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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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Expression of miR-21 in renal cancer cells.(A – C) Total RNAs from HK2 proximal tubular epithelial cells and ACHN renal cancer cells were used to detect mature miR-21 (panel A), pre-miR-21 (panel B) and pri-miR-21 (panel C) by real time qRT-PCR as described in the Materials and Methods. Mean ± SE of 4 measurements is shown. In panels A and B, *p  = 0.004 vs HK2 cells. In panel C, *p  = 0.02 vs HK2 cells. (D) HK2 and ACHN cells were transfected with miR-21-Luc reporter along with Renilla  plasmid. The cell lysates were used to determine luciferase activity as described in the Materials and Methods. Mean ± SE of 6 measurements is shown. *p  = 0.001 vs HK2 cells. (E) Increased phosphorylation of p65 NFkB in renal cancer cells. Lysates of HK2 and ACHN cells were immunoblotted with phospho-p65 (Ser-536), p65 and actin antibodies. (F) Mutant p65 S536A inhibits miR-21 transcription. ACHN renal cancer cells were cotransfected with miR-21-Luc and p65 S536A expression vector. The luciferase activity was determined in the cell lysates. Mean ± SE of quadruplicate measurements is shown. *p  = 0.02 vs vector. Bottom panels show expression of the HA-tagged p65 S536A and actin.
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pone-0037366-g001: Expression of miR-21 in renal cancer cells.(A – C) Total RNAs from HK2 proximal tubular epithelial cells and ACHN renal cancer cells were used to detect mature miR-21 (panel A), pre-miR-21 (panel B) and pri-miR-21 (panel C) by real time qRT-PCR as described in the Materials and Methods. Mean ± SE of 4 measurements is shown. In panels A and B, *p  = 0.004 vs HK2 cells. In panel C, *p  = 0.02 vs HK2 cells. (D) HK2 and ACHN cells were transfected with miR-21-Luc reporter along with Renilla plasmid. The cell lysates were used to determine luciferase activity as described in the Materials and Methods. Mean ± SE of 6 measurements is shown. *p  = 0.001 vs HK2 cells. (E) Increased phosphorylation of p65 NFkB in renal cancer cells. Lysates of HK2 and ACHN cells were immunoblotted with phospho-p65 (Ser-536), p65 and actin antibodies. (F) Mutant p65 S536A inhibits miR-21 transcription. ACHN renal cancer cells were cotransfected with miR-21-Luc and p65 S536A expression vector. The luciferase activity was determined in the cell lysates. Mean ± SE of quadruplicate measurements is shown. *p  = 0.02 vs vector. Bottom panels show expression of the HA-tagged p65 S536A and actin.

Mentions: miRNA expression profiling using tumor samples from patients with renal carcinoma has been reported. In three studies, the expression of miR-21 was found to be increased [14], [17], [19]. However, in another study in 16 clear cell renal carcinoma and 4 chromophobe renal carcinoma samples, miR-21 was not detected [16]. We used a panel of tumor tissues from clear cell renal carcinomas to detect the expression of mature miR-21. Real time qRT-PCR revealed markedly increased expression of mature miR-21 in all grade 2 (3 out of 3) and grade 3 (3 out of 3) renal cell carcinoma samples (Fig. S1A and S1B). Next, we investigated the expression of miR-21 in ACHN renal carcinoma cells. We found significantly increased expression of mature miR-21 in ACHN cells as compared to the HK2 normal proximal tubular epithelial cells (Fig. 1A). Similar results were obtained in another renal cancer cell line Caki-2 (Fig. S2). Recent reports showed that miR-21 is regulated at the level of maturation [20]. Therefore, we examined the levels of pre-miR-21. Real time qRT-PCR showed marked expression of pre-miR-21 in ACHN cells compared to HK2 cells (Fig. 1B). Similarly, increased expression of pri-miR-21 was also detected in the ACHN cells (Fig. 1C). This increase in pri-miR suggested a transcriptional mechanism of miR-21 expression. To directly examine the transcriptional regulation of miR-21 expression, we used a miR-21 promoter-driven firefly luciferase reporter construct (miR-21-Luc). Transient transfection assays in ACHN cells revealed significantly increased transcription of the reporter gene (Fig. 1D). These results indicate that enhanced levels of mature miR-21 in renal carcinoma cells may result from the increased transcription of miR-21 gene to produce pri-mir-21.


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)

Expression of miR-21 in renal cancer cells.(A – C) Total RNAs from HK2 proximal tubular epithelial cells and ACHN renal cancer cells were used to detect mature miR-21 (panel A), pre-miR-21 (panel B) and pri-miR-21 (panel C) by real time qRT-PCR as described in the Materials and Methods. Mean ± SE of 4 measurements is shown. In panels A and B, *p  = 0.004 vs HK2 cells. In panel C, *p  = 0.02 vs HK2 cells. (D) HK2 and ACHN cells were transfected with miR-21-Luc reporter along with Renilla  plasmid. The cell lysates were used to determine luciferase activity as described in the Materials and Methods. Mean ± SE of 6 measurements is shown. *p  = 0.001 vs HK2 cells. (E) Increased phosphorylation of p65 NFkB in renal cancer cells. Lysates of HK2 and ACHN cells were immunoblotted with phospho-p65 (Ser-536), p65 and actin antibodies. (F) Mutant p65 S536A inhibits miR-21 transcription. ACHN renal cancer cells were cotransfected with miR-21-Luc and p65 S536A expression vector. The luciferase activity was determined in the cell lysates. Mean ± SE of quadruplicate measurements is shown. *p  = 0.02 vs vector. Bottom panels show expression of the HA-tagged p65 S536A and actin.
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getmorefigures.php?uid=PMC3368259&req=5

pone-0037366-g001: Expression of miR-21 in renal cancer cells.(A – C) Total RNAs from HK2 proximal tubular epithelial cells and ACHN renal cancer cells were used to detect mature miR-21 (panel A), pre-miR-21 (panel B) and pri-miR-21 (panel C) by real time qRT-PCR as described in the Materials and Methods. Mean ± SE of 4 measurements is shown. In panels A and B, *p  = 0.004 vs HK2 cells. In panel C, *p  = 0.02 vs HK2 cells. (D) HK2 and ACHN cells were transfected with miR-21-Luc reporter along with Renilla plasmid. The cell lysates were used to determine luciferase activity as described in the Materials and Methods. Mean ± SE of 6 measurements is shown. *p  = 0.001 vs HK2 cells. (E) Increased phosphorylation of p65 NFkB in renal cancer cells. Lysates of HK2 and ACHN cells were immunoblotted with phospho-p65 (Ser-536), p65 and actin antibodies. (F) Mutant p65 S536A inhibits miR-21 transcription. ACHN renal cancer cells were cotransfected with miR-21-Luc and p65 S536A expression vector. The luciferase activity was determined in the cell lysates. Mean ± SE of quadruplicate measurements is shown. *p  = 0.02 vs vector. Bottom panels show expression of the HA-tagged p65 S536A and actin.
Mentions: miRNA expression profiling using tumor samples from patients with renal carcinoma has been reported. In three studies, the expression of miR-21 was found to be increased [14], [17], [19]. However, in another study in 16 clear cell renal carcinoma and 4 chromophobe renal carcinoma samples, miR-21 was not detected [16]. We used a panel of tumor tissues from clear cell renal carcinomas to detect the expression of mature miR-21. Real time qRT-PCR revealed markedly increased expression of mature miR-21 in all grade 2 (3 out of 3) and grade 3 (3 out of 3) renal cell carcinoma samples (Fig. S1A and S1B). Next, we investigated the expression of miR-21 in ACHN renal carcinoma cells. We found significantly increased expression of mature miR-21 in ACHN cells as compared to the HK2 normal proximal tubular epithelial cells (Fig. 1A). Similar results were obtained in another renal cancer cell line Caki-2 (Fig. S2). Recent reports showed that miR-21 is regulated at the level of maturation [20]. Therefore, we examined the levels of pre-miR-21. Real time qRT-PCR showed marked expression of pre-miR-21 in ACHN cells compared to HK2 cells (Fig. 1B). Similarly, increased expression of pri-miR-21 was also detected in the ACHN cells (Fig. 1C). This increase in pri-miR suggested a transcriptional mechanism of miR-21 expression. To directly examine the transcriptional regulation of miR-21 expression, we used a miR-21 promoter-driven firefly luciferase reporter construct (miR-21-Luc). Transient transfection assays in ACHN cells revealed significantly increased transcription of the reporter gene (Fig. 1D). These results indicate that enhanced levels of mature miR-21 in renal carcinoma cells may result from the increased transcription of miR-21 gene to produce pri-mir-21.

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