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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 expression of PTEN in renal cancer cells.(A and B) ACHN cells were transfected with pCMV-miR-21 expression plasmid (A) or miR-21 Sponge (B) and vector along with PTEN 3′UTR-Luc reporter construct. The cell lysates were used to determine luciferase activity as described in the Materials and Methods. Mean ± SE of 6 measurements is shown. In panel A, *p  = 0.02 vs vector alone. In panel B, *p  = 0.002 vs vector alone. (C and D) Lysates of miR-21 Sponge-transfected ACHN cells were immunoblotted with PTEN and actin antibodies (panel C), and phospho-Akt (Ser-473), phospho-Akt (Thr-308) and Akt antibodies (panel D).
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pone-0037366-g004: miR-21 regulates expression of PTEN in renal cancer cells.(A and B) ACHN cells were transfected with pCMV-miR-21 expression plasmid (A) or miR-21 Sponge (B) and vector along with PTEN 3′UTR-Luc reporter construct. The cell lysates were used to determine luciferase activity as described in the Materials and Methods. Mean ± SE of 6 measurements is shown. In panel A, *p  = 0.02 vs vector alone. In panel B, *p  = 0.002 vs vector alone. (C and D) Lysates of miR-21 Sponge-transfected ACHN cells were immunoblotted with PTEN and actin antibodies (panel C), and phospho-Akt (Ser-473), phospho-Akt (Thr-308) and Akt antibodies (panel D).

Mentions: Next, we examined whether miR-21 targets PTEN. We used a reporter construct in which the 3′UTR of PTEN mRNA is cloned downstream of firefly luciferase gene (PTEN 3′UTR-Luc) [25]. ACHN cells were transfected with this reporter and vector or miR-21 expression plasmid. Expression of miR-21 significantly inhibited the reporter activity (Fig. 4A and Fig. S6A). To confirm this observation, we used miR-21 Sponge. Expression of miR-21 Sponge markedly increased the luciferase activity of PTEN 3′UTR-Luc (Fig. 4B and Fig. S6B). These results suggest that in renal cancer cells, PTEN may be a downstream target of miR-21. To test this, we examined PTEN protein level in miR-21 Sponge-transfected ACHN cells. miR-21 Sponge increased the abundance of PTEN (Fig. 4C and Fig. S6C), concomitant with decreased phosphorylation of Akt at Thr-308 and Ser-473 (Fig. 4D).


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 expression of PTEN in renal cancer cells.(A and B) ACHN cells were transfected with pCMV-miR-21 expression plasmid (A) or miR-21 Sponge (B) and vector along with PTEN 3′UTR-Luc reporter construct. The cell lysates were used to determine luciferase activity as described in the Materials and Methods. Mean ± SE of 6 measurements is shown. In panel A, *p  = 0.02 vs vector alone. In panel B, *p  = 0.002 vs vector alone. (C and D) Lysates of miR-21 Sponge-transfected ACHN cells were immunoblotted with PTEN and actin antibodies (panel C), and phospho-Akt (Ser-473), phospho-Akt (Thr-308) and Akt antibodies (panel D).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368259&req=5

pone-0037366-g004: miR-21 regulates expression of PTEN in renal cancer cells.(A and B) ACHN cells were transfected with pCMV-miR-21 expression plasmid (A) or miR-21 Sponge (B) and vector along with PTEN 3′UTR-Luc reporter construct. The cell lysates were used to determine luciferase activity as described in the Materials and Methods. Mean ± SE of 6 measurements is shown. In panel A, *p  = 0.02 vs vector alone. In panel B, *p  = 0.002 vs vector alone. (C and D) Lysates of miR-21 Sponge-transfected ACHN cells were immunoblotted with PTEN and actin antibodies (panel C), and phospho-Akt (Ser-473), phospho-Akt (Thr-308) and Akt antibodies (panel D).
Mentions: Next, we examined whether miR-21 targets PTEN. We used a reporter construct in which the 3′UTR of PTEN mRNA is cloned downstream of firefly luciferase gene (PTEN 3′UTR-Luc) [25]. ACHN cells were transfected with this reporter and vector or miR-21 expression plasmid. Expression of miR-21 significantly inhibited the reporter activity (Fig. 4A and Fig. S6A). To confirm this observation, we used miR-21 Sponge. Expression of miR-21 Sponge markedly increased the luciferase activity of PTEN 3′UTR-Luc (Fig. 4B and Fig. S6B). These results suggest that in renal cancer cells, PTEN may be a downstream target of miR-21. To test this, we examined PTEN protein level in miR-21 Sponge-transfected ACHN cells. miR-21 Sponge increased the abundance of PTEN (Fig. 4C and Fig. S6C), concomitant with decreased phosphorylation of Akt at Thr-308 and Ser-473 (Fig. 4D).

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