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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

Forced miR122 expression sensitizes Hep3B cells to sorafenibtreatment(a, b) The effects of miR122 on the reporter constructscarrying miR122 responsive elements (a) and the SLC7A1 3′UTR (b)in control and miR122 precursor-expressing Hep3B cells. Control cellvalues were set to 1. Data represent the means ± s.d. of threeindependent experiments. *, p < 0.05.(c) SLC7A1 expression was decreased in miR122precursor-overexpressing Hep3B cells. Flow cytometry assessment ofSLC7A1 protein expression was performed. Representative results fromthree independent experiments are shown. (d) IntracellularNO levels were decreased by miR122 precursor-expression in Hep3B cells.This effect was reversed by stable overexpression of HA-tagged SLC7A1.Control cells (control), miR122 precursor-overexpressing cells (miR122precursor) and cells with stable overexpression of both the miR122precursor and HA-tagged SLC7A1 (miR122 precursor + SLC7A1) weretested. Control cells under arginine-depleted conditions and treatedwith PTIO were included. *, p < 0.05(n = 3). (e) The effects ofsorafenib were inversely correlated with the expression levels ofmiR122. Relative cell numbers were counted after treatment for 48 h with5 μM sorafenib with or without PTIO in miR122precursor-expressing Hep3B cells. The number of the control cells withDMSO treatment was set to 1 *, p < 0.05(n = 3). Control cells (control), miR122precursor-expressing cells (miR122 precursor), cells cultured inarginine-depleted media (Arg depleted), and miR122 precursor-expressingcells with expression of SLC7A1 (miR122 precursor + SLC7A1) weretested. Cells treated with PTIO to remove NO were included as a control.(f, g) The effects of sorafenib were inverselycorrelated with the expression levels of miR122, as determined by flowcytometry (f) and fluorescence microscopy (g). miR122-silenced Huh7cells and miR122 precursor-expressing Hep3B cells showed GFPco-expression. The percentage of GFP-expressing cells (~50% beforesorafenib treatment) increased in the miR122-silenced Huh7 cells anddecreased in the miR122 precursor-expressing Hep3B cells after 5μM sorafenib treatment for 48 h, as determined by flow cytometry.*, p < 0.05 (n = 3) (f).Representative results of GFP-positive cells from three independentexperiments are shown (g).
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Figure 4: Forced miR122 expression sensitizes Hep3B cells to sorafenibtreatment(a, b) The effects of miR122 on the reporter constructscarrying miR122 responsive elements (a) and the SLC7A1 3′UTR (b)in control and miR122 precursor-expressing Hep3B cells. Control cellvalues were set to 1. Data represent the means ± s.d. of threeindependent experiments. *, p < 0.05.(c) SLC7A1 expression was decreased in miR122precursor-overexpressing Hep3B cells. Flow cytometry assessment ofSLC7A1 protein expression was performed. Representative results fromthree independent experiments are shown. (d) IntracellularNO levels were decreased by miR122 precursor-expression in Hep3B cells.This effect was reversed by stable overexpression of HA-tagged SLC7A1.Control cells (control), miR122 precursor-overexpressing cells (miR122precursor) and cells with stable overexpression of both the miR122precursor and HA-tagged SLC7A1 (miR122 precursor + SLC7A1) weretested. Control cells under arginine-depleted conditions and treatedwith PTIO were included. *, p < 0.05(n = 3). (e) The effects ofsorafenib were inversely correlated with the expression levels ofmiR122. Relative cell numbers were counted after treatment for 48 h with5 μM sorafenib with or without PTIO in miR122precursor-expressing Hep3B cells. The number of the control cells withDMSO treatment was set to 1 *, p < 0.05(n = 3). Control cells (control), miR122precursor-expressing cells (miR122 precursor), cells cultured inarginine-depleted media (Arg depleted), and miR122 precursor-expressingcells with expression of SLC7A1 (miR122 precursor + SLC7A1) weretested. Cells treated with PTIO to remove NO were included as a control.(f, g) The effects of sorafenib were inverselycorrelated with the expression levels of miR122, as determined by flowcytometry (f) and fluorescence microscopy (g). miR122-silenced Huh7cells and miR122 precursor-expressing Hep3B cells showed GFPco-expression. The percentage of GFP-expressing cells (~50% beforesorafenib treatment) increased in the miR122-silenced Huh7 cells anddecreased in the miR122 precursor-expressing Hep3B cells after 5μM sorafenib treatment for 48 h, as determined by flow cytometry.*, p < 0.05 (n = 3) (f).Representative results of GFP-positive cells from three independentexperiments are shown (g).

Mentions: To determine the converse effects, the miR122 precursor was stably expressed inHep3B cells, because these cells naturally expressed only minimal levels ofmiR122 [5]. Hep3B cells expressing themiR122 precursor showed enhanced miR122 function (Figure 4a) and suppressed luciferase expression from a reportercarrying the 3′ UTR of the SLC7A1 gene (Figure 4b), with lower expression of SLC7A1 protein compared withthe control cells (Figure 4c). Furthermore,the cells showed lower intracellular NO levels, similar to those of cells grownunder arginine-depleted culture conditions or treated with PTIO (Figure 4d). Because stable overexpression of SLC7A1in the miR122 precursor-stably expressing Hep3B cells reversed the effects ofintracellular NO contents, changes in intracellular NO levels appeared dependenton changes in the SLC7A1 expression levels (Figure 4d). It was noted that miR122 expression had a positive effect onthe sorafenib-treated Hep3B cells, similar to the effects of removing NO withPTIO (Figure 4e). To exclude any possibleinfluences of stable cell line specificity or differences in the cultureconditions of the individual cell lines, Huh7 or Hep3B cells were transducedwith miR122-silencing or miR122-overexpressing lentiviruses with GFP, butwithout selection. GFP-positive (miR122-silenced) Huh7 cells and GFP-negative(miR122-non-overexpressing) Hep3B cells were more concentrated after sorafenibtreatment of mixtures of infected (GFP-expressing) and non-infected(non-GFP-expressing) cells cultured in bulk (Figure 4f and g), confirming that decreased miR122 function wasrelated to sorafenib resistance. These results suggest that miR122-silenced HCCcells are more resistant to chemotherapeutic drugs, and that depletion ofarginine in extracellular environments may be effective for the clinicalmanagement of an HCC subset with reduced miR122 expression levels.


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)

Forced miR122 expression sensitizes Hep3B cells to sorafenibtreatment(a, b) The effects of miR122 on the reporter constructscarrying miR122 responsive elements (a) and the SLC7A1 3′UTR (b)in control and miR122 precursor-expressing Hep3B cells. Control cellvalues were set to 1. Data represent the means ± s.d. of threeindependent experiments. *, p < 0.05.(c) SLC7A1 expression was decreased in miR122precursor-overexpressing Hep3B cells. Flow cytometry assessment ofSLC7A1 protein expression was performed. Representative results fromthree independent experiments are shown. (d) IntracellularNO levels were decreased by miR122 precursor-expression in Hep3B cells.This effect was reversed by stable overexpression of HA-tagged SLC7A1.Control cells (control), miR122 precursor-overexpressing cells (miR122precursor) and cells with stable overexpression of both the miR122precursor and HA-tagged SLC7A1 (miR122 precursor + SLC7A1) weretested. Control cells under arginine-depleted conditions and treatedwith PTIO were included. *, p < 0.05(n = 3). (e) The effects ofsorafenib were inversely correlated with the expression levels ofmiR122. Relative cell numbers were counted after treatment for 48 h with5 μM sorafenib with or without PTIO in miR122precursor-expressing Hep3B cells. The number of the control cells withDMSO treatment was set to 1 *, p < 0.05(n = 3). Control cells (control), miR122precursor-expressing cells (miR122 precursor), cells cultured inarginine-depleted media (Arg depleted), and miR122 precursor-expressingcells with expression of SLC7A1 (miR122 precursor + SLC7A1) weretested. Cells treated with PTIO to remove NO were included as a control.(f, g) The effects of sorafenib were inverselycorrelated with the expression levels of miR122, as determined by flowcytometry (f) and fluorescence microscopy (g). miR122-silenced Huh7cells and miR122 precursor-expressing Hep3B cells showed GFPco-expression. The percentage of GFP-expressing cells (~50% beforesorafenib treatment) increased in the miR122-silenced Huh7 cells anddecreased in the miR122 precursor-expressing Hep3B cells after 5μM sorafenib treatment for 48 h, as determined by flow cytometry.*, p < 0.05 (n = 3) (f).Representative results of GFP-positive cells from three independentexperiments are shown (g).
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Figure 4: Forced miR122 expression sensitizes Hep3B cells to sorafenibtreatment(a, b) The effects of miR122 on the reporter constructscarrying miR122 responsive elements (a) and the SLC7A1 3′UTR (b)in control and miR122 precursor-expressing Hep3B cells. Control cellvalues were set to 1. Data represent the means ± s.d. of threeindependent experiments. *, p < 0.05.(c) SLC7A1 expression was decreased in miR122precursor-overexpressing Hep3B cells. Flow cytometry assessment ofSLC7A1 protein expression was performed. Representative results fromthree independent experiments are shown. (d) IntracellularNO levels were decreased by miR122 precursor-expression in Hep3B cells.This effect was reversed by stable overexpression of HA-tagged SLC7A1.Control cells (control), miR122 precursor-overexpressing cells (miR122precursor) and cells with stable overexpression of both the miR122precursor and HA-tagged SLC7A1 (miR122 precursor + SLC7A1) weretested. Control cells under arginine-depleted conditions and treatedwith PTIO were included. *, p < 0.05(n = 3). (e) The effects ofsorafenib were inversely correlated with the expression levels ofmiR122. Relative cell numbers were counted after treatment for 48 h with5 μM sorafenib with or without PTIO in miR122precursor-expressing Hep3B cells. The number of the control cells withDMSO treatment was set to 1 *, p < 0.05(n = 3). Control cells (control), miR122precursor-expressing cells (miR122 precursor), cells cultured inarginine-depleted media (Arg depleted), and miR122 precursor-expressingcells with expression of SLC7A1 (miR122 precursor + SLC7A1) weretested. Cells treated with PTIO to remove NO were included as a control.(f, g) The effects of sorafenib were inverselycorrelated with the expression levels of miR122, as determined by flowcytometry (f) and fluorescence microscopy (g). miR122-silenced Huh7cells and miR122 precursor-expressing Hep3B cells showed GFPco-expression. The percentage of GFP-expressing cells (~50% beforesorafenib treatment) increased in the miR122-silenced Huh7 cells anddecreased in the miR122 precursor-expressing Hep3B cells after 5μM sorafenib treatment for 48 h, as determined by flow cytometry.*, p < 0.05 (n = 3) (f).Representative results of GFP-positive cells from three independentexperiments are shown (g).
Mentions: To determine the converse effects, the miR122 precursor was stably expressed inHep3B cells, because these cells naturally expressed only minimal levels ofmiR122 [5]. Hep3B cells expressing themiR122 precursor showed enhanced miR122 function (Figure 4a) and suppressed luciferase expression from a reportercarrying the 3′ UTR of the SLC7A1 gene (Figure 4b), with lower expression of SLC7A1 protein compared withthe control cells (Figure 4c). Furthermore,the cells showed lower intracellular NO levels, similar to those of cells grownunder arginine-depleted culture conditions or treated with PTIO (Figure 4d). Because stable overexpression of SLC7A1in the miR122 precursor-stably expressing Hep3B cells reversed the effects ofintracellular NO contents, changes in intracellular NO levels appeared dependenton changes in the SLC7A1 expression levels (Figure 4d). It was noted that miR122 expression had a positive effect onthe sorafenib-treated Hep3B cells, similar to the effects of removing NO withPTIO (Figure 4e). To exclude any possibleinfluences of stable cell line specificity or differences in the cultureconditions of the individual cell lines, Huh7 or Hep3B cells were transducedwith miR122-silencing or miR122-overexpressing lentiviruses with GFP, butwithout selection. GFP-positive (miR122-silenced) Huh7 cells and GFP-negative(miR122-non-overexpressing) Hep3B cells were more concentrated after sorafenibtreatment of mixtures of infected (GFP-expressing) and non-infected(non-GFP-expressing) cells cultured in bulk (Figure 4f and g), confirming that decreased miR122 function wasrelated to sorafenib resistance. These results suggest that miR122-silenced HCCcells are more resistant to chemotherapeutic drugs, and that depletion ofarginine in extracellular environments may be effective for the clinicalmanagement of an HCC subset with reduced miR122 expression levels.

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