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MicroRNA let-7c is downregulated in prostate cancer and suppresses prostate cancer growth.

Nadiminty N, Tummala R, Lou W, Zhu Y, Shi XB, Zou JX, Chen H, Zhang J, Chen X, Luo J, deVere White RW, Kung HJ, Evans CP, Gao AC - PLoS ONE (2012)

Bottom Line: Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts.Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro.Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, University of California Davis, Sacramento, California, United States of America. nnadiminty@ucdavis.edu

ABSTRACT

Purpose: Prostate cancer (PCa) is characterized by deregulated expression of several tumor suppressor or oncogenic miRNAs. The objective of this study was the identification and characterization of miR-let-7c as a potential tumor suppressor in PCa.

Experimental design: Levels of expression of miR-let-7c were examined in human PCa cell lines and tissues using qRT-PCR and in situ hybridization. Let-7c was overexpressed or suppressed to assess the effects on the growth of human PCa cell lines. Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts.

Results: We identified miR-let-7c as a potential tumor suppressor in PCa. Expression of let-7c is downregulated in castration-resistant prostate cancer (CRPC) cells. Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro. Suppression of let-7c expression enhanced the ability of androgen-sensitive PCa cells to grow in androgen-deprived conditions in vitro. Reconstitution of Let-7c by lentiviral-mediated intratumoral delivery significantly reduced tumor burden in xenografts of human PCa cells. Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens.

Conclusions: These results demonstrate that microRNA let-7c is downregulated in PCa and functions as a tumor suppressor, and is a potential therapeutic target for PCa.

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Let-7c is expressed in PCa cells.A) Total RNAs from LNCaP, PC-3, DU145, LNCaP-S17 and LN-IL6+ cells were analyzed by qRT-PCR. Results are presented as relative fold change compared to expression levels in LNCaP. Data points represent mean ± SD of triplicate samples from two independent experiments. B) 20 µg each of the above RNAs were also analyzed by northern blotting. U6 snRNA was used as the loading control. C) qRT-PCR showing the decrease in let-7c expression in LNCaP cells expressing Lin28 compared to LNCaP cells transfected with the empty vector (Con). Inset Western blot shows expression of Lin28. D) qRT-PCR showing the increase in let-7c expression in C4-2B cells transfected with shRNA against Lin28 compared to C4-2B cells transfected with control GFP shRNA. Inset Western blot shows downregulation of Lin28. Data points represent mean ± SD of triplicate samples from two independent experiments. Error bars denote ± SD (*p < 0.05). E) Western blot showing the expression levels of Lin28 in PCa cells. Actin is used as a loading control.
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pone-0032832-g001: Let-7c is expressed in PCa cells.A) Total RNAs from LNCaP, PC-3, DU145, LNCaP-S17 and LN-IL6+ cells were analyzed by qRT-PCR. Results are presented as relative fold change compared to expression levels in LNCaP. Data points represent mean ± SD of triplicate samples from two independent experiments. B) 20 µg each of the above RNAs were also analyzed by northern blotting. U6 snRNA was used as the loading control. C) qRT-PCR showing the decrease in let-7c expression in LNCaP cells expressing Lin28 compared to LNCaP cells transfected with the empty vector (Con). Inset Western blot shows expression of Lin28. D) qRT-PCR showing the increase in let-7c expression in C4-2B cells transfected with shRNA against Lin28 compared to C4-2B cells transfected with control GFP shRNA. Inset Western blot shows downregulation of Lin28. Data points represent mean ± SD of triplicate samples from two independent experiments. Error bars denote ± SD (*p < 0.05). E) Western blot showing the expression levels of Lin28 in PCa cells. Actin is used as a loading control.

Mentions: Our previous studies using miRNA microarrays showed that let-7c was among the most commonly modulated miRNAs in PCa cells (unpublished data). To determine the relative levels of expression of let-7c in PCa cells, we isolated total RNAs from LNCaP (androgen-dependent, AR-positive), PC-3, DU145 (castration-resistant, AR-negative) cells as well as LNCaP-S17 cells expressing IL-6 [30] and LN-IL6+ cells chronically treated with IL-6 [31]. cDNAs were analyzed by quantitative RT-PCR using primers amplifying the mature form of let-7c (Exiqon) specifically. Our results showed that let-7c is expressed at high levels in LNCaP cells compared to the hormone-refractory PC-3 and DU145 cells (Fig. 1A). Earlier reports showed that IL-6 and let-7 exhibit reciprocal regulation of expression. LNCaP-IL6+ and LNCaP-S17 cells (autocrine IL-6 signaling) showed reduction in let-7c levels, consistent with the report that IL-6 reduces let-7 expression in PCa cells and that let-7 regulates IL-6 expression [18]. These results were also confirmed by northern blotting using a probe against the mature let-7c sequence (Fig. 1B), suggesting that let-7c levels are reduced in more aggressive and castration-resistant PCa cells.


MicroRNA let-7c is downregulated in prostate cancer and suppresses prostate cancer growth.

Nadiminty N, Tummala R, Lou W, Zhu Y, Shi XB, Zou JX, Chen H, Zhang J, Chen X, Luo J, deVere White RW, Kung HJ, Evans CP, Gao AC - PLoS ONE (2012)

Let-7c is expressed in PCa cells.A) Total RNAs from LNCaP, PC-3, DU145, LNCaP-S17 and LN-IL6+ cells were analyzed by qRT-PCR. Results are presented as relative fold change compared to expression levels in LNCaP. Data points represent mean ± SD of triplicate samples from two independent experiments. B) 20 µg each of the above RNAs were also analyzed by northern blotting. U6 snRNA was used as the loading control. C) qRT-PCR showing the decrease in let-7c expression in LNCaP cells expressing Lin28 compared to LNCaP cells transfected with the empty vector (Con). Inset Western blot shows expression of Lin28. D) qRT-PCR showing the increase in let-7c expression in C4-2B cells transfected with shRNA against Lin28 compared to C4-2B cells transfected with control GFP shRNA. Inset Western blot shows downregulation of Lin28. Data points represent mean ± SD of triplicate samples from two independent experiments. Error bars denote ± SD (*p < 0.05). E) Western blot showing the expression levels of Lin28 in PCa cells. Actin is used as a loading control.
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pone-0032832-g001: Let-7c is expressed in PCa cells.A) Total RNAs from LNCaP, PC-3, DU145, LNCaP-S17 and LN-IL6+ cells were analyzed by qRT-PCR. Results are presented as relative fold change compared to expression levels in LNCaP. Data points represent mean ± SD of triplicate samples from two independent experiments. B) 20 µg each of the above RNAs were also analyzed by northern blotting. U6 snRNA was used as the loading control. C) qRT-PCR showing the decrease in let-7c expression in LNCaP cells expressing Lin28 compared to LNCaP cells transfected with the empty vector (Con). Inset Western blot shows expression of Lin28. D) qRT-PCR showing the increase in let-7c expression in C4-2B cells transfected with shRNA against Lin28 compared to C4-2B cells transfected with control GFP shRNA. Inset Western blot shows downregulation of Lin28. Data points represent mean ± SD of triplicate samples from two independent experiments. Error bars denote ± SD (*p < 0.05). E) Western blot showing the expression levels of Lin28 in PCa cells. Actin is used as a loading control.
Mentions: Our previous studies using miRNA microarrays showed that let-7c was among the most commonly modulated miRNAs in PCa cells (unpublished data). To determine the relative levels of expression of let-7c in PCa cells, we isolated total RNAs from LNCaP (androgen-dependent, AR-positive), PC-3, DU145 (castration-resistant, AR-negative) cells as well as LNCaP-S17 cells expressing IL-6 [30] and LN-IL6+ cells chronically treated with IL-6 [31]. cDNAs were analyzed by quantitative RT-PCR using primers amplifying the mature form of let-7c (Exiqon) specifically. Our results showed that let-7c is expressed at high levels in LNCaP cells compared to the hormone-refractory PC-3 and DU145 cells (Fig. 1A). Earlier reports showed that IL-6 and let-7 exhibit reciprocal regulation of expression. LNCaP-IL6+ and LNCaP-S17 cells (autocrine IL-6 signaling) showed reduction in let-7c levels, consistent with the report that IL-6 reduces let-7 expression in PCa cells and that let-7 regulates IL-6 expression [18]. These results were also confirmed by northern blotting using a probe against the mature let-7c sequence (Fig. 1B), suggesting that let-7c levels are reduced in more aggressive and castration-resistant PCa cells.

Bottom Line: Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts.Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro.Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, University of California Davis, Sacramento, California, United States of America. nnadiminty@ucdavis.edu

ABSTRACT

Purpose: Prostate cancer (PCa) is characterized by deregulated expression of several tumor suppressor or oncogenic miRNAs. The objective of this study was the identification and characterization of miR-let-7c as a potential tumor suppressor in PCa.

Experimental design: Levels of expression of miR-let-7c were examined in human PCa cell lines and tissues using qRT-PCR and in situ hybridization. Let-7c was overexpressed or suppressed to assess the effects on the growth of human PCa cell lines. Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts.

Results: We identified miR-let-7c as a potential tumor suppressor in PCa. Expression of let-7c is downregulated in castration-resistant prostate cancer (CRPC) cells. Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro. Suppression of let-7c expression enhanced the ability of androgen-sensitive PCa cells to grow in androgen-deprived conditions in vitro. Reconstitution of Let-7c by lentiviral-mediated intratumoral delivery significantly reduced tumor burden in xenografts of human PCa cells. Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens.

Conclusions: These results demonstrate that microRNA let-7c is downregulated in PCa and functions as a tumor suppressor, and is a potential therapeutic target for PCa.

Show MeSH
Related in: MedlinePlus