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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 represses CD44 variants splicing. (A) Schematic diagram of the primers. (B) Expression profile of CD44 variants in HepG2 cells. (C) pcDNA3.1(His/Myc)-PCBP1 or control vector were transfected into HepG2 cells for 24 hours and total RNA were extracted for semi-quantitative RT-PCR using the primers indicated in (A). 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. Statistical analysis was performed and the results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 and ** p ≤ 0.001 (D). (E) Knockdown of PCBP1. HepG2 cells were transfected with a negative control siRNA (N.C) or PCBP1 specific siRNA oligos (siPCBP1-1 and siPCBP1-2) for 48 hours. The level of PCBP1 was examined by Real-time PCR analysis (upper panel) and Western blotting (bottom panel). Cells with mock transfection were used as control (normal). For Real-time PCR, GAPDH was used as internal control. For Western blotting, the blots were scanned for densitometry analysis with the value obtained from control cells set as 1. The values were normalized with those of Actin. Statistical analysis was performed and the results represented mean ± SD of 3 independent experiments. (F). The levels of CD44 variants were examined by semi-quantitative RT-PCR (G). The PCR bands were scanned for densitometry analysis with the value obtained from N.C cells set as 1 (H).
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Figure 1: PCBP1 represses CD44 variants splicing. (A) Schematic diagram of the primers. (B) Expression profile of CD44 variants in HepG2 cells. (C) pcDNA3.1(His/Myc)-PCBP1 or control vector were transfected into HepG2 cells for 24 hours and total RNA were extracted for semi-quantitative RT-PCR using the primers indicated in (A). 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. Statistical analysis was performed and the results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 and ** p ≤ 0.001 (D). (E) Knockdown of PCBP1. HepG2 cells were transfected with a negative control siRNA (N.C) or PCBP1 specific siRNA oligos (siPCBP1-1 and siPCBP1-2) for 48 hours. The level of PCBP1 was examined by Real-time PCR analysis (upper panel) and Western blotting (bottom panel). Cells with mock transfection were used as control (normal). For Real-time PCR, GAPDH was used as internal control. For Western blotting, the blots were scanned for densitometry analysis with the value obtained from control cells set as 1. The values were normalized with those of Actin. Statistical analysis was performed and the results represented mean ± SD of 3 independent experiments. (F). The levels of CD44 variants were examined by semi-quantitative RT-PCR (G). The PCR bands were scanned for densitometry analysis with the value obtained from N.C cells set as 1 (H).

Mentions: To investigate whether PCBP1 regulates CD44 variants, HepG2 cells were transfected with pcDNA3.1(His/Myc)-PCBP1 or control vector. A GFP-expression plasmid was cotransfected either with the PCBP1 expressing vector or control vector and all of the transfections displayed a similar efficiency of about 45-48% (data not shown). At 24 hrs after transfection, the levels of CD44 variants were monitored by RT-PCR using the primers described as previously [11] and indicated as Figure 1A. In addition, the CD44 standard form was detected using primers that base-pair to the constitutive exons. The expression profile of CD44 variants in HepG2 cells was shown in Figure 1B. As Figure 1C shows, enforced expression of PCBP1 in HepG2 cells resulted in significant down-regulation of v2, v3, v6, v7, v8, and v10 expression. In contrast, the levels of CD44 v9 and standard form (std) were not affected by overexpression of PCBP1.


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 represses CD44 variants splicing. (A) Schematic diagram of the primers. (B) Expression profile of CD44 variants in HepG2 cells. (C) pcDNA3.1(His/Myc)-PCBP1 or control vector were transfected into HepG2 cells for 24 hours and total RNA were extracted for semi-quantitative RT-PCR using the primers indicated in (A). 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. Statistical analysis was performed and the results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 and ** p ≤ 0.001 (D). (E) Knockdown of PCBP1. HepG2 cells were transfected with a negative control siRNA (N.C) or PCBP1 specific siRNA oligos (siPCBP1-1 and siPCBP1-2) for 48 hours. The level of PCBP1 was examined by Real-time PCR analysis (upper panel) and Western blotting (bottom panel). Cells with mock transfection were used as control (normal). For Real-time PCR, GAPDH was used as internal control. For Western blotting, the blots were scanned for densitometry analysis with the value obtained from control cells set as 1. The values were normalized with those of Actin. Statistical analysis was performed and the results represented mean ± SD of 3 independent experiments. (F). The levels of CD44 variants were examined by semi-quantitative RT-PCR (G). The PCR bands were scanned for densitometry analysis with the value obtained from N.C cells set as 1 (H).
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Figure 1: PCBP1 represses CD44 variants splicing. (A) Schematic diagram of the primers. (B) Expression profile of CD44 variants in HepG2 cells. (C) pcDNA3.1(His/Myc)-PCBP1 or control vector were transfected into HepG2 cells for 24 hours and total RNA were extracted for semi-quantitative RT-PCR using the primers indicated in (A). 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. Statistical analysis was performed and the results represented mean ± SD of 3 independent experiments. The statistical difference between the samples was demonstrated as *p ≤ 0.05 and ** p ≤ 0.001 (D). (E) Knockdown of PCBP1. HepG2 cells were transfected with a negative control siRNA (N.C) or PCBP1 specific siRNA oligos (siPCBP1-1 and siPCBP1-2) for 48 hours. The level of PCBP1 was examined by Real-time PCR analysis (upper panel) and Western blotting (bottom panel). Cells with mock transfection were used as control (normal). For Real-time PCR, GAPDH was used as internal control. For Western blotting, the blots were scanned for densitometry analysis with the value obtained from control cells set as 1. The values were normalized with those of Actin. Statistical analysis was performed and the results represented mean ± SD of 3 independent experiments. (F). The levels of CD44 variants were examined by semi-quantitative RT-PCR (G). The PCR bands were scanned for densitometry analysis with the value obtained from N.C cells set as 1 (H).
Mentions: To investigate whether PCBP1 regulates CD44 variants, HepG2 cells were transfected with pcDNA3.1(His/Myc)-PCBP1 or control vector. A GFP-expression plasmid was cotransfected either with the PCBP1 expressing vector or control vector and all of the transfections displayed a similar efficiency of about 45-48% (data not shown). At 24 hrs after transfection, the levels of CD44 variants were monitored by RT-PCR using the primers described as previously [11] and indicated as Figure 1A. In addition, the CD44 standard form was detected using primers that base-pair to the constitutive exons. The expression profile of CD44 variants in HepG2 cells was shown in Figure 1B. As Figure 1C shows, enforced expression of PCBP1 in HepG2 cells resulted in significant down-regulation of v2, v3, v6, v7, v8, and v10 expression. In contrast, the levels of CD44 v9 and standard form (std) were not affected by overexpression of PCBP1.

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