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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

Arginine depletion decreases NO levels in miR122-silenced Huh7cells(a) Intracellular arginine levels decreased inmiR122-silenced cells under arginine-depleted conditions. *,p < 0.05 (n = 3).(b) Intracellular NO levels decreased inmiR122-silenced cells under arginine-depleted conditions. This effectwas reversed by knockdown of SLC7A1. Control cells (control),miR122-silecend cells (miR122-silenced), miR122-silenced cells culturedin arginine-depleted media (miR122-silenced + Arg depleted), andmiR122-silenced cells with expression of shSLC7A1 (miR122-silenced+ shSLC7A1) were tested. Cells treated with PTIO to remove NOwere included as a positive control. *, p < 0.05(n = 3).
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Figure 3: Arginine depletion decreases NO levels in miR122-silenced Huh7cells(a) Intracellular arginine levels decreased inmiR122-silenced cells under arginine-depleted conditions. *,p < 0.05 (n = 3).(b) Intracellular NO levels decreased inmiR122-silenced cells under arginine-depleted conditions. This effectwas reversed by knockdown of SLC7A1. Control cells (control),miR122-silecend cells (miR122-silenced), miR122-silenced cells culturedin arginine-depleted media (miR122-silenced + Arg depleted), andmiR122-silenced cells with expression of shSLC7A1 (miR122-silenced+ shSLC7A1) were tested. Cells treated with PTIO to remove NOwere included as a positive control. *, p < 0.05(n = 3).

Mentions: Because intracellular arginine levels are largely dependent on the transport ofextracellular arginine into cells [36],the effects of arginine depletion in the culture media were examined. Althoughintracellular arginine levels were increased in miR122-silenced cells in thearginine-content media, this increase was not seen when the cells were culturedunder arginine-depleted conditions (Figure 3a). Accordingly, culture under arginine-depleted conditionsprevented an increase in intracellular NO, with levels similar to those of thecontrol cells treated with a NO remover (Figure 3b). In addition, because knockdown of SLC7A1 in miR122-silencedcells through expression of shSLC7A1 also prevented an increase in intracellularNO, it was considered that the increased NO levels in miR122-silenced cells weredue to increased SLC7A1 levels (Figure 3b).Concordantly, although we could not test the effects of sorafenib underarginine-depleted media because most cells died when maintained in serum-freemedia (to remove the effects of arginine in serum) and treated for 48 h withsorafenib, the expression levels of HCC cancer-progenitor cell-markers weredecreased by arginine depletion (Supplementary Figure 3b). These results showed that reducedintracellular arginine and NO levels in miR122-silecend cells can be achieved bythe depletion of arginine in the extracellular media.


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)

Arginine depletion decreases NO levels in miR122-silenced Huh7cells(a) Intracellular arginine levels decreased inmiR122-silenced cells under arginine-depleted conditions. *,p < 0.05 (n = 3).(b) Intracellular NO levels decreased inmiR122-silenced cells under arginine-depleted conditions. This effectwas reversed by knockdown of SLC7A1. Control cells (control),miR122-silecend cells (miR122-silenced), miR122-silenced cells culturedin arginine-depleted media (miR122-silenced + Arg depleted), andmiR122-silenced cells with expression of shSLC7A1 (miR122-silenced+ shSLC7A1) were tested. Cells treated with PTIO to remove NOwere included as a positive control. *, p < 0.05(n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480756&req=5

Figure 3: Arginine depletion decreases NO levels in miR122-silenced Huh7cells(a) Intracellular arginine levels decreased inmiR122-silenced cells under arginine-depleted conditions. *,p < 0.05 (n = 3).(b) Intracellular NO levels decreased inmiR122-silenced cells under arginine-depleted conditions. This effectwas reversed by knockdown of SLC7A1. Control cells (control),miR122-silecend cells (miR122-silenced), miR122-silenced cells culturedin arginine-depleted media (miR122-silenced + Arg depleted), andmiR122-silenced cells with expression of shSLC7A1 (miR122-silenced+ shSLC7A1) were tested. Cells treated with PTIO to remove NOwere included as a positive control. *, p < 0.05(n = 3).
Mentions: Because intracellular arginine levels are largely dependent on the transport ofextracellular arginine into cells [36],the effects of arginine depletion in the culture media were examined. Althoughintracellular arginine levels were increased in miR122-silenced cells in thearginine-content media, this increase was not seen when the cells were culturedunder arginine-depleted conditions (Figure 3a). Accordingly, culture under arginine-depleted conditionsprevented an increase in intracellular NO, with levels similar to those of thecontrol cells treated with a NO remover (Figure 3b). In addition, because knockdown of SLC7A1 in miR122-silencedcells through expression of shSLC7A1 also prevented an increase in intracellularNO, it was considered that the increased NO levels in miR122-silenced cells weredue to increased SLC7A1 levels (Figure 3b).Concordantly, although we could not test the effects of sorafenib underarginine-depleted media because most cells died when maintained in serum-freemedia (to remove the effects of arginine in serum) and treated for 48 h withsorafenib, the expression levels of HCC cancer-progenitor cell-markers weredecreased by arginine depletion (Supplementary Figure 3b). These results showed that reducedintracellular arginine and NO levels in miR122-silecend cells can be achieved bythe depletion of arginine in the extracellular media.

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