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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

Reduced miR122 is linked with resistance against sorafenib(a) The firefly luciferase-based reporter with miR1223′UTR responsive elements was transfected along with the Renillaluciferase-based control reporter. Firefly luciferase values werenormalized to those of Renilla luciferase (as an internal control) tocalculate the relative luciferase values. Control cell values were setto 1. Data represent the means ± s.d. of three independentexperiments. *, p < 0.05. (b) TheRenilla luciferase-based reporter with the SLC7A1 3′UTR(pRL-catA) was transfected as well as the CMV-driven fireflyluciferase-based control reporter, to determine the effect of miR122 onthe SLC7A1 3′UTR. Renilla luciferase values were normalized tothose of firefly luciferase (as an internal control) to calculate therelative luciferase values. Control cell values were set to 1. Datarepresent the means ± s.d. of three independent experiments. *,p < 0.05. (c) SLC7A1 expressionwas increased in miR122-silenced Huh7 cells. Flow cytometry assessmentof SLC7A1 protein expression in control cells (black line) andmiR122-silenced cells (pink line) was performed. Gray-shaded histogramsrepresent the isotype IgG background staining. Representative resultsfrom three independent experiments are shown. (d)Intracellular levels of arginine, but not phenylalanine, were increasedin miR122-silenced cells. Data represent the means ± s.d. ofthree independent experiments. *, p < 0.05.(e) Intracellular NO levels were increased inmiR122-silenced cells. Data represent the means ± s.d. of threeindependent experiments. *, p < 0.05.(f) The effects of sorafenib were inversely correlatedwith the expression levels of miR122. Relative cell numbers were countedafter treatment for 48 h with 5 μM sorafenib with or without PTIOin miR122-silenced Huh7 cells. The value of control cells with DMSOtreatment was set to 1 *, p < 0.05(n = 3).
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Figure 2: Reduced miR122 is linked with resistance against sorafenib(a) The firefly luciferase-based reporter with miR1223′UTR responsive elements was transfected along with the Renillaluciferase-based control reporter. Firefly luciferase values werenormalized to those of Renilla luciferase (as an internal control) tocalculate the relative luciferase values. Control cell values were setto 1. Data represent the means ± s.d. of three independentexperiments. *, p < 0.05. (b) TheRenilla luciferase-based reporter with the SLC7A1 3′UTR(pRL-catA) was transfected as well as the CMV-driven fireflyluciferase-based control reporter, to determine the effect of miR122 onthe SLC7A1 3′UTR. Renilla luciferase values were normalized tothose of firefly luciferase (as an internal control) to calculate therelative luciferase values. Control cell values were set to 1. Datarepresent the means ± s.d. of three independent experiments. *,p < 0.05. (c) SLC7A1 expressionwas increased in miR122-silenced Huh7 cells. Flow cytometry assessmentof SLC7A1 protein expression in control cells (black line) andmiR122-silenced cells (pink line) was performed. Gray-shaded histogramsrepresent the isotype IgG background staining. Representative resultsfrom three independent experiments are shown. (d)Intracellular levels of arginine, but not phenylalanine, were increasedin miR122-silenced cells. Data represent the means ± s.d. ofthree independent experiments. *, p < 0.05.(e) Intracellular NO levels were increased inmiR122-silenced cells. Data represent the means ± s.d. of threeindependent experiments. *, p < 0.05.(f) The effects of sorafenib were inversely correlatedwith the expression levels of miR122. Relative cell numbers were countedafter treatment for 48 h with 5 μM sorafenib with or without PTIOin miR122-silenced Huh7 cells. The value of control cells with DMSOtreatment was set to 1 *, p < 0.05(n = 3).

Mentions: To confirm the above screening results in vitro, we usedmiR122-silenced Huh7 cells, well-differentiated HCC cells which stably expressmiR122 antisense constructs and have impaired miR122 function [5] (Figure 2a). Huh7 cells were chosen for the experiments using the antisensemiR122 construct because they have endogenously high miR122 expression levels[27] and the effects ofmiR122-silecing are more easily observed. Consistent with previous reports[27], luciferase expression from areporter construct containing the 3′ UTR of the SLC7A1 gene wasincreased, and SLC7A1 expression was increased by miR122-silecing (Figure 2b and 2c). Moreover, while intracellularlevels of phenylalanine did not change, intracellular arginine levels increasedsignificantly, confirming the initial screening results (Figure 2d).


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)

Reduced miR122 is linked with resistance against sorafenib(a) The firefly luciferase-based reporter with miR1223′UTR responsive elements was transfected along with the Renillaluciferase-based control reporter. Firefly luciferase values werenormalized to those of Renilla luciferase (as an internal control) tocalculate the relative luciferase values. Control cell values were setto 1. Data represent the means ± s.d. of three independentexperiments. *, p < 0.05. (b) TheRenilla luciferase-based reporter with the SLC7A1 3′UTR(pRL-catA) was transfected as well as the CMV-driven fireflyluciferase-based control reporter, to determine the effect of miR122 onthe SLC7A1 3′UTR. Renilla luciferase values were normalized tothose of firefly luciferase (as an internal control) to calculate therelative luciferase values. Control cell values were set to 1. Datarepresent the means ± s.d. of three independent experiments. *,p < 0.05. (c) SLC7A1 expressionwas increased in miR122-silenced Huh7 cells. Flow cytometry assessmentof SLC7A1 protein expression in control cells (black line) andmiR122-silenced cells (pink line) was performed. Gray-shaded histogramsrepresent the isotype IgG background staining. Representative resultsfrom three independent experiments are shown. (d)Intracellular levels of arginine, but not phenylalanine, were increasedin miR122-silenced cells. Data represent the means ± s.d. ofthree independent experiments. *, p < 0.05.(e) Intracellular NO levels were increased inmiR122-silenced cells. Data represent the means ± s.d. of threeindependent experiments. *, p < 0.05.(f) The effects of sorafenib were inversely correlatedwith the expression levels of miR122. Relative cell numbers were countedafter treatment for 48 h with 5 μM sorafenib with or without PTIOin miR122-silenced Huh7 cells. The value of control cells with DMSOtreatment was set to 1 *, p < 0.05(n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Reduced miR122 is linked with resistance against sorafenib(a) The firefly luciferase-based reporter with miR1223′UTR responsive elements was transfected along with the Renillaluciferase-based control reporter. Firefly luciferase values werenormalized to those of Renilla luciferase (as an internal control) tocalculate the relative luciferase values. Control cell values were setto 1. Data represent the means ± s.d. of three independentexperiments. *, p < 0.05. (b) TheRenilla luciferase-based reporter with the SLC7A1 3′UTR(pRL-catA) was transfected as well as the CMV-driven fireflyluciferase-based control reporter, to determine the effect of miR122 onthe SLC7A1 3′UTR. Renilla luciferase values were normalized tothose of firefly luciferase (as an internal control) to calculate therelative luciferase values. Control cell values were set to 1. Datarepresent the means ± s.d. of three independent experiments. *,p < 0.05. (c) SLC7A1 expressionwas increased in miR122-silenced Huh7 cells. Flow cytometry assessmentof SLC7A1 protein expression in control cells (black line) andmiR122-silenced cells (pink line) was performed. Gray-shaded histogramsrepresent the isotype IgG background staining. Representative resultsfrom three independent experiments are shown. (d)Intracellular levels of arginine, but not phenylalanine, were increasedin miR122-silenced cells. Data represent the means ± s.d. ofthree independent experiments. *, p < 0.05.(e) Intracellular NO levels were increased inmiR122-silenced cells. Data represent the means ± s.d. of threeindependent experiments. *, p < 0.05.(f) The effects of sorafenib were inversely correlatedwith the expression levels of miR122. Relative cell numbers were countedafter treatment for 48 h with 5 μM sorafenib with or without PTIOin miR122-silenced Huh7 cells. The value of control cells with DMSOtreatment was set to 1 *, p < 0.05(n = 3).
Mentions: To confirm the above screening results in vitro, we usedmiR122-silenced Huh7 cells, well-differentiated HCC cells which stably expressmiR122 antisense constructs and have impaired miR122 function [5] (Figure 2a). Huh7 cells were chosen for the experiments using the antisensemiR122 construct because they have endogenously high miR122 expression levels[27] and the effects ofmiR122-silecing are more easily observed. Consistent with previous reports[27], luciferase expression from areporter construct containing the 3′ UTR of the SLC7A1 gene wasincreased, and SLC7A1 expression was increased by miR122-silecing (Figure 2b and 2c). Moreover, while intracellularlevels of phenylalanine did not change, intracellular arginine levels increasedsignificantly, confirming the initial screening results (Figure 2d).

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