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microRNA-21 governs TORC1 activation in renal cancer cell proliferation and invasion.

Dey N, Das F, Ghosh-Choudhury N, Mandal CC, Parekh DJ, Block K, Kasinath BS, Abboud HE, Choudhury GG - PLoS ONE (2012)

Bottom Line: Metastatic renal cancer manifests multiple signatures of gene expression.Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation.Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT
Metastatic renal cancer manifests multiple signatures of gene expression. Deviation in expression of mature miRNAs has been linked to human cancers. Importance of miR-21 in renal cell carcinomas is proposed from profiling studies using tumor tissue samples. However, the role of miR-21 function in causing renal cancer cell proliferation and invasion has not yet been shown. Using cultured renal carcinoma cells, we demonstrate enhanced expression of mature miR-21 along with pre-and pri-miR-21 by increased transcription compared to normal proximal tubular epithelial cells. Overexpression of miR-21 Sponge to quench endogenous miR-21 levels inhibited proliferation, migration and invasion of renal cancer cells. In the absence of mutation in the PTEN tumor suppressor gene, PTEN protein levels are frequently downregulated in renal cancer. We show that miR-21 targets PTEN mRNA 3'untranslated region to decrease PTEN protein expression and augments Akt phosphorylation in renal cancer cells. Downregulation of PTEN as well as overexpression of constitutively active Akt kinase prevented miR-21 Sponge-induced inhibition of renal cancer cell proliferation and migration. Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation. Finally, we demonstrate that expression of constitutively active TORC1 attenuated miR-21 Sponge-mediated suppression of proliferation and migration of renal cancer cells. Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.

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miR-21 stimulates Akt kinase to induce proliferation and migration of renal cancer cells.ACHN cells were transfected with miR-21 Sponge along with constitutively active Gag Akt plasmids as indicated. (A) 3H-thymidine incorporation was determined as described in the Materials and Methods[115]. Mean ± SE of 6 measurements is shown. *p<0.01 vs vector; **p<0.001 vs miR-21 Sponge alone. (B) Transfected cells were counted at indicated time periods. The symbols diamond, square and cross represent vector, miR-21 Sponge and miR-21 Sponge plus Gag Akt expression plasmids, respectively. *p<0.01 vs vector alone; **p<0.001 vs miR-21 Sponge alone. (C) Transfected cells were seeded onto membrane in trans-well chambers and the migrated cells were stained as described in the Materials and Methods[111]. (D) Stains from the membranes in panel C were eluted and absorbance was measured. Mean ± SE of 3 independent chambers is shown. *p<0.001 vs vector alone; **p<0.001 vs miR-21 Sponge.
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pone-0037366-g006: miR-21 stimulates Akt kinase to induce proliferation and migration of renal cancer cells.ACHN cells were transfected with miR-21 Sponge along with constitutively active Gag Akt plasmids as indicated. (A) 3H-thymidine incorporation was determined as described in the Materials and Methods[115]. Mean ± SE of 6 measurements is shown. *p<0.01 vs vector; **p<0.001 vs miR-21 Sponge alone. (B) Transfected cells were counted at indicated time periods. The symbols diamond, square and cross represent vector, miR-21 Sponge and miR-21 Sponge plus Gag Akt expression plasmids, respectively. *p<0.01 vs vector alone; **p<0.001 vs miR-21 Sponge alone. (C) Transfected cells were seeded onto membrane in trans-well chambers and the migrated cells were stained as described in the Materials and Methods[111]. (D) Stains from the membranes in panel C were eluted and absorbance was measured. Mean ± SE of 3 independent chambers is shown. *p<0.001 vs vector alone; **p<0.001 vs miR-21 Sponge.

Mentions: The lipid phosphatase activity of PTEN is known to regulate the Akt phosphorylation [27]. We have shown above that reduced PTEN level in renal cancer cells is associated with increased phosphorylation of Akt (Fig. 3 and Fig. S5). Since miR-21 regulates PTEN protein level, which in turn activates Akt, we tested the role of this kinase in renal cancer cell proliferation in relation to miR-21. We transfected ACHN and Caki-2 cells with constitutively active Gag-Akt along with miR-21 Sponge [33]. Expression of Gag Akt significantly reversed the inhibition of DNA synthesis induced by miR-21 Sponge (Fig. 6A, Fig. S11A and Fig. S12). Expression of Gag Akt also reversed the cell proliferation by miR-21 Sponge (6B and Figs. S13A). Similarly, constitutively active Akt significantly prevented the inhibition of migration of ACHN cells in response to miR-21 Sponge (Figs. 6C, 6D and Fig. S13B). Identical results were obtained with Caki-2 renal cancer cells (Figs. S14A, S14B and S14C). These results indicate that miR-21 targets PTEN mRNA to suppress its protein expression, which leads to Akt-dependent proliferation and migration of renal cancer cells.


microRNA-21 governs TORC1 activation in renal cancer cell proliferation and invasion.

Dey N, Das F, Ghosh-Choudhury N, Mandal CC, Parekh DJ, Block K, Kasinath BS, Abboud HE, Choudhury GG - PLoS ONE (2012)

miR-21 stimulates Akt kinase to induce proliferation and migration of renal cancer cells.ACHN cells were transfected with miR-21 Sponge along with constitutively active Gag Akt plasmids as indicated. (A) 3H-thymidine incorporation was determined as described in the Materials and Methods[115]. Mean ± SE of 6 measurements is shown. *p<0.01 vs vector; **p<0.001 vs miR-21 Sponge alone. (B) Transfected cells were counted at indicated time periods. The symbols diamond, square and cross represent vector, miR-21 Sponge and miR-21 Sponge plus Gag Akt expression plasmids, respectively. *p<0.01 vs vector alone; **p<0.001 vs miR-21 Sponge alone. (C) Transfected cells were seeded onto membrane in trans-well chambers and the migrated cells were stained as described in the Materials and Methods[111]. (D) Stains from the membranes in panel C were eluted and absorbance was measured. Mean ± SE of 3 independent chambers is shown. *p<0.001 vs vector alone; **p<0.001 vs miR-21 Sponge.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368259&req=5

pone-0037366-g006: miR-21 stimulates Akt kinase to induce proliferation and migration of renal cancer cells.ACHN cells were transfected with miR-21 Sponge along with constitutively active Gag Akt plasmids as indicated. (A) 3H-thymidine incorporation was determined as described in the Materials and Methods[115]. Mean ± SE of 6 measurements is shown. *p<0.01 vs vector; **p<0.001 vs miR-21 Sponge alone. (B) Transfected cells were counted at indicated time periods. The symbols diamond, square and cross represent vector, miR-21 Sponge and miR-21 Sponge plus Gag Akt expression plasmids, respectively. *p<0.01 vs vector alone; **p<0.001 vs miR-21 Sponge alone. (C) Transfected cells were seeded onto membrane in trans-well chambers and the migrated cells were stained as described in the Materials and Methods[111]. (D) Stains from the membranes in panel C were eluted and absorbance was measured. Mean ± SE of 3 independent chambers is shown. *p<0.001 vs vector alone; **p<0.001 vs miR-21 Sponge.
Mentions: The lipid phosphatase activity of PTEN is known to regulate the Akt phosphorylation [27]. We have shown above that reduced PTEN level in renal cancer cells is associated with increased phosphorylation of Akt (Fig. 3 and Fig. S5). Since miR-21 regulates PTEN protein level, which in turn activates Akt, we tested the role of this kinase in renal cancer cell proliferation in relation to miR-21. We transfected ACHN and Caki-2 cells with constitutively active Gag-Akt along with miR-21 Sponge [33]. Expression of Gag Akt significantly reversed the inhibition of DNA synthesis induced by miR-21 Sponge (Fig. 6A, Fig. S11A and Fig. S12). Expression of Gag Akt also reversed the cell proliferation by miR-21 Sponge (6B and Figs. S13A). Similarly, constitutively active Akt significantly prevented the inhibition of migration of ACHN cells in response to miR-21 Sponge (Figs. 6C, 6D and Fig. S13B). Identical results were obtained with Caki-2 renal cancer cells (Figs. S14A, S14B and S14C). These results indicate that miR-21 targets PTEN mRNA to suppress its protein expression, which leads to Akt-dependent proliferation and migration of renal cancer cells.

Bottom Line: Metastatic renal cancer manifests multiple signatures of gene expression.Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation.Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT
Metastatic renal cancer manifests multiple signatures of gene expression. Deviation in expression of mature miRNAs has been linked to human cancers. Importance of miR-21 in renal cell carcinomas is proposed from profiling studies using tumor tissue samples. However, the role of miR-21 function in causing renal cancer cell proliferation and invasion has not yet been shown. Using cultured renal carcinoma cells, we demonstrate enhanced expression of mature miR-21 along with pre-and pri-miR-21 by increased transcription compared to normal proximal tubular epithelial cells. Overexpression of miR-21 Sponge to quench endogenous miR-21 levels inhibited proliferation, migration and invasion of renal cancer cells. In the absence of mutation in the PTEN tumor suppressor gene, PTEN protein levels are frequently downregulated in renal cancer. We show that miR-21 targets PTEN mRNA 3'untranslated region to decrease PTEN protein expression and augments Akt phosphorylation in renal cancer cells. Downregulation of PTEN as well as overexpression of constitutively active Akt kinase prevented miR-21 Sponge-induced inhibition of renal cancer cell proliferation and migration. Moreover, we show that miR-21 Sponge inhibited the inactivating phosphorylation of the tumor suppressor protein tuberin and attenuated TORC1 activation. Finally, we demonstrate that expression of constitutively active TORC1 attenuated miR-21 Sponge-mediated suppression of proliferation and migration of renal cancer cells. Our results uncover a layer of post-transcriptional regulation of PTEN by transcriptional activation of miR-21 to force the canonical oncogenic Akt/TORC1 signaling conduit to drive renal cancer cell proliferation and invasion.

Show MeSH
Related in: MedlinePlus