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Loss of miR-200c up-regulates CYP1B1 and confers docetaxel resistance in renal cell carcinoma.

Chang I, Mitsui Y, Fukuhara S, Gill A, Wong DK, Yamamura S, Shahryari V, Tabatabai ZL, Dahiya R, Shin DM, Tanaka Y - Oncotarget (2015)

Bottom Line: Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity.These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel.Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Division of Urology, Veterans Affairs Medical Center, San Francisco, California, United States of America.

ABSTRACT
Despite high protein expression and enzymatic activity of cytochrome P450 1B1 (CYP1B1) in renal cell cancer (RCC), its functional significance has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

No MeSH data available.


Related in: MedlinePlus

MiR-200c directly targets the CYP1B1 3′-UTR(A) Sequence and location of MCS1 and 2 in the 3′-UTR of human CYP1B1 mRNA. (B) Diagram of reporter constructs containing the wild type or MCS-deleted 3′-UTR of human CYP1B1. (C) Relative luciferase activity after transfection of reporter constructs containing the wild type or MCS-deleted 3′-UTR of CYP1B1. **P < 0.01; ***P < 0.001; n.s.: non-significant (D) Relative luciferase activity after co-transfection of reporter constructs containing the wild type or MCS-deleted 3′-UTR of CYP1B1with either miR-200c inhibitor or precursor. *P < 0.05; **P < 0.01; n.s.: non-significant (E-G) After miR-200c inhibitor transfection, relative miR-200c expression was analyzed by RT-PCR (E), CYP1B1 protein level was determined by Western blot (F) and enzyme activity was measured with P450-Glo assay (G) in 786-O cells. *P < 0.05; ***P < 0.001 (H-J) After miR-200c precursor transfection, relative miR-200c expression was analyzed by RT-PCR (H), CYP1B1 protein level was determined by Western blot (I) and enzyme activity was measured with P450-Glo assay (J) in A498 cells. **P < 0.01.
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Figure 5: MiR-200c directly targets the CYP1B1 3′-UTR(A) Sequence and location of MCS1 and 2 in the 3′-UTR of human CYP1B1 mRNA. (B) Diagram of reporter constructs containing the wild type or MCS-deleted 3′-UTR of human CYP1B1. (C) Relative luciferase activity after transfection of reporter constructs containing the wild type or MCS-deleted 3′-UTR of CYP1B1. **P < 0.01; ***P < 0.001; n.s.: non-significant (D) Relative luciferase activity after co-transfection of reporter constructs containing the wild type or MCS-deleted 3′-UTR of CYP1B1with either miR-200c inhibitor or precursor. *P < 0.05; **P < 0.01; n.s.: non-significant (E-G) After miR-200c inhibitor transfection, relative miR-200c expression was analyzed by RT-PCR (E), CYP1B1 protein level was determined by Western blot (F) and enzyme activity was measured with P450-Glo assay (G) in 786-O cells. *P < 0.05; ***P < 0.001 (H-J) After miR-200c precursor transfection, relative miR-200c expression was analyzed by RT-PCR (H), CYP1B1 protein level was determined by Western blot (I) and enzyme activity was measured with P450-Glo assay (J) in A498 cells. **P < 0.01.

Mentions: In silico analysis (http://www.microrna.org/ and http://targetscan.org/) showed that CYP1B1 3′-UTR contains two potential complementary miR-200c binding sequences which are termed miR-200c complementary sequence 1 and 2, respectively (MCS1 and MCS2) (Fig. 5A). To examine whether CYP1B1 expression is regulated by miR-200c, we cloned various 3′UTR reporter constructs containing the MCSs or controls (Fig. 5B) and conducted luciferase assays with 786-O and A498 cells, respectively. With 786-O cells, the luciferase activity of the construct containing two copies (pMir-1B1/MCS1-MCS2) or one copy (pMir-1B1/MCS1 or pMir-1B1-MCS2) of MCS was significantly lower than that of the control plasmid. However, the luciferase activity of the construct containing no MCS (pMir-1B1/ΔMCS1-ΔMCS2) showed no change compared to the control vector. In A498 cells, only the luciferase activity of pMir-1B1/MCS1-MCS2 was significantly decreased probably due to the low endogenous miR-200c expression. To investigate the direct regulation of CYP1B1 expression by miR-200c, luciferase activities were measured after miR-200c inhibition or overexpression. As shown in Fig. 5D, inhibition of endogenous miR-200c increased the luciferase activities of the pMir-1B1/MCS1-MCS2 but not that of pMir-1B1/ΔMCS1-ΔMCS2. In reciprocal experiments, exogenous miR-200c expression decreased the luciferase activities of the pMir-1B1/MCS1-MCS2 but not that of pMir-1B1/ΔMCS1-ΔMCS2. These results suggest that miR-200c directly targets the MCS on the CYP1B1 3′-UTR and regulates its expression.


Loss of miR-200c up-regulates CYP1B1 and confers docetaxel resistance in renal cell carcinoma.

Chang I, Mitsui Y, Fukuhara S, Gill A, Wong DK, Yamamura S, Shahryari V, Tabatabai ZL, Dahiya R, Shin DM, Tanaka Y - Oncotarget (2015)

MiR-200c directly targets the CYP1B1 3′-UTR(A) Sequence and location of MCS1 and 2 in the 3′-UTR of human CYP1B1 mRNA. (B) Diagram of reporter constructs containing the wild type or MCS-deleted 3′-UTR of human CYP1B1. (C) Relative luciferase activity after transfection of reporter constructs containing the wild type or MCS-deleted 3′-UTR of CYP1B1. **P < 0.01; ***P < 0.001; n.s.: non-significant (D) Relative luciferase activity after co-transfection of reporter constructs containing the wild type or MCS-deleted 3′-UTR of CYP1B1with either miR-200c inhibitor or precursor. *P < 0.05; **P < 0.01; n.s.: non-significant (E-G) After miR-200c inhibitor transfection, relative miR-200c expression was analyzed by RT-PCR (E), CYP1B1 protein level was determined by Western blot (F) and enzyme activity was measured with P450-Glo assay (G) in 786-O cells. *P < 0.05; ***P < 0.001 (H-J) After miR-200c precursor transfection, relative miR-200c expression was analyzed by RT-PCR (H), CYP1B1 protein level was determined by Western blot (I) and enzyme activity was measured with P450-Glo assay (J) in A498 cells. **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: MiR-200c directly targets the CYP1B1 3′-UTR(A) Sequence and location of MCS1 and 2 in the 3′-UTR of human CYP1B1 mRNA. (B) Diagram of reporter constructs containing the wild type or MCS-deleted 3′-UTR of human CYP1B1. (C) Relative luciferase activity after transfection of reporter constructs containing the wild type or MCS-deleted 3′-UTR of CYP1B1. **P < 0.01; ***P < 0.001; n.s.: non-significant (D) Relative luciferase activity after co-transfection of reporter constructs containing the wild type or MCS-deleted 3′-UTR of CYP1B1with either miR-200c inhibitor or precursor. *P < 0.05; **P < 0.01; n.s.: non-significant (E-G) After miR-200c inhibitor transfection, relative miR-200c expression was analyzed by RT-PCR (E), CYP1B1 protein level was determined by Western blot (F) and enzyme activity was measured with P450-Glo assay (G) in 786-O cells. *P < 0.05; ***P < 0.001 (H-J) After miR-200c precursor transfection, relative miR-200c expression was analyzed by RT-PCR (H), CYP1B1 protein level was determined by Western blot (I) and enzyme activity was measured with P450-Glo assay (J) in A498 cells. **P < 0.01.
Mentions: In silico analysis (http://www.microrna.org/ and http://targetscan.org/) showed that CYP1B1 3′-UTR contains two potential complementary miR-200c binding sequences which are termed miR-200c complementary sequence 1 and 2, respectively (MCS1 and MCS2) (Fig. 5A). To examine whether CYP1B1 expression is regulated by miR-200c, we cloned various 3′UTR reporter constructs containing the MCSs or controls (Fig. 5B) and conducted luciferase assays with 786-O and A498 cells, respectively. With 786-O cells, the luciferase activity of the construct containing two copies (pMir-1B1/MCS1-MCS2) or one copy (pMir-1B1/MCS1 or pMir-1B1-MCS2) of MCS was significantly lower than that of the control plasmid. However, the luciferase activity of the construct containing no MCS (pMir-1B1/ΔMCS1-ΔMCS2) showed no change compared to the control vector. In A498 cells, only the luciferase activity of pMir-1B1/MCS1-MCS2 was significantly decreased probably due to the low endogenous miR-200c expression. To investigate the direct regulation of CYP1B1 expression by miR-200c, luciferase activities were measured after miR-200c inhibition or overexpression. As shown in Fig. 5D, inhibition of endogenous miR-200c increased the luciferase activities of the pMir-1B1/MCS1-MCS2 but not that of pMir-1B1/ΔMCS1-ΔMCS2. In reciprocal experiments, exogenous miR-200c expression decreased the luciferase activities of the pMir-1B1/MCS1-MCS2 but not that of pMir-1B1/ΔMCS1-ΔMCS2. These results suggest that miR-200c directly targets the MCS on the CYP1B1 3′-UTR and regulates its expression.

Bottom Line: Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity.These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel.Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Division of Urology, Veterans Affairs Medical Center, San Francisco, California, United States of America.

ABSTRACT
Despite high protein expression and enzymatic activity of cytochrome P450 1B1 (CYP1B1) in renal cell cancer (RCC), its functional significance has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

No MeSH data available.


Related in: MedlinePlus