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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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Effect of miR-21 Sponge on proliferation, migration and invasion of renal cancer cells.ACHN cells were transfected with miR-21 Sponge or vector plasmids. (A) Serum-starved cells were incubated with 3H-thymidine and its incorporation into DNA was determined as described in the Materials and Methods[115]. Mean ± SE of 12 measurements is shown. *p  = 0.004 vs vector alone. (B) The cells were counted at indicated time periods as described in the Materials and Methods. Diamond and square symbols represent vector and miR-21 Sponge-transfected cells, respectively. Means ± SE of triplicate measurements are shown. *p<0.05 vs vector alone. (C) Serum-starved cells were seeded on to membrane in a trans-well chamber. Migration assay was performed and the migrated cells at the opposite side of the membrane were stained as described in the Materials and Methods[111]. (D) The stains in the migrated cells in panel C were eluted as described in the Materials and Methods[111]. Mean ± SE of triplicate measurements is shown. *p<0.0001 vs vector-transfected cells. (E) Transfected serum-starved cells were seeded on to collagen-embedded membrane in a trans-well chamber. Invasion assay was performed and the invaded cells at the opposite side of the membrane were stained as described in the Materials and Methods[111]. (F) The stains in the invaded cells in panel E were eluted as described in the Materials and Methods[111]. Mean ± SE of triplicate measurements is shown. *p<0.001 vs vector-transfected cells.
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pone-0037366-g002: Effect of miR-21 Sponge on proliferation, migration and invasion of renal cancer cells.ACHN cells were transfected with miR-21 Sponge or vector plasmids. (A) Serum-starved cells were incubated with 3H-thymidine and its incorporation into DNA was determined as described in the Materials and Methods[115]. Mean ± SE of 12 measurements is shown. *p  = 0.004 vs vector alone. (B) The cells were counted at indicated time periods as described in the Materials and Methods. Diamond and square symbols represent vector and miR-21 Sponge-transfected cells, respectively. Means ± SE of triplicate measurements are shown. *p<0.05 vs vector alone. (C) Serum-starved cells were seeded on to membrane in a trans-well chamber. Migration assay was performed and the migrated cells at the opposite side of the membrane were stained as described in the Materials and Methods[111]. (D) The stains in the migrated cells in panel C were eluted as described in the Materials and Methods[111]. Mean ± SE of triplicate measurements is shown. *p<0.0001 vs vector-transfected cells. (E) Transfected serum-starved cells were seeded on to collagen-embedded membrane in a trans-well chamber. Invasion assay was performed and the invaded cells at the opposite side of the membrane were stained as described in the Materials and Methods[111]. (F) The stains in the invaded cells in panel E were eluted as described in the Materials and Methods[111]. Mean ± SE of triplicate measurements is shown. *p<0.001 vs vector-transfected cells.

Mentions: miR-21 is considered to be an onco-miR in many cancers. However, its role in renal cell carcinoma has not been explored. Therefore, we tested the involvement of miR-21 in mitogenesis of ACHN cells, using a plasmid vector expressing seven copies of the bulged miR-21 binding site placed in the 3′ end of CMV promoter-driven GFP mRNA (Fig. S3) [25]. Expression of this construct serves as a “Sponge” that quenches the levels of endogenous miR-21 [25], [26]. ACHN cells were transfected with this construct (miR-21 Sponge). DNA synthesis was measured as 3H-thymidine incorporation. Expression of miR-21 Sponge significantly inhibited DNA synthesis in ACHN cells (Fig. 2A and Fig. S4A). To confirm this observation, we performed proliferation assay by counting the number of cells after transfecting miR-21 Sponge. Expression of miR-21 Sponge suppressed cell growth (Fig. 2B and Fig. S4B).


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)

Effect of miR-21 Sponge on proliferation, migration and invasion of renal cancer cells.ACHN cells were transfected with miR-21 Sponge or vector plasmids. (A) Serum-starved cells were incubated with 3H-thymidine and its incorporation into DNA was determined as described in the Materials and Methods[115]. Mean ± SE of 12 measurements is shown. *p  = 0.004 vs vector alone. (B) The cells were counted at indicated time periods as described in the Materials and Methods. Diamond and square symbols represent vector and miR-21 Sponge-transfected cells, respectively. Means ± SE of triplicate measurements are shown. *p<0.05 vs vector alone. (C) Serum-starved cells were seeded on to membrane in a trans-well chamber. Migration assay was performed and the migrated cells at the opposite side of the membrane were stained as described in the Materials and Methods[111]. (D) The stains in the migrated cells in panel C were eluted as described in the Materials and Methods[111]. Mean ± SE of triplicate measurements is shown. *p<0.0001 vs vector-transfected cells. (E) Transfected serum-starved cells were seeded on to collagen-embedded membrane in a trans-well chamber. Invasion assay was performed and the invaded cells at the opposite side of the membrane were stained as described in the Materials and Methods[111]. (F) The stains in the invaded cells in panel E were eluted as described in the Materials and Methods[111]. Mean ± SE of triplicate measurements is shown. *p<0.001 vs vector-transfected cells.
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pone-0037366-g002: Effect of miR-21 Sponge on proliferation, migration and invasion of renal cancer cells.ACHN cells were transfected with miR-21 Sponge or vector plasmids. (A) Serum-starved cells were incubated with 3H-thymidine and its incorporation into DNA was determined as described in the Materials and Methods[115]. Mean ± SE of 12 measurements is shown. *p  = 0.004 vs vector alone. (B) The cells were counted at indicated time periods as described in the Materials and Methods. Diamond and square symbols represent vector and miR-21 Sponge-transfected cells, respectively. Means ± SE of triplicate measurements are shown. *p<0.05 vs vector alone. (C) Serum-starved cells were seeded on to membrane in a trans-well chamber. Migration assay was performed and the migrated cells at the opposite side of the membrane were stained as described in the Materials and Methods[111]. (D) The stains in the migrated cells in panel C were eluted as described in the Materials and Methods[111]. Mean ± SE of triplicate measurements is shown. *p<0.0001 vs vector-transfected cells. (E) Transfected serum-starved cells were seeded on to collagen-embedded membrane in a trans-well chamber. Invasion assay was performed and the invaded cells at the opposite side of the membrane were stained as described in the Materials and Methods[111]. (F) The stains in the invaded cells in panel E were eluted as described in the Materials and Methods[111]. Mean ± SE of triplicate measurements is shown. *p<0.001 vs vector-transfected cells.
Mentions: miR-21 is considered to be an onco-miR in many cancers. However, its role in renal cell carcinoma has not been explored. Therefore, we tested the involvement of miR-21 in mitogenesis of ACHN cells, using a plasmid vector expressing seven copies of the bulged miR-21 binding site placed in the 3′ end of CMV promoter-driven GFP mRNA (Fig. S3) [25]. Expression of this construct serves as a “Sponge” that quenches the levels of endogenous miR-21 [25], [26]. ACHN cells were transfected with this construct (miR-21 Sponge). DNA synthesis was measured as 3H-thymidine incorporation. Expression of miR-21 Sponge significantly inhibited DNA synthesis in ACHN cells (Fig. 2A and Fig. S4A). To confirm this observation, we performed proliferation assay by counting the number of cells after transfecting miR-21 Sponge. Expression of miR-21 Sponge suppressed cell growth (Fig. 2B and Fig. S4B).

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