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Decreased miR122 in hepatocellular carcinoma leads to chemoresistance with increased arginine.

Kishikawa T, Otsuka M, Tan PS, Ohno M, Sun X, Yoshikawa T, Shibata C, Takata A, Kojima K, Takehana K, Ohishi M, Ota S, Noyama T, Kondo Y, Sato M, Soga T, Hoshida Y, Koike K - Oncotarget (2015)

Bottom Line: Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib.Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib.These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

ABSTRACT
Reduced expression of microRNA122 (miR122), a liver-specific microRNA, is frequent in hepatocellular carcinoma (HCC). However, its biological significances remain poorly understood. Because deregulated amino acid levels in cancers can affect their biological behavior, we determined the amino acid levels in miR122-silenced mouse liver tissues, in which intracellular arginine levels were significantly increased. The increased intracellular arginine levels were through upregulation of the solute carrier family 7 (SLC7A1), a transporter of arginine and a direct target of miR122. Arginine is the substrate for nitric oxide (NO) synthetase, and intracellular NO levels were increased in miR122-silenced HCC cells, with increased resistance to sorafenib, a multikinase inhibitor. Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib. Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib. These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset.

No MeSH data available.


Related in: MedlinePlus

PD407824 increases miR122 expression and sensitizes Hep3B cells tosorafenib treatment(a) A schematic of the knock-in reporter at the miR122pri-precursor locus. The arrow indicates the possible transcriptionstart site. The gene-editing target site by Cas9 is located just beforethe miR122 precursor locus. Below, the knock-in construct is shown whichconsists of IRES, GFP fused with a firefly luciferase gene reporter, theEF1 promoter, and the puromycin resistant gene. (b, c)Mature miR122 levels in Hep3B cells after the treatment of PD407824 (PD)and Ellipticine for 24 h were measured by qRT-PCR. The levels from thecontrol cells were set as 1.0. *, p < 0.05(n = 3). nt, nucleotides. c, Flow cytometryresults determining SLC7A1 protein expression levels. Representativeresults from three independent experiments are shown. SLC7A1 expressionwas decreased in Hep3B cells treated with PD407824 (PD), which wasantagonized by expressing the antisense miR122 construct (PD +miR122-silenced). (d) Intracellular NO levels weredecreased in Hep3B cells treated with PD407824. This effect was reversedby stable overexpression of HA-tagged SLC7A1. Control cells (Control),PD407824 treated cells (PD), and PD407824 treated cells with stableexpression of HA-tagged SLC7A1 (PD + SLC7A1) or the antisensemiR122 construct (PD + miR122-silenced) were tested. *,p < 0.05 (n = 3).(e) The effects of sorafenib were augmented bycombination treatment with PD407824. Relative Hep3B cell numbers werecounted after treatment with or without PD407824 (PD) for 48 h. Controlcells (Control), sorafenib treated cells (Sorafenib), sorafenib-treatedcells with stable expression of SLC7A1 (Sorafenib + SLC7A1) orstable expression of the antisense miR122 construct (Sorafenib +miR122-silenced) were tested. The number of control cells with DMSO(without sorafenib and PD407824) treatment was set to 1. *,p < 0.05 (n =3).
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Figure 5: PD407824 increases miR122 expression and sensitizes Hep3B cells tosorafenib treatment(a) A schematic of the knock-in reporter at the miR122pri-precursor locus. The arrow indicates the possible transcriptionstart site. The gene-editing target site by Cas9 is located just beforethe miR122 precursor locus. Below, the knock-in construct is shown whichconsists of IRES, GFP fused with a firefly luciferase gene reporter, theEF1 promoter, and the puromycin resistant gene. (b, c)Mature miR122 levels in Hep3B cells after the treatment of PD407824 (PD)and Ellipticine for 24 h were measured by qRT-PCR. The levels from thecontrol cells were set as 1.0. *, p < 0.05(n = 3). nt, nucleotides. c, Flow cytometryresults determining SLC7A1 protein expression levels. Representativeresults from three independent experiments are shown. SLC7A1 expressionwas decreased in Hep3B cells treated with PD407824 (PD), which wasantagonized by expressing the antisense miR122 construct (PD +miR122-silenced). (d) Intracellular NO levels weredecreased in Hep3B cells treated with PD407824. This effect was reversedby stable overexpression of HA-tagged SLC7A1. Control cells (Control),PD407824 treated cells (PD), and PD407824 treated cells with stableexpression of HA-tagged SLC7A1 (PD + SLC7A1) or the antisensemiR122 construct (PD + miR122-silenced) were tested. *,p < 0.05 (n = 3).(e) The effects of sorafenib were augmented bycombination treatment with PD407824. Relative Hep3B cell numbers werecounted after treatment with or without PD407824 (PD) for 48 h. Controlcells (Control), sorafenib treated cells (Sorafenib), sorafenib-treatedcells with stable expression of SLC7A1 (Sorafenib + SLC7A1) orstable expression of the antisense miR122 construct (Sorafenib +miR122-silenced) were tested. The number of control cells with DMSO(without sorafenib and PD407824) treatment was set to 1. *,p < 0.05 (n =3).

Mentions: The results obtained led us to hypothesize that if it were possible to enhancemiR122 expression levels in HCC cells using chemical compounds, such compoundscould augment the chemosensitivity of HCCs to sorafenib. To undertake acomprehensive screen of possible compounds, a knock-in reporter construct wascreated by inserting a firefly luciferase gene in the miR122 precursor genomiclocus by genome-editing using the CRISPR-Cas9 system (Figure 5a and Supplementary Figure 4).These cells were screened with over 1,200 chemical compounds to identifymolecules which potentially enhance miR122 pri-precursor transcription. Ofseveral compounds that increased the reporter values (Supplementary Table 1),PD407824, a Wee1 kinase inhibitor, and Ellipticine, a DNA topoisomeraseinhibitor, returned the highest values.


Decreased miR122 in hepatocellular carcinoma leads to chemoresistance with increased arginine.

Kishikawa T, Otsuka M, Tan PS, Ohno M, Sun X, Yoshikawa T, Shibata C, Takata A, Kojima K, Takehana K, Ohishi M, Ota S, Noyama T, Kondo Y, Sato M, Soga T, Hoshida Y, Koike K - Oncotarget (2015)

PD407824 increases miR122 expression and sensitizes Hep3B cells tosorafenib treatment(a) A schematic of the knock-in reporter at the miR122pri-precursor locus. The arrow indicates the possible transcriptionstart site. The gene-editing target site by Cas9 is located just beforethe miR122 precursor locus. Below, the knock-in construct is shown whichconsists of IRES, GFP fused with a firefly luciferase gene reporter, theEF1 promoter, and the puromycin resistant gene. (b, c)Mature miR122 levels in Hep3B cells after the treatment of PD407824 (PD)and Ellipticine for 24 h were measured by qRT-PCR. The levels from thecontrol cells were set as 1.0. *, p < 0.05(n = 3). nt, nucleotides. c, Flow cytometryresults determining SLC7A1 protein expression levels. Representativeresults from three independent experiments are shown. SLC7A1 expressionwas decreased in Hep3B cells treated with PD407824 (PD), which wasantagonized by expressing the antisense miR122 construct (PD +miR122-silenced). (d) Intracellular NO levels weredecreased in Hep3B cells treated with PD407824. This effect was reversedby stable overexpression of HA-tagged SLC7A1. Control cells (Control),PD407824 treated cells (PD), and PD407824 treated cells with stableexpression of HA-tagged SLC7A1 (PD + SLC7A1) or the antisensemiR122 construct (PD + miR122-silenced) were tested. *,p < 0.05 (n = 3).(e) The effects of sorafenib were augmented bycombination treatment with PD407824. Relative Hep3B cell numbers werecounted after treatment with or without PD407824 (PD) for 48 h. Controlcells (Control), sorafenib treated cells (Sorafenib), sorafenib-treatedcells with stable expression of SLC7A1 (Sorafenib + SLC7A1) orstable expression of the antisense miR122 construct (Sorafenib +miR122-silenced) were tested. The number of control cells with DMSO(without sorafenib and PD407824) treatment was set to 1. *,p < 0.05 (n =3).
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Figure 5: PD407824 increases miR122 expression and sensitizes Hep3B cells tosorafenib treatment(a) A schematic of the knock-in reporter at the miR122pri-precursor locus. The arrow indicates the possible transcriptionstart site. The gene-editing target site by Cas9 is located just beforethe miR122 precursor locus. Below, the knock-in construct is shown whichconsists of IRES, GFP fused with a firefly luciferase gene reporter, theEF1 promoter, and the puromycin resistant gene. (b, c)Mature miR122 levels in Hep3B cells after the treatment of PD407824 (PD)and Ellipticine for 24 h were measured by qRT-PCR. The levels from thecontrol cells were set as 1.0. *, p < 0.05(n = 3). nt, nucleotides. c, Flow cytometryresults determining SLC7A1 protein expression levels. Representativeresults from three independent experiments are shown. SLC7A1 expressionwas decreased in Hep3B cells treated with PD407824 (PD), which wasantagonized by expressing the antisense miR122 construct (PD +miR122-silenced). (d) Intracellular NO levels weredecreased in Hep3B cells treated with PD407824. This effect was reversedby stable overexpression of HA-tagged SLC7A1. Control cells (Control),PD407824 treated cells (PD), and PD407824 treated cells with stableexpression of HA-tagged SLC7A1 (PD + SLC7A1) or the antisensemiR122 construct (PD + miR122-silenced) were tested. *,p < 0.05 (n = 3).(e) The effects of sorafenib were augmented bycombination treatment with PD407824. Relative Hep3B cell numbers werecounted after treatment with or without PD407824 (PD) for 48 h. Controlcells (Control), sorafenib treated cells (Sorafenib), sorafenib-treatedcells with stable expression of SLC7A1 (Sorafenib + SLC7A1) orstable expression of the antisense miR122 construct (Sorafenib +miR122-silenced) were tested. The number of control cells with DMSO(without sorafenib and PD407824) treatment was set to 1. *,p < 0.05 (n =3).
Mentions: The results obtained led us to hypothesize that if it were possible to enhancemiR122 expression levels in HCC cells using chemical compounds, such compoundscould augment the chemosensitivity of HCCs to sorafenib. To undertake acomprehensive screen of possible compounds, a knock-in reporter construct wascreated by inserting a firefly luciferase gene in the miR122 precursor genomiclocus by genome-editing using the CRISPR-Cas9 system (Figure 5a and Supplementary Figure 4).These cells were screened with over 1,200 chemical compounds to identifymolecules which potentially enhance miR122 pri-precursor transcription. Ofseveral compounds that increased the reporter values (Supplementary Table 1),PD407824, a Wee1 kinase inhibitor, and Ellipticine, a DNA topoisomeraseinhibitor, returned the highest values.

Bottom Line: Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib.Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib.These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

ABSTRACT
Reduced expression of microRNA122 (miR122), a liver-specific microRNA, is frequent in hepatocellular carcinoma (HCC). However, its biological significances remain poorly understood. Because deregulated amino acid levels in cancers can affect their biological behavior, we determined the amino acid levels in miR122-silenced mouse liver tissues, in which intracellular arginine levels were significantly increased. The increased intracellular arginine levels were through upregulation of the solute carrier family 7 (SLC7A1), a transporter of arginine and a direct target of miR122. Arginine is the substrate for nitric oxide (NO) synthetase, and intracellular NO levels were increased in miR122-silenced HCC cells, with increased resistance to sorafenib, a multikinase inhibitor. Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib. Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib. These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset.

No MeSH data available.


Related in: MedlinePlus