Limits...
MicroRNA-21 is a candidate driver gene for 17q23-25 amplification in ovarian clear cell carcinoma.

Hirata Y, Murai N, Yanaihara N, Saito M, Saito M, Urashima M, Murakami Y, Matsufuji S, Okamoto A - BMC Cancer (2014)

Bottom Line: The patients with 17q23-25 amplification had significantly shorter progression-free and overall survival than those without 17q23-25 amplification (log-rank test: p = 0.0496; p = 0.0469, respectively).A significant correlation was observed between miR-21 overexpression and endometriosis.Aberrant expression of miR-21 by chromosomal amplification might play an important role in CCC carcinogenesis through the regulation of the PTEN tumor suppressor gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan. yanazou@jikei.ac.jp.

ABSTRACT

Background: Epithelial ovarian cancer (EOC) is the most common cause of gynecological malignancy-related mortality. Ovarian clear cell carcinoma (CCC) has unique clinical characteristics and behaviors that differ from other histological types of EOC, including a frequent association with endometriosis and a highly chemoresistant nature, resulting in poor prognosis. However, factors underlying its malignant behavior are still poorly understood. Aberrant expression of microRNAs has been shown to be involved in oncogenesis, and microRNA-21 (miR-21) is frequently overexpressed in many types of cancers. The aim of this study was to investigate the role of miR-21 in 17q23-25 amplification associated with CCC oncogenesis.

Methods: We identified 17q23-25 copy number aberrations among 28 primary CCC tumors by using a comparative genomic hybridization method. Next, we measured expression levels of the candidate target genes, miR-21 and PPM1D, for 17q23-25 amplification by real-time RT-PCR analysis and compared those data with copy number status and clinicopathological features. In addition, immunohistochemical analysis of PTEN (a potential target of miR-21) was performed using the same primary CCC cases. We investigated the biological significance of miR-21 overexpression in CCC using a loss-of-function antisense approach.

Results: 17q23-25 amplification with both miR-21 overexpression and PTEN protein loss was detected in 4/28 CCC cases (14.2%). The patients with 17q23-25 amplification had significantly shorter progression-free and overall survival than those without 17q23-25 amplification (log-rank test: p = 0.0496; p = 0.0469, respectively). A significant correlation was observed between miR-21 overexpression and endometriosis. Both PTEN mRNA and PTEN protein expression were increased by miR-21 knockdown in CCC cells. We also confirmed that miR-21 directly bound to the 3'-untranslated region of PTEN mRNA using a dual-luciferase reporter assay.

Conclusions: MiR-21 is a possible driver gene other than PPM1D for 17q23-25 amplification in CCC. Aberrant expression of miR-21 by chromosomal amplification might play an important role in CCC carcinogenesis through the regulation of the PTEN tumor suppressor gene.

Show MeSH

Related in: MedlinePlus

miR-21modulates PTEN tumor suppressor gene expression. To evaluate the biological significance of miR-21 overexpression in CCC, we used a loss-of-function antisense approach. An antisense miR-21 oligonucleotide (ODN) was used to knock down miR-21 expression in RMG-II cells. (A) Efficiency of RMG-II cell transfection was confirmed by real- time RT PCR. PTEN mRNA expression was increased by knockdown of miR-21 in RMG-II cells. Western blot analysis showing that PTEN expression was increased in RMG-II cells upon inhibition of miR-21. (B)MiR-21 directly targets the 3'-UTR of PTEN mRNA. The activity of luciferase in the pGL3 wild-type PTEN 3′-UTR was downregulated compared to pGL3 mutant-type PTEN 3’-UTR and the pGL3 control in RMG-II cells. P <0.05 according to the t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4289307&req=5

Fig3: miR-21modulates PTEN tumor suppressor gene expression. To evaluate the biological significance of miR-21 overexpression in CCC, we used a loss-of-function antisense approach. An antisense miR-21 oligonucleotide (ODN) was used to knock down miR-21 expression in RMG-II cells. (A) Efficiency of RMG-II cell transfection was confirmed by real- time RT PCR. PTEN mRNA expression was increased by knockdown of miR-21 in RMG-II cells. Western blot analysis showing that PTEN expression was increased in RMG-II cells upon inhibition of miR-21. (B)MiR-21 directly targets the 3'-UTR of PTEN mRNA. The activity of luciferase in the pGL3 wild-type PTEN 3′-UTR was downregulated compared to pGL3 mutant-type PTEN 3’-UTR and the pGL3 control in RMG-II cells. P <0.05 according to the t-test.

Mentions: To investigate the regulation of PTEN expression by miR-21 in CCC, we used a loss-of-function antisense approach in RMG-II cells. Knockdown efficiency was confirmed by real-time RT-PCR analysis of miR-21 (Figure 3A). In RMG-II cells, we found that miR-21 knockdown caused a significant increase in PTEN protein expression as indicated by Western blot analysis, along with increased PTEN mRNA expression (Figure 3A). However, suppression of miR-21 expression did not inhibit cell proliferation or invasion (date not shown). We next investigated the direct binding of miR-21 to the 3’UTR of PTEN mRNA by luciferase assay using a pGL3 plasmid harboring either the wild- or mutant-type PTEN 3’-UTR. The activity of the luciferase reporter was significantly decreased when fused to the wild-type PTEN 3′-UTR. Deletion mutations in the miR-21–interacting seed region rescued the luciferase activity. Taken together, these data suggest that PTEN is a direct functional target of miR-21, and its expression is regulated by miR-21 in CCC (Figure 3B). Several potential miR21 targets that could have implications in CCC were identified using web-based computational approaches to predict gene targets (miRBase Targets BETA Version 1.0, PicTar predictions, and TargetScan). Three putative target genes, PDCD4, SMARCA4, and SPRY2, were predicted by 3 different programs. This result indicates that tumor suppressor genes are potentially regulated by miR21. Therefore, we performed real-time RT-PCR for PDCD4, SMARCA4, SPRY2 in the miR21 knockdown experiments in RMG-II cells. We found that miR-21 knockdown increased the expression of these mRNAs (Additional file 5: Figure S5). To investigate the regulation of PTEN expression by miR-21 in JHOC9 cells, we overexpressed miR21 using miR21 mimics in JHOC9 cell. Quantitative real-time PCR analysis confirmed the level of miR21 was significantly overexpressed. As expected, the level of PTEN mRNA was downregulated in JHOC9 cells. Expression of PDCD4, SMARCA4, and SPRY2 mRNA was also decreased by the overexpression of miR-21 in response to miR-21 mimics in JHOC9 cells (Additional file 6: Figure S6).Figure 3


MicroRNA-21 is a candidate driver gene for 17q23-25 amplification in ovarian clear cell carcinoma.

Hirata Y, Murai N, Yanaihara N, Saito M, Saito M, Urashima M, Murakami Y, Matsufuji S, Okamoto A - BMC Cancer (2014)

miR-21modulates PTEN tumor suppressor gene expression. To evaluate the biological significance of miR-21 overexpression in CCC, we used a loss-of-function antisense approach. An antisense miR-21 oligonucleotide (ODN) was used to knock down miR-21 expression in RMG-II cells. (A) Efficiency of RMG-II cell transfection was confirmed by real- time RT PCR. PTEN mRNA expression was increased by knockdown of miR-21 in RMG-II cells. Western blot analysis showing that PTEN expression was increased in RMG-II cells upon inhibition of miR-21. (B)MiR-21 directly targets the 3'-UTR of PTEN mRNA. The activity of luciferase in the pGL3 wild-type PTEN 3′-UTR was downregulated compared to pGL3 mutant-type PTEN 3’-UTR and the pGL3 control in RMG-II cells. P <0.05 according to the t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4289307&req=5

Fig3: miR-21modulates PTEN tumor suppressor gene expression. To evaluate the biological significance of miR-21 overexpression in CCC, we used a loss-of-function antisense approach. An antisense miR-21 oligonucleotide (ODN) was used to knock down miR-21 expression in RMG-II cells. (A) Efficiency of RMG-II cell transfection was confirmed by real- time RT PCR. PTEN mRNA expression was increased by knockdown of miR-21 in RMG-II cells. Western blot analysis showing that PTEN expression was increased in RMG-II cells upon inhibition of miR-21. (B)MiR-21 directly targets the 3'-UTR of PTEN mRNA. The activity of luciferase in the pGL3 wild-type PTEN 3′-UTR was downregulated compared to pGL3 mutant-type PTEN 3’-UTR and the pGL3 control in RMG-II cells. P <0.05 according to the t-test.
Mentions: To investigate the regulation of PTEN expression by miR-21 in CCC, we used a loss-of-function antisense approach in RMG-II cells. Knockdown efficiency was confirmed by real-time RT-PCR analysis of miR-21 (Figure 3A). In RMG-II cells, we found that miR-21 knockdown caused a significant increase in PTEN protein expression as indicated by Western blot analysis, along with increased PTEN mRNA expression (Figure 3A). However, suppression of miR-21 expression did not inhibit cell proliferation or invasion (date not shown). We next investigated the direct binding of miR-21 to the 3’UTR of PTEN mRNA by luciferase assay using a pGL3 plasmid harboring either the wild- or mutant-type PTEN 3’-UTR. The activity of the luciferase reporter was significantly decreased when fused to the wild-type PTEN 3′-UTR. Deletion mutations in the miR-21–interacting seed region rescued the luciferase activity. Taken together, these data suggest that PTEN is a direct functional target of miR-21, and its expression is regulated by miR-21 in CCC (Figure 3B). Several potential miR21 targets that could have implications in CCC were identified using web-based computational approaches to predict gene targets (miRBase Targets BETA Version 1.0, PicTar predictions, and TargetScan). Three putative target genes, PDCD4, SMARCA4, and SPRY2, were predicted by 3 different programs. This result indicates that tumor suppressor genes are potentially regulated by miR21. Therefore, we performed real-time RT-PCR for PDCD4, SMARCA4, SPRY2 in the miR21 knockdown experiments in RMG-II cells. We found that miR-21 knockdown increased the expression of these mRNAs (Additional file 5: Figure S5). To investigate the regulation of PTEN expression by miR-21 in JHOC9 cells, we overexpressed miR21 using miR21 mimics in JHOC9 cell. Quantitative real-time PCR analysis confirmed the level of miR21 was significantly overexpressed. As expected, the level of PTEN mRNA was downregulated in JHOC9 cells. Expression of PDCD4, SMARCA4, and SPRY2 mRNA was also decreased by the overexpression of miR-21 in response to miR-21 mimics in JHOC9 cells (Additional file 6: Figure S6).Figure 3

Bottom Line: The patients with 17q23-25 amplification had significantly shorter progression-free and overall survival than those without 17q23-25 amplification (log-rank test: p = 0.0496; p = 0.0469, respectively).A significant correlation was observed between miR-21 overexpression and endometriosis.Aberrant expression of miR-21 by chromosomal amplification might play an important role in CCC carcinogenesis through the regulation of the PTEN tumor suppressor gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan. yanazou@jikei.ac.jp.

ABSTRACT

Background: Epithelial ovarian cancer (EOC) is the most common cause of gynecological malignancy-related mortality. Ovarian clear cell carcinoma (CCC) has unique clinical characteristics and behaviors that differ from other histological types of EOC, including a frequent association with endometriosis and a highly chemoresistant nature, resulting in poor prognosis. However, factors underlying its malignant behavior are still poorly understood. Aberrant expression of microRNAs has been shown to be involved in oncogenesis, and microRNA-21 (miR-21) is frequently overexpressed in many types of cancers. The aim of this study was to investigate the role of miR-21 in 17q23-25 amplification associated with CCC oncogenesis.

Methods: We identified 17q23-25 copy number aberrations among 28 primary CCC tumors by using a comparative genomic hybridization method. Next, we measured expression levels of the candidate target genes, miR-21 and PPM1D, for 17q23-25 amplification by real-time RT-PCR analysis and compared those data with copy number status and clinicopathological features. In addition, immunohistochemical analysis of PTEN (a potential target of miR-21) was performed using the same primary CCC cases. We investigated the biological significance of miR-21 overexpression in CCC using a loss-of-function antisense approach.

Results: 17q23-25 amplification with both miR-21 overexpression and PTEN protein loss was detected in 4/28 CCC cases (14.2%). The patients with 17q23-25 amplification had significantly shorter progression-free and overall survival than those without 17q23-25 amplification (log-rank test: p = 0.0496; p = 0.0469, respectively). A significant correlation was observed between miR-21 overexpression and endometriosis. Both PTEN mRNA and PTEN protein expression were increased by miR-21 knockdown in CCC cells. We also confirmed that miR-21 directly bound to the 3'-untranslated region of PTEN mRNA using a dual-luciferase reporter assay.

Conclusions: MiR-21 is a possible driver gene other than PPM1D for 17q23-25 amplification in CCC. Aberrant expression of miR-21 by chromosomal amplification might play an important role in CCC carcinogenesis through the regulation of the PTEN tumor suppressor gene.

Show MeSH
Related in: MedlinePlus