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Abstract
It has been shown that CA repeats in the 3′-untranslated region (UTR) of bcl-2 mRNA contribute the constitutive decay of bcl-2 mRNA and that hnRNP L (heterogenous nuclear ribonucleoprotein L) interacts with CA repeats in the 3′-UTR of bcl-2 mRNA, both in vitro and in vivo. The aim of this study was to determine whether the alteration of hnRNP L affects the stability of bcl-2 mRNA in vivo. Human breast carcinoma MCF-7 cells were transfected with hnRNP L-specific shRNA or hnRNP L-expressing vector to decrease or increase hnRNP L levels, respectively, followed by an actinomycin D chase. An RT-PCR analysis showed that the rate of degradation of endogenous bcl-2 mRNA was not affected by the decrease or increase in the hnRNP L levels. Furthermore, during apoptosis or autophagy, in which bcl-2 expression has been reported to decrease, no difference in the degradation of bcl-2 mRNA was observed between control and hnRNP L-knock down MCF-7 Cells. On the other hand, the levels of AUF-1 and nucleolin, transacting factors for ARE in the 3'UTR of bcl-2 mRNA, were not significantly affected by the decrease in hnRNP L, suggesting that a disturbance in the quantitative balance between these transacting factors is not likely to interfere with the effect of hnRNP L. Collectively, the findings indicate that the decay of bcl-2 mRNA does not appear to be directly controlled by hnRNP L in vivo.
References
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Fig. 1.
Down-regulation of hnRNP L expression using shRNA does not affect the degradation of bcl-2 mRNA. (A) MCF-7 cells were transfected with pSUPER.puro vector (vector) or shRNA-hnRNP L (sh-hnRNP L) for 72 h and hnRNP L expression as well as Bcl-2 expression was then examined by western blot. The expression of β-actin was used as a loading control. (B, C) Real-time RT-PCR analysis of hnRNP L mRNA expression or bcl-2 mRNA expression after transfection of sh-hnRNP L for 72 h. The relative levels were normalized to 18s rRNA. The value of vector-transfected MCF-7 cells was arbitrarily designated as 1.0. (D) The decay of endogenous bcl-2 mRNA was measured by Real-time RT-PCR after treatment of actinomycin D. At the indicated time points (0, 2, 4, 6, and 8 h) after addition of actinomycin D (5μg/ml), total RNA was extracted from MCF-7 cells transfected with vector (▪) or with sh-hnRNP L (Δ) and processed for real time PCR as described in Methods. Data were normalized to 18s rRNA and the remaining bcl-2 mRNA is presented as % of initial levels. Values are the mean of three independent experiments±SD.
Fig. 2.
Effect of overexpression of hnRNP L on the stability of bcl-2 mRNA in MCF-7 cells. (A) Western blot for hnRNP L expression after transfection of hnRNP L-expressing vector for 48 h. The expression of β-actin was used as a loading control. (B) bcl-2 mRNA half-life was calculated from decay curves by linear regression between 0 and 8 h as in Fig. 1. Values, shown as mean±SD, are based on two independent experiments.
Fig. 3.
Decay of bcl-2 mRNA in vector and sh-hnRNP L-transfected MCF-7 cells during apoptosis or autophagy. After the down-regulation of hnRNP L, MCF-7 cells were exposed to 200 nM of staurospoin (A) or incubated with HBSS (B). Total RNA was isolated at the indicated time points and real-time RT-PCR was performed with bcl-2 and 18s rRNA primers as in Fig. 1. The remaining bcl-2 mRNA at each time point was plotted against time as a percentage of its initial value. Results are shown as means±SD of three independent experiments.
Fig. 4.
Effect of hnRNP L expression on AUF-1 and nucleolin levels. Total protein lysates were prepared from MCF-7 cells transfected with sh-hnRNP L or hnRNP L-expressing vector. A western blot was performed for hnRNP L, AUF-1 and nucleolin levels. The expression of β-actin was included as a loading control.
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