferation. Transfection of antisense miR-31 (AS-miR-31) attenuated anti-growth effect of miR-31. The cell viability was determined by measuring MTT absorbance at A570. Cell growth was measured at every 24 hours. N.C represents negative control microRNA (means ± SD; **P<0.005, ***P<0.001 compared to control, Student's t test). (B) After transfection of miR-31 mimics or co-transfection with AS-miR-31 to SNU-449 and SKHep-1, the DNA content of PI-stained cells was analyzed by flow-cytometry (means ± SD; **P<0.005, ***P<0.001 compared to control, Student's t test). The stained cell number ratios are presented in bar graph (means ± SD; *P<0.05, **P<0.005, Student's t test). (C) Evaluation of apoptosis by annexin V-PITC (AV) and propidium iodide (PI) staining and analysis by flow-cytometry in SNU-449 and SKHep-1 cells after transfection of miR-31 mimics or negative control miRNA (N.C). (D) SNU-449 and SKHep-1 cells were transfected with miR-31 mimics or co-transfected with AS-miR-31. si-HDAC2 and si-CDK2 were used for knockdown of miR-31 target genes, respectively. The protein expression levels of G1/S regulatory molecules were analyzed by immunoblotting. N.C represents negative control miRNA. (E) Co-transfection of miR-31 with 3′UTR-deleted HDAC2 plasmid (pME18s-HDAC2-FLAG) rescued the expressions of G1/S regulatory molecules. The expressions were analyzed by immunoblotting.

Mentions: Next, to investigate biological functions of miR-31 in hepatocellular malignant proliferation and transformation, we attempted ectopic expression of miR-31 and studied in the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenytetrazolium bromide (MTT) assay for the measurement of cell growth rate of two different liver cancer cell lines, SNU-449 and SKHep-1. Ectopic overexpression of miR-31 resulted in reduced growth rates of these two different liver cancer cell lines, whereas co-transfection with AS-miR-31 (an antisense inhibitor of miR-31) significantly blocked this anti-growth effect (Fig. 3A). In contrast, overexpression of miR-31 in MIHA and L-O2 (immortalized normal hepatic cell lines) did not effect on cell growth rates of these two different cell lines (Supplementary Fig. S2). The anti-growth effect could be partially explained by the disruption of cell growth regulation on miR-31 targeting, such as cell cycle arrest, cellular senescence or apoptosis. Thus, we next explored the effects of miR-31 overexpression on cell death and cell cycle regulation. Flow cytometric cell cycle analysis indicated that miR-31 overexpression led to an increase in the number of cells in the G1 phase with a concomitant decrease in the number of cells in the S phase and G2/M phase, but AS-miR-31 co-transfection attenuated this effect in the same cells (Fig. 3B). We then stained the cells with annexin V-FITC and PI after transfection of miR-31 mimics for apoptosis analysis. However, miR-31 overexpression showed no significant induction of apoptotic cells compared to miRNA control (Fig. 3C).


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 inhibits liver cancer cell growth by targeting G1/S transition regulatory molecules(A) Ectopic expression of miR-31 suppressed SNU-449 and SKHep-1 cell proliferation. Transfection of antisense miR-31 (AS-miR-31) attenuated anti-growth effect of miR-31. The cell viability was determined by measuring MTT absorbance at A570. Cell growth was measured at every 24 hours. N.C represents negative control microRNA (means ± SD; **P<0.005, ***P<0.001 compared to control, Student's t test). (B) After transfection of miR-31 mimics or co-transfection with AS-miR-31 to SNU-449 and SKHep-1, the DNA content of PI-stained cells was analyzed by flow-cytometry (means ± SD; **P<0.005, ***P<0.001 compared to control, Student's t test). The stained cell number ratios are presented in bar graph (means ± SD; *P<0.05, **P<0.005, Student's t test). (C) Evaluation of apoptosis by annexin V-PITC (AV) and propidium iodide (PI) staining and analysis by flow-cytometry in SNU-449 and SKHep-1 cells after transfection of miR-31 mimics or negative control miRNA (N.C). (D) SNU-449 and SKHep-1 cells were transfected with miR-31 mimics or co-transfected with AS-miR-31. si-HDAC2 and si-CDK2 were used for knockdown of miR-31 target genes, respectively. The protein expression levels of G1/S regulatory molecules were analyzed by immunoblotting. N.C represents negative control miRNA. (E) Co-transfection of miR-31 with 3′UTR-deleted HDAC2 plasmid (pME18s-HDAC2-FLAG) rescued the expressions of G1/S regulatory molecules. The expressions were analyzed by immunoblotting.
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Figure 3: MiR-31 inhibits liver cancer cell growth by targeting G1/S transition regulatory molecules(A) Ectopic expression of miR-31 suppressed SNU-449 and SKHep-1 cell proliferation. Transfection of antisense miR-31 (AS-miR-31) attenuated anti-growth effect of miR-31. The cell viability was determined by measuring MTT absorbance at A570. Cell growth was measured at every 24 hours. N.C represents negative control microRNA (means ± SD; **P<0.005, ***P<0.001 compared to control, Student's t test). (B) After transfection of miR-31 mimics or co-transfection with AS-miR-31 to SNU-449 and SKHep-1, the DNA content of PI-stained cells was analyzed by flow-cytometry (means ± SD; **P<0.005, ***P<0.001 compared to control, Student's t test). The stained cell number ratios are presented in bar graph (means ± SD; *P<0.05, **P<0.005, Student's t test). (C) Evaluation of apoptosis by annexin V-PITC (AV) and propidium iodide (PI) staining and analysis by flow-cytometry in SNU-449 and SKHep-1 cells after transfection of miR-31 mimics or negative control miRNA (N.C). (D) SNU-449 and SKHep-1 cells were transfected with miR-31 mimics or co-transfected with AS-miR-31. si-HDAC2 and si-CDK2 were used for knockdown of miR-31 target genes, respectively. The protein expression levels of G1/S regulatory molecules were analyzed by immunoblotting. N.C represents negative control miRNA. (E) Co-transfection of miR-31 with 3′UTR-deleted HDAC2 plasmid (pME18s-HDAC2-FLAG) rescued the expressions of G1/S regulatory molecules. The expressions were analyzed by immunoblotting.
Mentions: Next, to investigate biological functions of miR-31 in hepatocellular malignant proliferation and transformation, we attempted ectopic expression of miR-31 and studied in the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenytetrazolium bromide (MTT) assay for the measurement of cell growth rate of two different liver cancer cell lines, SNU-449 and SKHep-1. Ectopic overexpression of miR-31 resulted in reduced growth rates of these two different liver cancer cell lines, whereas co-transfection with AS-miR-31 (an antisense inhibitor of miR-31) significantly blocked this anti-growth effect (Fig. 3A). In contrast, overexpression of miR-31 in MIHA and L-O2 (immortalized normal hepatic cell lines) did not effect on cell growth rates of these two different cell lines (Supplementary Fig. S2). The anti-growth effect could be partially explained by the disruption of cell growth regulation on miR-31 targeting, such as cell cycle arrest, cellular senescence or apoptosis. Thus, we next explored the effects of miR-31 overexpression on cell death and cell cycle regulation. Flow cytometric cell cycle analysis indicated that miR-31 overexpression led to an increase in the number of cells in the G1 phase with a concomitant decrease in the number of cells in the S phase and G2/M phase, but AS-miR-31 co-transfection attenuated this effect in the same cells (Fig. 3B). We then stained the cells with annexin V-FITC and PI after transfection of miR-31 mimics for apoptosis analysis. However, miR-31 overexpression showed no significant induction of apoptotic cells compared to miRNA control (Fig. 3C).

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