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PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells.

Zhang T, Huang XH, Dong L, Hu D, Ge C, Zhan YQ, Xu WX, Yu M, Li W, Wang X, Tang L, Li CY, Yang XM - Mol. Cancer (2010)

Bottom Line: We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing.A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues.We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.

View Article: PubMed Central - HTML - PubMed

Affiliation: Beijing Institute of Radiation Medicine, Beijing, 100850, China.

ABSTRACT

Background: PCBP1 (or alpha CP1 or hnRNP E1), a member of the PCBP family, is widely expressed in many human tissues and involved in regulation of transcription, transportation process, and function of RNA molecules. However, the role of PCBP1 in CD44 variants splicing still remains elusive.

Results: We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing. Invasion assay suggested that PCBP1 played a negative role in tumor invasion and re-expression of v6 partly reversed the inhibition effect by PCBP1. A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues.

Conclusions: We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.

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PCBP1 is a negative regulator of CD44 v6. (A) Different doses of PCBP1 expression vector as indicated were transfected into HepG2 cells. Cells were harvested 24 hours later and total RNA were extracted for semi-quantitative RT-PCR and Real-time PCR analysis (B) to determine the relative amounts of CD44 v6. (C) HepG2 cells were transfected with a negative control siRNA (N.C) or PCBP1 specific siRNA oligos for 48 hours and then total RNA were extracted. The mRNA level of v6 was examined by semi-quantitative RT-PCR and Real-time PCR analysis (D). (E) PCBP1 inhibits the up-regulation of CD44 v6 induced by Ras activation. HepG2 cells were transfected with a constitutively activated mutant H-Ras V12 construct along with PCBP1 expression vector just as indicated. 24 hours later, total RNA were extracted for semi-quantitative PCR and Real-time PCR analysis (F). (G-H) PCBP1 inhibits the upregulation of CD44 v6 induced by HGF. HepG2 cells were transfected with control vector or PCBP1 expression vector, and then 24 hours later the cells were stimulated with 20 ng/ml HGF for 8 hours. Total RNA was extracted and RT-PCR (G) or Real-time PCR (H) was performed to detect the level of CD44 v6. All the PCR bands were scanned for densitometry analysis with the value obtained from control cells set as 1. The values were normalized with those of GAPDH. Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as * p ≤ 0.05 or ** p ≤ 0.001.
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Figure 3: PCBP1 is a negative regulator of CD44 v6. (A) Different doses of PCBP1 expression vector as indicated were transfected into HepG2 cells. Cells were harvested 24 hours later and total RNA were extracted for semi-quantitative RT-PCR and Real-time PCR analysis (B) to determine the relative amounts of CD44 v6. (C) HepG2 cells were transfected with a negative control siRNA (N.C) or PCBP1 specific siRNA oligos for 48 hours and then total RNA were extracted. The mRNA level of v6 was examined by semi-quantitative RT-PCR and Real-time PCR analysis (D). (E) PCBP1 inhibits the up-regulation of CD44 v6 induced by Ras activation. HepG2 cells were transfected with a constitutively activated mutant H-Ras V12 construct along with PCBP1 expression vector just as indicated. 24 hours later, total RNA were extracted for semi-quantitative PCR and Real-time PCR analysis (F). (G-H) PCBP1 inhibits the upregulation of CD44 v6 induced by HGF. HepG2 cells were transfected with control vector or PCBP1 expression vector, and then 24 hours later the cells were stimulated with 20 ng/ml HGF for 8 hours. Total RNA was extracted and RT-PCR (G) or Real-time PCR (H) was performed to detect the level of CD44 v6. All the PCR bands were scanned for densitometry analysis with the value obtained from control cells set as 1. The values were normalized with those of GAPDH. Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as * p ≤ 0.05 or ** p ≤ 0.001.

Mentions: We further investigated the role of PCBP1 in the regulation of CD44 v6 splicing in more detail. HepG2 cells were transfected with different doses of PCBP1 expression vector, and 24 hours later total RNA was extracted for RT-PCR and Real-time PCR analysis. As shown in Figure 3A-B, transiently transfection with increasing amounts of PCBP1 expression vector led to inhibition of CD44 v6 expression in a dose-dependent manner. Moreover, we performed RNA interference assays as described above, and the results suggested that knockdown of endogenous PCBP1 resulted in significant induction of CD44 v6 expression (Figure 3C-D). To confirm the inhibition of CD44 v6 expression by PCBP1, we performed the assays in another hepatocellular carcinoma cell line SMMC-7721 cells and the similar results were obtained (Figure S2; Additional file 1).


PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells.

Zhang T, Huang XH, Dong L, Hu D, Ge C, Zhan YQ, Xu WX, Yu M, Li W, Wang X, Tang L, Li CY, Yang XM - Mol. Cancer (2010)

PCBP1 is a negative regulator of CD44 v6. (A) Different doses of PCBP1 expression vector as indicated were transfected into HepG2 cells. Cells were harvested 24 hours later and total RNA were extracted for semi-quantitative RT-PCR and Real-time PCR analysis (B) to determine the relative amounts of CD44 v6. (C) HepG2 cells were transfected with a negative control siRNA (N.C) or PCBP1 specific siRNA oligos for 48 hours and then total RNA were extracted. The mRNA level of v6 was examined by semi-quantitative RT-PCR and Real-time PCR analysis (D). (E) PCBP1 inhibits the up-regulation of CD44 v6 induced by Ras activation. HepG2 cells were transfected with a constitutively activated mutant H-Ras V12 construct along with PCBP1 expression vector just as indicated. 24 hours later, total RNA were extracted for semi-quantitative PCR and Real-time PCR analysis (F). (G-H) PCBP1 inhibits the upregulation of CD44 v6 induced by HGF. HepG2 cells were transfected with control vector or PCBP1 expression vector, and then 24 hours later the cells were stimulated with 20 ng/ml HGF for 8 hours. Total RNA was extracted and RT-PCR (G) or Real-time PCR (H) was performed to detect the level of CD44 v6. All the PCR bands were scanned for densitometry analysis with the value obtained from control cells set as 1. The values were normalized with those of GAPDH. Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as * p ≤ 0.05 or ** p ≤ 0.001.
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Related In: Results  -  Collection

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Figure 3: PCBP1 is a negative regulator of CD44 v6. (A) Different doses of PCBP1 expression vector as indicated were transfected into HepG2 cells. Cells were harvested 24 hours later and total RNA were extracted for semi-quantitative RT-PCR and Real-time PCR analysis (B) to determine the relative amounts of CD44 v6. (C) HepG2 cells were transfected with a negative control siRNA (N.C) or PCBP1 specific siRNA oligos for 48 hours and then total RNA were extracted. The mRNA level of v6 was examined by semi-quantitative RT-PCR and Real-time PCR analysis (D). (E) PCBP1 inhibits the up-regulation of CD44 v6 induced by Ras activation. HepG2 cells were transfected with a constitutively activated mutant H-Ras V12 construct along with PCBP1 expression vector just as indicated. 24 hours later, total RNA were extracted for semi-quantitative PCR and Real-time PCR analysis (F). (G-H) PCBP1 inhibits the upregulation of CD44 v6 induced by HGF. HepG2 cells were transfected with control vector or PCBP1 expression vector, and then 24 hours later the cells were stimulated with 20 ng/ml HGF for 8 hours. Total RNA was extracted and RT-PCR (G) or Real-time PCR (H) was performed to detect the level of CD44 v6. All the PCR bands were scanned for densitometry analysis with the value obtained from control cells set as 1. The values were normalized with those of GAPDH. Results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as * p ≤ 0.05 or ** p ≤ 0.001.
Mentions: We further investigated the role of PCBP1 in the regulation of CD44 v6 splicing in more detail. HepG2 cells were transfected with different doses of PCBP1 expression vector, and 24 hours later total RNA was extracted for RT-PCR and Real-time PCR analysis. As shown in Figure 3A-B, transiently transfection with increasing amounts of PCBP1 expression vector led to inhibition of CD44 v6 expression in a dose-dependent manner. Moreover, we performed RNA interference assays as described above, and the results suggested that knockdown of endogenous PCBP1 resulted in significant induction of CD44 v6 expression (Figure 3C-D). To confirm the inhibition of CD44 v6 expression by PCBP1, we performed the assays in another hepatocellular carcinoma cell line SMMC-7721 cells and the similar results were obtained (Figure S2; Additional file 1).

Bottom Line: We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing.A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues.We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.

View Article: PubMed Central - HTML - PubMed

Affiliation: Beijing Institute of Radiation Medicine, Beijing, 100850, China.

ABSTRACT

Background: PCBP1 (or alpha CP1 or hnRNP E1), a member of the PCBP family, is widely expressed in many human tissues and involved in regulation of transcription, transportation process, and function of RNA molecules. However, the role of PCBP1 in CD44 variants splicing still remains elusive.

Results: We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing. Invasion assay suggested that PCBP1 played a negative role in tumor invasion and re-expression of v6 partly reversed the inhibition effect by PCBP1. A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues.

Conclusions: We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.

Show MeSH
Related in: MedlinePlus