miR-185 Inhibits Hepatocellular Carcinoma Growth by Targeting the DNMT1/PTEN/Akt Pathway

Abstract

miRNAs have recently been implicated in hepatocarcinogenesis, although the actions and mechanisms of individual miRNAs remain incompletely understood. We examined the biological functions and molecular mechanisms of miR-185 in hepatocellular carcinoma (HCC). The expression of miR-185 is decreased in human HCC tissues compared with the nonneoplastic liver parenchyma. Quantitative RT-PCR showed a reduction of miR-185 in human HCC cells compared with primary hepatocytes. miR-185 overexpression in human HCC cells inhibited cell proliferation and invasion in vitro and prevented tumor growth in SCID mice. miR-185 overexpression inhibited DNMT1 3′ untranslated region luciferase reporter activity in HCC cells; this effect was abolished when the miR-185 binding site was mutated. miR-185 mimic or overexpression decreased the level of DNMT1 protein in HCC cells. These findings establish DNMT1 as a bona fide target of miR-185 in HCC cells. The role of DNMT1 in miR-185–induced inhibition of HCC growth was further supported by the fact that DNMT1 overexpression prevented miR-185–induced inhibition of HCC cell proliferation/invasion. miR-185 mimic or overexpression reduced PTEN promoter DNA methylation and enhanced PTEN expression, leading to the inhibition of Akt phosphorylation; these effects were partially reversed by DNMT1 overexpression. These results provide novel evidence that miR-185 inhibits HCC cell growth by targeting DNMT1, leading to PTEN induction and Akt inhibition. Thus, reactivation or induction of miR-185 may represent a novel therapeutic strategy for HCC treatment. miRNAs have recently been implicated in hepatocarcinogenesis, although the actions and mechanisms of individual miRNAs remain incompletely understood. We examined the biological functions and molecular mechanisms of miR-185 in hepatocellular carcinoma (HCC). The expression of miR-185 is decreased in human HCC tissues compared with the nonneoplastic liver parenchyma. Quantitative RT-PCR showed a reduction of miR-185 in human HCC cells compared with primary hepatocytes. miR-185 overexpression in human HCC cells inhibited cell proliferation and invasion in vitro and prevented tumor growth in SCID mice. miR-185 overexpression inhibited DNMT1 3′ untranslated region luciferase reporter activity in HCC cells; this effect was abolished when the miR-185 binding site was mutated. miR-185 mimic or overexpression decreased the level of DNMT1 protein in HCC cells. These findings establish DNMT1 as a bona fide target of miR-185 in HCC cells. The role of DNMT1 in miR-185–induced inhibition of HCC growth was further supported by the fact that DNMT1 overexpression prevented miR-185–induced inhibition of HCC cell proliferation/invasion. miR-185 mimic or overexpression reduced PTEN promoter DNA methylation and enhanced PTEN expression, leading to the inhibition of Akt phosphorylation; these effects were partially reversed by DNMT1 overexpression. These results provide novel evidence that miR-185 inhibits HCC cell growth by targeting DNMT1, leading to PTEN induction and Akt inhibition. Thus, reactivation or induction of miR-185 may represent a novel therapeutic strategy for HCC treatment. Hepatocellular carcinoma (HCC) is a highly malignant primary liver cancer with a poor prognosis.1Farazi P.A. DePinho R.A. Hepatocellular carcinoma pathogenesis: from genes to environment.Nat Rev Cancer. 2006; 6: 674-687Crossref PubMed Scopus (1671) Google Scholar, 2Seeff L.B. Hoofnagle J.H. 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Thus, further studies are needed to define the mechanisms of hepatocarcinogenesis and develop new target therapies. miRNAs are small noncoding RNA molecules that regulate gene expression post-transcriptionally through complementary base pairing with messenger RNAs.9Valencia-Sanchez M.A. Liu J. Hannon G.J. Parker R. Control of translation and mRNA degradation by miRNAs and siRNAs.Genes Dev. 2006; 20: 515-524Crossref PubMed Scopus (1745) Google Scholar Recent studies have shown an important role of miRNAs in the regulation of HCC growth.10Thorgeirsson S.S. Lee J.S. Grisham J.W. Functional genomics of hepatocellular carcinoma.Hepatology. 2006; 43: S145-S150Crossref PubMed Scopus (125) Google Scholar, 11Aravalli R.N. Steer C.J. Cressman E.N. Molecular mechanisms of hepatocellular carcinoma.Hepatology. 2008; 48: 2047-2063Crossref PubMed Scopus (569) Google Scholar, 12Giordano S. Columbano A. 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MicroRNA silencing and the development of novel therapies for liver disease.J Hepatol. 2012; 57: 462-466Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Profiling of HCC miRNA expression has identified signatures associated with diagnosis, staging, progression, prognosis, and response to treatment. For example, Budhu et al17Budhu A. Jia H.L. Forgues M. Liu C.G. Goldstein D. Lam A. Zanetti K.A. Ye Q.H. Qin L.X. Croce C.M. Tang Z.Y. Wang X.W. Identification of metastasis-related microRNAs in hepatocellular carcinoma.Hepatology. 2008; 47: 897-907Crossref PubMed Scopus (632) Google Scholar identified a 20-miRNA tumor signature associated with HCC venous metastasis that can predict HCC survival and recurrence in patients with multinodular or solitary tumors; one of these miRNAs is miR-185. A subsequent study by Zhi et al18Zhi Q. Zhu J. Guo X. He S. Xue X. Zhou J. Hu B. Li H. Chen S. Zhao H. Kuang Y. Metastasis-related miR-185 is a potential prognostic biomarker for hepatocellular carcinoma in early stage.Biomed Pharmacother. 2013; 67: 393-398Crossref PubMed Scopus (42) Google Scholar found that low miR-185 expression correlates with more tumor recurrence and a lower patient survival rate compared with the high miR-185 group and that miR-185 reduces HCC cell growth and invasion in vitro, suggesting that miR-185 may represent a tumor-suppressive miRNA in HCC. However, to date, the biological functions of miR-185 in hepatocarcinogenesis remain largely unknown, and its mechanisms of action have not yet been defined. This study was designed to further examine the effect and mechanism of miR-185 in human HCC. We show herein that miR-185 is a tumor suppressor miRNA in HCC and that its expression is down-regulated in human HCC tissues and cells. Our findings indicate that miR-185 inhibits HCC cell growth in vitro and in SCID mice by targeting the DNMT1/PTEN/Akt signaling pathway. Human hepatocellular cancer cell lines (HepG2, Huh7, and Hep3B cells) were obtained from ATCC (Manassas, VA). All the cell lines were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO). Primary human hepatocytes were purchased from Lonza Inc. (Walkersville, MD) and were cultured in collagen I–coated plates (BD Biosciences, Bedford, MA) with hepatocyte basal medium supplemented with HCM SingleQuots growth factors (Lonza Inc.). The cells were maintained at 37°C and 5% CO2. The plasmids, including pcDNA3.1/DNMT1 and pcDNA3.1, were purchased from Addgene Inc. (Cambridge, MA). DNMT1 3′ untranslated region (3′-UTR) luciferase reporter plasmid was purchased from OriGene Technologies Inc. (Rockville, MD). Lentivirus encoding human miR-185 (L/miR-185) and control lentivirus expressing scramble control miRNA (L/control) were purchased from GeneCopoeia Inc. (Rockville, MD). The antibodies against PTEN, total Akt, and phospho-Akt (p-Akt) were obtained from Cell Signaling Technology Inc. (Beverly, CA). The antibody against β-actin was purchased from Sigma-Aldrich. The anti-DNMT1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 5-Aza-2′-deoxycytidine (5-Aza-CdR) and trichostatin A were obtained from Sigma-Aldrich. Other chemicals were of analytical grade. In situ hybridization for miR-185 was performed in human HCC tissue microarray (serial # LV8013; US Biomax Inc., Rockville, MD). The tissue microarray consisted of 80 tissue core samples, including 20 cases of HCC, 10 cases of liver cirrhosis, and 10 cases of normal liver tissue (each case with duplicate samples) (more detailed information is available from US Biomax Inc.). The tissue microarray was prepared using the UVP HB-1000 Hybridizer hybridization oven (UVP LLC, Upland, CA), designed to create the ideal environment for nucleic acid hybridization. Human miR-185 miRCURY LNA probes were purchased from Exiqon Inc. (Woburn, MA), and the in situ hybridization procedure was performed according to the manufacturer's instruction. A scramble miRNA control probe having with in the was as a A small RNA control probe was as a The control and the scramble control were to a and for and The was by from to and was as or RNA was using a RNA Technologies Inc., MD) and were to using Technologies Inc.). The were for RT-PCR in to the expression level of miR-185 using a Inc., CA) a CA). were according to the manufacturer's The cells were with and in a with and from Cell were at for 20 at and were to protein for 10 in the of 10% cells or tissue were by a and in 10% with 20 for at The were with primary antibody at the were with antibody Biosciences, in with 20 for at or were using the The antibodies the DNMT1, PTEN, total Akt, and antibodies from studies were performed using the cell invasion MA) for the of tumor cells. medium was to the of the to the for to A cell of in medium was was from the and of prepared cell was to the In of medium 10% fetal bovine serum was to the lower This was for in a tissue a cells and were from the The cells the lower of were in for 20 The were in and to were by the through the were in plates in in growth the the cells were from the using and were in of growth were with and were using a cell Cell were at and was performed for and the results are as cells were with or to establish the cell lines in the of of We by protein signaling a with overexpression of miR-185 were by a cells were in of modified Eagle's medium 10% fetal bovine serum and These cells were with miR-185 mimic or its control RNA with a of using in medium from for these cells were for and cells with miR-185 or control lentivirus were at cells in a and were with or using for The cell were for DNMT1 DNMT1 luciferase reporter plasmid was obtained from OriGene Technologies Inc. DNMT1 luciferase reporter plasmid was by DNMT1 of the of the For cells were at cells in a the cells were with the or DNMT1 luciferase reporter plasmid using The cells were and the luciferase reporter were by using the reporter according to the manufacturer's PTEN tumor suppressor gene at site has a region from the site of PTEN and in the PTEN promoter region by with a total of The region in the PTEN promoter was by using that a region has been shown to of PTEN promoter Liu Chen Hu J.M. Li Li F. of PTEN methylation in tissue by 2013; PubMed Scopus Google The methylation was by using the DNA methylation with the PTEN human from are in the PTEN promoter region with a 10: of The was of the DNA with a a and The of and DNA were by the in with that of a using a SCID mice from The were for in studies tumor A of was with high (BD in a total for a of cell was for liver using a The high protein to the to its liver the mice. This the tumor cells to remain for The were of tumor were in 10% for were for and for the proliferation was of the liver tumor were in by were performed using a The were performed according to the by the and were performed using for Inc., and were using the In situ hybridization results were using the was We performed in situ hybridization for miR-185 in the human liver tissue microarray of 80 tissue cases of HCC, 10 cases of and 10 cases of normal duplicate for miR-185 is in nonneoplastic its level of expression is reduced in HCC cells a reduction of miR-185 in HCCs from chronic hepatitis C chronic hepatitis B compared with the normal liver tissues was normal and liver This is by the results of decreased miR-185 expression in cultured human HCC cells and compared with human primary hepatocytes We performed in for target gene by using Human and the from to the of DNA as one of the genes of miR-185 performed further to the effect of miR-185 DNMT1 in HCC cells. of miR-185 mimic decreased the level of DNMT1 in and cells of cells with an decreased DNMT1 protein overexpression of miR-185 was by A and cells miR-185 showed a reduction of cell proliferation and invasion reduction of cell an of cell C and These results that miR-185 DNMT1 and inhibits HCC cell growth in DNMT1 and inhibits HCC proliferation and invasion in of miR-185 showed a in cells with compared with cells with for DNMT1 in cells with or overexpression of miR-185. and The level of DNMT1 protein is decreased in cells expressing miR-185 compared with control cells Cell proliferation was at and in cells in miR-185 overexpression decreased cell proliferation at the A cell invasion was performed in cells. miR-185 cells showed an reduction in the of cells the compared with control The cells. are as and We identified a miR-185 binding site in the of DNMT1 this binding site is at to of DNMT1 this site is in miR-185 targeting of DNMT1 in HCC luciferase reporter DNMT1 or miR-185 binding site cells or miR-185 miR-185 overexpression reduced the DNMT1 luciferase reporter activity and the effect was abolished when the miR-185 binding site was Thus, DNMT1 is a bona fide target of miR-185 in HCC cells. further the role of DNMT1 in miR-185–induced inhibition of HCC miR-185 cells with a DNMT1 expression of the cells or miR-185 were with a DNMT1 expression in or a control and the cells were for proliferation and DNMT1 overexpression prevented miR-185–induced inhibition of cell proliferation and invasion DNMT1 is for DNA methylation during and is important for cancer cell survival and T. S. F. He T. B. Y. Li of DNMT1 to in human cancer PubMed Scopus Google Scholar, Lee J.H. Lee in hepatocarcinogenesis by hepatitis B Full Text Full Text PDF PubMed Scopus Google Scholar, H. regulation of cancer cell by growth 2010; PubMed Scopus Google Scholar The effect of is in through methylation of of target gene silencing the expression of tumor suppressor genes R. H. T. A. S. T. T. T. M. of DNA by protein to promoter of PTEN gene in 2009; PubMed Scopus Google Scholar, Lee J.H. of PTEN promoter methylation in cancer 2011; PubMed Scopus Google Scholar We performed a to the of DNA methylation in the PTEN promoter overexpression of miR-185 in cells reduced PTEN promoter DNA methylation miR-185 mimic enhanced the expression of PTEN protein in cells and cells with the inhibition of Akt by PTEN, decreased of in miR-185 cells. overexpression of miR-185 enhanced PTEN protein and inhibited Akt phosphorylation; these effects were partially reversed by overexpression of DNMT1 of the We that inhibition of by decreased PTEN promoter enhanced PTEN and reduced Akt are with by miR-185 mimic or overexpression. these findings that miR-185 inhibits HCC cell growth by targeting DNMT1, to induction of PTEN expression and subsequent inhibition of Akt This is further by the role of PTEN and Akt in M. M. PTEN in liver and 2010; PubMed Scopus Google Scholar, Lee J.M. of and cell invasion and through in hepatocellular carcinoma and has 2011; PubMed Scopus Google Scholar, F. R. H. Patel T. expression of the PTEN tumor suppressor gene in human hepatocellular Full Text Full Text PDF PubMed Scopus Google Scholar, T. Guo H. Wang Wang X. PTEN inhibits the and invasion of cells by expression the 2010; Google Scholar, J.S. Wang Q. Chen Chen Li J. of in invasion and metastasis in hepatocellular carcinoma: with 2009; PubMed Scopus Google Scholar the tumor suppressor genes in patients with HCC and of and in Gastroenterol Hepatol. 2006; PubMed Scopus Google Scholar further examined the of these in We that the protein of and were in cells with compared with cells with control However, viral of miR-185 in cells not the protein of and the protein of and in miR-185 were not compared with the control Thus, miR-185 not to the of and in that miR-185 The for the is and remains We the that miR-185 these molecules through mechanisms that are yet to is that the may cell the findings in this study the of PTEN in inhibition of HCC cell the effect of miR-185 HCC growth in cells expressing miR-185 or scramble control miRNA the of SCID mice was by cell with (BD the were for to for tumor growth in the The cells miR-185 tumor in the liver compared with the in tumor for the cell proliferation showed a reduction of cells in miR-185 a reduction of with of PTEN and a of in miR-185 tumor tissues These results that miR-185 inhibits HCC growth in This study novel evidence that miR-185 is a tumor-suppressive miRNA that inhibits HCC cell growth by targeting This is the expression of miR-185 is decreased in human HCC tissues and cell lines compared with the nonneoplastic overexpression of miR-185 in human HCC cells inhibits tumor cell growth in vitro and in SCID miR-185 DNMT1, a that the expression of tumor suppressor genes overexpression of DNMT1 of the miR-185–induced inhibition of HCC cell proliferation and miR-185 mimic or overexpression reduces PTEN promoter DNA methylation and PTEN expression to Akt and these effects are partially reversed by overexpression of and the effects of miR-185 mimic or overexpression PTEN in HCC cells are to by the These findings that miR-185 inhibits HCC cell growth by targeting DNMT1, to induction of PTEN and inhibition of Akt Budhu et al17Budhu A. Jia H.L. Forgues M. Liu C.G. Goldstein D. Lam A. Zanetti K.A. Ye Q.H. Qin L.X. Croce C.M. Tang Z.Y. Wang X.W. Identification of metastasis-related microRNAs in hepatocellular carcinoma.Hepatology. 2008; 47: 897-907Crossref PubMed Scopus (632) Google Scholar that miR-185 is one of the 20 miRNAs associated with HCC venous metastasis that can predict HCC survival and identified miR-185 as one of the miRNAs that are in HCCs with venous metastasis compared with HCCs venous metastasis miRNAs were found to down-regulated in HCCs with venous metastasis compared with HCCs venous these miRNAs HCC and liver tissues was not Zhi and Q. Zhu J. Guo X. He S. Xue X. Zhou J. Hu B. Li H. Chen S. Zhao H. Kuang Y. Metastasis-related miR-185 is a potential prognostic biomarker for hepatocellular carcinoma in early stage.Biomed Pharmacother. 2013; 67: 393-398Crossref PubMed Scopus (42) Google Scholar found that low miR-185 expression in HCC with more tumor recurrence and patient survival compared with the high miR-185 the study not miR-185 HCC and liver the role of miR-185 in venous metastasis and in HCC recurrence with high miR-185 expression found to associated with venous metastasis tumor recurrence and patient is that factors have to the patient tumor and study an important is that in of the the of miR-185 in HCC tissues were by of in cell in the tumor This is an important that miRNAs in cell in the tumor may G. Bala S. MicroRNAs in liver disease.Nat Rev Gastroenterol Hepatol. 2013; 10: 542-552Crossref PubMed Scopus (471) Google Scholar, 16Szabo G. Sarnow P. Bala S. MicroRNA silencing and the development of novel therapies for liver disease.J Hepatol. 2012; 57: 462-466Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar In this the in situ hybridization in the study of miR-185 in HCC and nonneoplastic liver These indicate that in nonneoplastic liver miR-185 is in its expression is reduced in hepatocytes. This is further by the decreased miR-185 expression in cultured human HCC cell lines compared with primary human hepatocytes. These with the and studies that miR-185 is a tumor suppressor miRNA that inhibits HCC important of the study is the of DNMT1 as the target of miR-185 in human HCC cells. DNMT1 is the in cells that the methylation of at the is for and methylation of tumor suppressor genes in cancer C. F. for the of DNA as new strategy for modulation of tumor response to and Cancer. 2010; PubMed Scopus Google Scholar, X. F. H. P.A. DNA methylation for 2010; Full Text Full Text PDF PubMed Scopus Google Scholar The that miR-185 DNMT1 is supported by the that a miR-185 binding site is in the of miR-185 mimic or overexpression decreased DNMT1 protein in HCC cells; miR-185 overexpression reduced the DNMT1 luciferase reporter activity in HCC and the effect was abolished when the miR-185 binding site was and of DNMT1 prevented miR-185–induced inhibition of HCC cell proliferation and the methylation of at the of tumor suppressor genes Y. S. of DNA methylation associated with of DNA in human during from a to a malignant PubMed Scopus Google Scholar are in an activity to to the in of gene A. and the regulation of Dev. 2011; 25: PubMed Scopus Google Scholar of tumor suppressor gene has been as an important mechanism in D. P. B. R. of the gene in hepatocellular PubMed Scopus Google Scholar, J. M. J. H. B. J. Chen J. M. S. Zhu J. of of genes may to hepatocellular Cancer. PubMed Scopus Google Scholar In the found that miR-185 mimic or overexpression reduced DNA methylation in the PTEN promoter and enhanced PTEN protein expression, and these effects were reversed by overexpression of In that the effects of miR-185 mimic or overexpression PTEN promoter DNA methylation and protein expression in HCC cells are with the effect by the These findings that miR-185 HCC growth through inhibition of protein PTEN with the role of PTEN in the of Akt that miR-185 mimic or overexpression decreased Akt in HCC cells. These findings are with the of PTEN and of Akt in M. M. PTEN in liver and 2010; PubMed Scopus Google Scholar, Lee J.M. of and cell invasion and through in hepatocellular carcinoma and has 2011; PubMed Scopus Google Scholar, F. R. H. Patel T. expression of the PTEN tumor suppressor gene in human hepatocellular Full Text Full Text PDF PubMed Scopus Google Scholar, T. Guo H. Wang Wang X. PTEN inhibits the and invasion of cells by expression the 2010; Google Scholar, J.S. Wang Q. Chen Chen Li J. of in invasion and metastasis in hepatocellular carcinoma: with 2009; PubMed Scopus Google Scholar The and miRNA regulation has been a of the development and of human M. M. in 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, M. and miRNAs in human 2010; PubMed Scopus Google Scholar DNA and mechanisms in miRNAs are In this is that the study novel evidence for an important tumor-suppressive role of miR-185 in HCC through targeting a in cancer In this study novel evidence that miR-185 is a tumor suppressor miRNA that inhibits HCC cell growth by targeting DNMT1 and PTEN reactivation or induction of miR-185 may represent a novel therapeutic strategy for HCC that further remains to miR-185 and can targeted for with PTEN protein expression and reduces Akt in cells. cells were with miR-185 mimic or scramble control and the cell were obtained for to the of PTEN, and The were with effect of A PTEN promoter methylation and PTEN protein for PTEN, and Akt in cells with and the The were cells with showed decreased methylation in the PTEN promoter are as with for and in cells. cells were with or trichostatin A or were with or miR-185 The cell were and to and for and The were