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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 regulates proliferation and migration of renal cancer cells through activation of TORC1.ACHN cells were transfected with miR-21 Sponge along with constitutively active mTOR 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<0.001 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 constitutively active (CA) mTOR 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 at 590 nm 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-g008: miR-21 regulates proliferation and migration of renal cancer cells through activation of TORC1.ACHN cells were transfected with miR-21 Sponge along with constitutively active mTOR 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<0.001 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 constitutively active (CA) mTOR 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 at 590 nm was measured. Mean ± SE of 3 independent chambers is shown. *p<0.001 vs vector alone; **p<0.001 vs miR-21 Sponge.

Mentions: To elucidate whether miR-21-activated TORC1 contributes to proliferation of renal cancer cells, we cotransfected ACHN and Caki-2 cells with miR-21 Sponge and a constitutively active mTOR expression vector. 3H-thymidine incorporation was determined. Expression of constitutively active mTOR significantly prevented the inhibition of DNA synthesis by miR-21 Sponge (Fig. 8A, Fig. S16 and Fig. S17). Similarly, constitutively active mTOR inhibited the miR-21 Sponge-mediated decrease in ACHN cell proliferation (Fig. 8B and Fig. S18A). Next we examined the effect of constitutively active mTOR on renal cancer cell migration. miR-21 Sponge abolished migration of ACHN and Caki-2 cells (Fig. 8C, Fig. S18B and Fig. S19). However, coexpression of constitutively active mTOR with miR-21 Sponge significantly reversed the inhibition in migration of both renal cancer cell lines in response to miR-21 Sponge (Figs. 8C, 8D and Fig. S19). Taken together these results demonstrate that miR-21 contributes to renal cancer cell proliferation and migration via TORC1.


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 regulates proliferation and migration of renal cancer cells through activation of TORC1.ACHN cells were transfected with miR-21 Sponge along with constitutively active mTOR 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<0.001 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 constitutively active (CA) mTOR 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 at 590 nm 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-g008: miR-21 regulates proliferation and migration of renal cancer cells through activation of TORC1.ACHN cells were transfected with miR-21 Sponge along with constitutively active mTOR 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<0.001 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 constitutively active (CA) mTOR 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 at 590 nm was measured. Mean ± SE of 3 independent chambers is shown. *p<0.001 vs vector alone; **p<0.001 vs miR-21 Sponge.
Mentions: To elucidate whether miR-21-activated TORC1 contributes to proliferation of renal cancer cells, we cotransfected ACHN and Caki-2 cells with miR-21 Sponge and a constitutively active mTOR expression vector. 3H-thymidine incorporation was determined. Expression of constitutively active mTOR significantly prevented the inhibition of DNA synthesis by miR-21 Sponge (Fig. 8A, Fig. S16 and Fig. S17). Similarly, constitutively active mTOR inhibited the miR-21 Sponge-mediated decrease in ACHN cell proliferation (Fig. 8B and Fig. S18A). Next we examined the effect of constitutively active mTOR on renal cancer cell migration. miR-21 Sponge abolished migration of ACHN and Caki-2 cells (Fig. 8C, Fig. S18B and Fig. S19). However, coexpression of constitutively active mTOR with miR-21 Sponge significantly reversed the inhibition in migration of both renal cancer cell lines in response to miR-21 Sponge (Figs. 8C, 8D and Fig. S19). Taken together these results demonstrate that miR-21 contributes to renal cancer cell proliferation and migration via TORC1.

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