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Estrogen receptor (ER) was regulated by RNPC1 stabilizing mRNA in ER positive breast cancer.

Shi L, Xia TS, Wei XL, Zhou W, Xue J, Cheng L, Lou P, Li C, Wang Y, Wei JF, Ding Q - Oncotarget (2015)

Bottom Line: Endocrine therapy is the most effective and safety adjunctive therapy for ER positive breast cancers.Furthermore, overexpression of ERα could decrease the level of RNPC1 transcript and protein.A regulatory feedback loop between RNPC1 and ERα was proved.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Breast Disease Center, The First Affiliated Hospital with Nanjing Medical University, Nanjing, China.

ABSTRACT
Estrogen receptors (ERs), including ERα and ERβ, mainly mediate the genotype effect of estrogen. ERα is highly expressed in most breast cancers. Endocrine therapy is the most effective and safety adjunctive therapy for ER positive breast cancers. RNPC1, an RNA binding protein (RBP), post-transcriptionally regulating gene expression, is emerging as a critical mechanism for gene regulation in mammalian cells. In this study, we revealed RNPC1's capability of regulating ERα expression. There was a significant correlation between RNPC1 and ERα expression in breast cancer tissues. Ectopic expression of RNPC1 could increase ERα transcript and expression in breast cancer cells, and vice versa. Consistent with this, RNPC1 was able to bind to ERα transcript to increase its stability. Furthermore, overexpression of ERα could decrease the level of RNPC1 transcript and protein. It suggested a novel mechanism by which ERα expression was regulated via stabilizing mRNA. A regulatory feedback loop between RNPC1 and ERα was proved. It indicated that RNPC1 played a crucial role in ERα regulation in ER-positive breast cancers via binding to ERα mRNA. These findings might provide new insights into breast cancer endocrine therapy and ERα research.

No MeSH data available.


Related in: MedlinePlus

ERα could reversely regulate endogenous RNPC1 expression(A-D) The expression of RNPC1a was reduced by ERα overexpression in ER negative breast cancer cells. (A, B) MDA-MB-231 was transfected with ERα overexpression (ERα) and the control (LV5NC) lentivirus. (A) Western blot and (B) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (C, D) The experiment shown in panel A was also performed in SUM 1315 cells. (C) Western blot and (D) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (E-H) The expression of RNPC1a was reduced by ERα overexpression in ER positive breast cancer cells. (E, F) MCF-7 was transfected with ERα overexpression (ERα) and the control (LV5NC) lentivirus. (E) Western blot and (F) qRT-PCR were used to analyze the expression of ERα and RNPC1a. The experiment shown in panel E was also performed in BT474 cells. (G) Western blot and (H) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (I-L) The expression of RNPC1a was increased with ERα knockdown in ER positive breast cancer cells. (I, J) MCF-7 was transfected with ERα knockdown (shERα1, shERα2) and the control (SNC) lentivirus. (I) Western blot and (J) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (K, L) The experiment shown in panel I was also performed in BT474 cells. (K) Western blot and (L) qRT-PCR were used to analyze the expression of ERα and RNPC1a. The relative quantification was calculated by the ΔΔCt method and normalized based on β-actin. Data were means of three separate experiments and performed as mean ± SEM, **p < 0.01.
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Figure 5: ERα could reversely regulate endogenous RNPC1 expression(A-D) The expression of RNPC1a was reduced by ERα overexpression in ER negative breast cancer cells. (A, B) MDA-MB-231 was transfected with ERα overexpression (ERα) and the control (LV5NC) lentivirus. (A) Western blot and (B) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (C, D) The experiment shown in panel A was also performed in SUM 1315 cells. (C) Western blot and (D) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (E-H) The expression of RNPC1a was reduced by ERα overexpression in ER positive breast cancer cells. (E, F) MCF-7 was transfected with ERα overexpression (ERα) and the control (LV5NC) lentivirus. (E) Western blot and (F) qRT-PCR were used to analyze the expression of ERα and RNPC1a. The experiment shown in panel E was also performed in BT474 cells. (G) Western blot and (H) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (I-L) The expression of RNPC1a was increased with ERα knockdown in ER positive breast cancer cells. (I, J) MCF-7 was transfected with ERα knockdown (shERα1, shERα2) and the control (SNC) lentivirus. (I) Western blot and (J) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (K, L) The experiment shown in panel I was also performed in BT474 cells. (K) Western blot and (L) qRT-PCR were used to analyze the expression of ERα and RNPC1a. The relative quantification was calculated by the ΔΔCt method and normalized based on β-actin. Data were means of three separate experiments and performed as mean ± SEM, **p < 0.01.

Mentions: RNA electrophoretic mobility shift assay (REMSA) was performed to detect the binding site(s) of RNPC1a in ERα transcript. The recombinant His-tagged RNPC1a protein formed a complex with probe A, B and D, respectively (Figure 4B, comparing lanes 4, 7, 13 with 5, 8, 14, respectively), compared with the negative control (NC) (Figure 4B). Probes C and E were unable to connect with recombinant His-tagged RNPC1a (Figure 4B, comparing lane 10, 16 with 11, 17, respectively). The combination of RNA-protein was increased with protein density (Figure 4B, comparing lanes 5, 8, 14 with 6, 9, 15, respectively). It suggested that RNPC1a could bind to ERα mRNA 3′UTR. To functionally confirm the AU/U-rich elements were required for RNPC1a binding to the ERα transcript, we performed a dual-luciferase assay using pGL3 reporters that carried various region of ERα 3′UTR, including 3′UTR-A, B, C, D and E, whose sequences were identical to probes A, B, C, D and E, respectively (Figure 4C). The luciferase activity for a reporter carrying ERα 3′UTR-A, B and D was significantly increased by RNPC1a. By contrast, the ERα 3′UTR-C and E were not responsive to RNPC1a (Figure 5D). Taken together, these data suggested that ERα 3′UTR-A, B and D were responsive to RNPC1a and that each region was sufficient for RNPC1a to increase ERα expression.


Estrogen receptor (ER) was regulated by RNPC1 stabilizing mRNA in ER positive breast cancer.

Shi L, Xia TS, Wei XL, Zhou W, Xue J, Cheng L, Lou P, Li C, Wang Y, Wei JF, Ding Q - Oncotarget (2015)

ERα could reversely regulate endogenous RNPC1 expression(A-D) The expression of RNPC1a was reduced by ERα overexpression in ER negative breast cancer cells. (A, B) MDA-MB-231 was transfected with ERα overexpression (ERα) and the control (LV5NC) lentivirus. (A) Western blot and (B) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (C, D) The experiment shown in panel A was also performed in SUM 1315 cells. (C) Western blot and (D) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (E-H) The expression of RNPC1a was reduced by ERα overexpression in ER positive breast cancer cells. (E, F) MCF-7 was transfected with ERα overexpression (ERα) and the control (LV5NC) lentivirus. (E) Western blot and (F) qRT-PCR were used to analyze the expression of ERα and RNPC1a. The experiment shown in panel E was also performed in BT474 cells. (G) Western blot and (H) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (I-L) The expression of RNPC1a was increased with ERα knockdown in ER positive breast cancer cells. (I, J) MCF-7 was transfected with ERα knockdown (shERα1, shERα2) and the control (SNC) lentivirus. (I) Western blot and (J) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (K, L) The experiment shown in panel I was also performed in BT474 cells. (K) Western blot and (L) qRT-PCR were used to analyze the expression of ERα and RNPC1a. The relative quantification was calculated by the ΔΔCt method and normalized based on β-actin. Data were means of three separate experiments and performed as mean ± SEM, **p < 0.01.
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Figure 5: ERα could reversely regulate endogenous RNPC1 expression(A-D) The expression of RNPC1a was reduced by ERα overexpression in ER negative breast cancer cells. (A, B) MDA-MB-231 was transfected with ERα overexpression (ERα) and the control (LV5NC) lentivirus. (A) Western blot and (B) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (C, D) The experiment shown in panel A was also performed in SUM 1315 cells. (C) Western blot and (D) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (E-H) The expression of RNPC1a was reduced by ERα overexpression in ER positive breast cancer cells. (E, F) MCF-7 was transfected with ERα overexpression (ERα) and the control (LV5NC) lentivirus. (E) Western blot and (F) qRT-PCR were used to analyze the expression of ERα and RNPC1a. The experiment shown in panel E was also performed in BT474 cells. (G) Western blot and (H) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (I-L) The expression of RNPC1a was increased with ERα knockdown in ER positive breast cancer cells. (I, J) MCF-7 was transfected with ERα knockdown (shERα1, shERα2) and the control (SNC) lentivirus. (I) Western blot and (J) qRT-PCR were used to analyze the expression of ERα and RNPC1a. (K, L) The experiment shown in panel I was also performed in BT474 cells. (K) Western blot and (L) qRT-PCR were used to analyze the expression of ERα and RNPC1a. The relative quantification was calculated by the ΔΔCt method and normalized based on β-actin. Data were means of three separate experiments and performed as mean ± SEM, **p < 0.01.
Mentions: RNA electrophoretic mobility shift assay (REMSA) was performed to detect the binding site(s) of RNPC1a in ERα transcript. The recombinant His-tagged RNPC1a protein formed a complex with probe A, B and D, respectively (Figure 4B, comparing lanes 4, 7, 13 with 5, 8, 14, respectively), compared with the negative control (NC) (Figure 4B). Probes C and E were unable to connect with recombinant His-tagged RNPC1a (Figure 4B, comparing lane 10, 16 with 11, 17, respectively). The combination of RNA-protein was increased with protein density (Figure 4B, comparing lanes 5, 8, 14 with 6, 9, 15, respectively). It suggested that RNPC1a could bind to ERα mRNA 3′UTR. To functionally confirm the AU/U-rich elements were required for RNPC1a binding to the ERα transcript, we performed a dual-luciferase assay using pGL3 reporters that carried various region of ERα 3′UTR, including 3′UTR-A, B, C, D and E, whose sequences were identical to probes A, B, C, D and E, respectively (Figure 4C). The luciferase activity for a reporter carrying ERα 3′UTR-A, B and D was significantly increased by RNPC1a. By contrast, the ERα 3′UTR-C and E were not responsive to RNPC1a (Figure 5D). Taken together, these data suggested that ERα 3′UTR-A, B and D were responsive to RNPC1a and that each region was sufficient for RNPC1a to increase ERα expression.

Bottom Line: Endocrine therapy is the most effective and safety adjunctive therapy for ER positive breast cancers.Furthermore, overexpression of ERα could decrease the level of RNPC1 transcript and protein.A regulatory feedback loop between RNPC1 and ERα was proved.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Breast Disease Center, The First Affiliated Hospital with Nanjing Medical University, Nanjing, China.

ABSTRACT
Estrogen receptors (ERs), including ERα and ERβ, mainly mediate the genotype effect of estrogen. ERα is highly expressed in most breast cancers. Endocrine therapy is the most effective and safety adjunctive therapy for ER positive breast cancers. RNPC1, an RNA binding protein (RBP), post-transcriptionally regulating gene expression, is emerging as a critical mechanism for gene regulation in mammalian cells. In this study, we revealed RNPC1's capability of regulating ERα expression. There was a significant correlation between RNPC1 and ERα expression in breast cancer tissues. Ectopic expression of RNPC1 could increase ERα transcript and expression in breast cancer cells, and vice versa. Consistent with this, RNPC1 was able to bind to ERα transcript to increase its stability. Furthermore, overexpression of ERα could decrease the level of RNPC1 transcript and protein. It suggested a novel mechanism by which ERα expression was regulated via stabilizing mRNA. A regulatory feedback loop between RNPC1 and ERα was proved. It indicated that RNPC1 played a crucial role in ERα regulation in ER-positive breast cancers via binding to ERα mRNA. These findings might provide new insights into breast cancer endocrine therapy and ERα research.

No MeSH data available.


Related in: MedlinePlus