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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 targets PTEN to induce proliferation and migration of renal cancer cells.ACHN cells were transfected with miR-21 Sponge along with siRNA pools targeting PTEN mRNA as indicated. (A) 3H-thymidine incorporation was determined as described in the Materials and Methods[115]. Mean ± SE of 6 measurements is shown. *p<0.05 vs vector; **p<0.05 vs miR-21 Sponge-transfected cells. (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 siRNA pool against PTEN, respectively. Mean ± SE of triplicate measurements is shown. *p<0.05 vs vector alone; **p<0.01 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-g005: miR-21 targets PTEN to induce proliferation and migration of renal cancer cells.ACHN cells were transfected with miR-21 Sponge along with siRNA pools targeting PTEN mRNA as indicated. (A) 3H-thymidine incorporation was determined as described in the Materials and Methods[115]. Mean ± SE of 6 measurements is shown. *p<0.05 vs vector; **p<0.05 vs miR-21 Sponge-transfected cells. (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 siRNA pool against PTEN, respectively. Mean ± SE of triplicate measurements is shown. *p<0.05 vs vector alone; **p<0.01 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: Our results above suggest that miR-21 promotes Akt activation by decreasing PTEN levels in renal cancer cells (Fig. 4). In order to directly investigate the role of this signaling pathway in renal cancer cell proliferation, we transfected ACHN and Caki-2 cells with miR-21 Sponge and siRNAs targeting PTEN (siPTEN). As expected, miR-21 Sponge inhibited DNA synthesis (Fig. 5A and Fig. S7A). In contrast, transfection of siPTEN significantly prevented miR-21 Sponge-induced inhibition of DNA synthesis (Fig. 5A and Figs. S7A, S7B and S7C). Similarly, siPTEN reversed the decrease in proliferation of ACHN cells induced by miR-21 Sponge (Fig. 5B and Fig. S8). Next, we determined the effect of siPTEN on ACHN and Caki-2 cell migration. Expression of miR-21 Sponge decreased the migration of both these renal cancer cell lines. siPTEN significantly suppressed the inhibitory effect of miR-21 Sponge on cell migration (Figs. 5C, 5D and Figs. S9 and S10).


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 targets PTEN to induce proliferation and migration of renal cancer cells.ACHN cells were transfected with miR-21 Sponge along with siRNA pools targeting PTEN mRNA as indicated. (A) 3H-thymidine incorporation was determined as described in the Materials and Methods[115]. Mean ± SE of 6 measurements is shown. *p<0.05 vs vector; **p<0.05 vs miR-21 Sponge-transfected cells. (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 siRNA pool against PTEN, respectively. Mean ± SE of triplicate measurements is shown. *p<0.05 vs vector alone; **p<0.01 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-g005: miR-21 targets PTEN to induce proliferation and migration of renal cancer cells.ACHN cells were transfected with miR-21 Sponge along with siRNA pools targeting PTEN mRNA as indicated. (A) 3H-thymidine incorporation was determined as described in the Materials and Methods[115]. Mean ± SE of 6 measurements is shown. *p<0.05 vs vector; **p<0.05 vs miR-21 Sponge-transfected cells. (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 siRNA pool against PTEN, respectively. Mean ± SE of triplicate measurements is shown. *p<0.05 vs vector alone; **p<0.01 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: Our results above suggest that miR-21 promotes Akt activation by decreasing PTEN levels in renal cancer cells (Fig. 4). In order to directly investigate the role of this signaling pathway in renal cancer cell proliferation, we transfected ACHN and Caki-2 cells with miR-21 Sponge and siRNAs targeting PTEN (siPTEN). As expected, miR-21 Sponge inhibited DNA synthesis (Fig. 5A and Fig. S7A). In contrast, transfection of siPTEN significantly prevented miR-21 Sponge-induced inhibition of DNA synthesis (Fig. 5A and Figs. S7A, S7B and S7C). Similarly, siPTEN reversed the decrease in proliferation of ACHN cells induced by miR-21 Sponge (Fig. 5B and Fig. S8). Next, we determined the effect of siPTEN on ACHN and Caki-2 cell migration. Expression of miR-21 Sponge decreased the migration of both these renal cancer cell lines. siPTEN significantly suppressed the inhibitory effect of miR-21 Sponge on cell migration (Figs. 5C, 5D and Figs. S9 and S10).

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