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Identification of FN1BP1 as a novel cell cycle regulator through modulating G1 checkpoint in human hepatocarcinoma Hep3B cells.

Liu M, Wu R, Yang F, Wang T, Zhang P, Gu J, Wan D, Yang S - PLoS ONE (2013)

Bottom Line: Some interesting genes (p21cip1, ID2, GMSF, ERCC5, and RPA1), which changed in the cDNA microarray analysis, were confirmed by semi-qRT-PCR, and similar changes in expression were observed.FCM cell-cycle analysis indicated that FN1BP1 over-expression could result in G1 phase arrest.These results indicate the potential role of FN1BP1 as a treatment target for hepatocellular carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Jingsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, Jiangsu, China.

ABSTRACT
A novel human gene, FN1BP1 (fibronectin 1 binding protein 1), was identified using the human placenta cDNA library. Northern blotting showed a transcript of ∼2.8 kb in human placenta, liver, and skeletal muscle tissues. This mRNA transcript length was similar to the full FN1BP1 sequence obtained previously. We established a conditionally induced stable cell line of Hep3B-Tet-on-FN1BP1 to investigate the preliminary function and mechanism of the secretory FN1BP1 protein. Cell-proliferation and colony-conformation assays demonstrated that FN1BP1 protein suppressed Hep3B cell growth and colonization in vitro. Analysis of Atlas human cDNA expression indicated that after FN1BP1 Dox-inducing expression for 24 h, 19 genes were up-regulated and 22 genes were down-regulated more than two-fold. Most of these gene changes were related to cell-cycle-arrest proteins (p21cip1, p15, and cyclin E1), transcription factors (general transcription factors, zinc finger proteins, transcriptional enhancer factors), SWI/SNF (SWItch/Sucrose NonFermentable) complex units, early-response proteins, and nerve growth or neurotrophic factors. Down-regulated genes were subject to colony-stimulating factors (e.g., GMSFs), and many repair genes were involved in DNA damage (RAD, ERCC, DNA topoisomerase, polymerase, and ligase). Some interesting genes (p21cip1, ID2, GMSF, ERCC5, and RPA1), which changed in the cDNA microarray analysis, were confirmed by semi-qRT-PCR, and similar changes in expression were observed. FCM cell-cycle analysis indicated that FN1BP1 over-expression could result in G1 phase arrest. FN1BP1 might inhibit cell growth and/or colony conformation through G1 phase arrest of the Hep3B cell cycle. These results indicate the potential role of FN1BP1 as a treatment target for hepatocellular carcinoma.

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Inducing FN1BP1 expression in Hep3B cells reduced cell proliferation and colony formation.After screening in the medium containing hygromycin for more than 8 wk, a total of 33 independent Hyg-resistant cell lines were obtained from the Hep3B-Tet-on cells transfected with pTRE2hyg-FN1BP1. (A) The 11th sense clone of Hep3B Tet-On-FN1BP1 (marked as Hep3B-Tet-On-FN1BP1/S11) showed a positive band identified by either anti-HA tag antibody (a) or anti-FN1BP1 antibody (b) when the cells were induced by Dox. S10 and S18 represent the 10th and 18th sense clones of Hep3B Tet-On-FN1BP1. AS3 represents the third antisense clone of Hep3B Tet-On-FN1BP1, respectively. (B) The cell proliferation activity curve of 6 days shows that expression of FN1BP1 suppressed the Hep3B cell proliferation activity significantly from day 3 to day 6. The data are represented as mean ± SD, * P<0.05 vs non-induced S11 cells, n = 4. (C) The data indicate that the expression of FN1BP1 induced by Dox distinctly decreased the cell colony formation compared to non-induced Hep3B cells, *P<0.05, n = 4. The upper panel shows the representative figure, while the statistical result is shown in the lower panel.
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pone-0057574-g003: Inducing FN1BP1 expression in Hep3B cells reduced cell proliferation and colony formation.After screening in the medium containing hygromycin for more than 8 wk, a total of 33 independent Hyg-resistant cell lines were obtained from the Hep3B-Tet-on cells transfected with pTRE2hyg-FN1BP1. (A) The 11th sense clone of Hep3B Tet-On-FN1BP1 (marked as Hep3B-Tet-On-FN1BP1/S11) showed a positive band identified by either anti-HA tag antibody (a) or anti-FN1BP1 antibody (b) when the cells were induced by Dox. S10 and S18 represent the 10th and 18th sense clones of Hep3B Tet-On-FN1BP1. AS3 represents the third antisense clone of Hep3B Tet-On-FN1BP1, respectively. (B) The cell proliferation activity curve of 6 days shows that expression of FN1BP1 suppressed the Hep3B cell proliferation activity significantly from day 3 to day 6. The data are represented as mean ± SD, * P<0.05 vs non-induced S11 cells, n = 4. (C) The data indicate that the expression of FN1BP1 induced by Dox distinctly decreased the cell colony formation compared to non-induced Hep3B cells, *P<0.05, n = 4. The upper panel shows the representative figure, while the statistical result is shown in the lower panel.

Mentions: After it was screened in the medium containing hygromycin for more than 8 wk and identified by western blotting, the 11th clone (named the Hep3B Tet-On FN1BP1/S11 cells) showed a positive band while the cells were induced by Dox using either HA tag antibody or FN1BP1 polyclonal antibody (Fig. 3A). The cell proliferation activity of Hep3B Tet-On FN1BP1/S11 cells with or without Dox treatment is shown in Fig. 3B, the data demonstrate that the over-expression of FN1BP1 suppressed Hep3B cell growth. A marked decline in growth began on the third day of culture. In the colony formation assay, the number of colonies in the group of Hep3B Tet-On FN1BP1/S11 cells with Dox induction was greatly decreased (Fig. 3C) compared to the non–Dox-induced Hep3B cells. These results prove that FN1BP1 can repress cell colony formation.


Identification of FN1BP1 as a novel cell cycle regulator through modulating G1 checkpoint in human hepatocarcinoma Hep3B cells.

Liu M, Wu R, Yang F, Wang T, Zhang P, Gu J, Wan D, Yang S - PLoS ONE (2013)

Inducing FN1BP1 expression in Hep3B cells reduced cell proliferation and colony formation.After screening in the medium containing hygromycin for more than 8 wk, a total of 33 independent Hyg-resistant cell lines were obtained from the Hep3B-Tet-on cells transfected with pTRE2hyg-FN1BP1. (A) The 11th sense clone of Hep3B Tet-On-FN1BP1 (marked as Hep3B-Tet-On-FN1BP1/S11) showed a positive band identified by either anti-HA tag antibody (a) or anti-FN1BP1 antibody (b) when the cells were induced by Dox. S10 and S18 represent the 10th and 18th sense clones of Hep3B Tet-On-FN1BP1. AS3 represents the third antisense clone of Hep3B Tet-On-FN1BP1, respectively. (B) The cell proliferation activity curve of 6 days shows that expression of FN1BP1 suppressed the Hep3B cell proliferation activity significantly from day 3 to day 6. The data are represented as mean ± SD, * P<0.05 vs non-induced S11 cells, n = 4. (C) The data indicate that the expression of FN1BP1 induced by Dox distinctly decreased the cell colony formation compared to non-induced Hep3B cells, *P<0.05, n = 4. The upper panel shows the representative figure, while the statistical result is shown in the lower panel.
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Related In: Results  -  Collection

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pone-0057574-g003: Inducing FN1BP1 expression in Hep3B cells reduced cell proliferation and colony formation.After screening in the medium containing hygromycin for more than 8 wk, a total of 33 independent Hyg-resistant cell lines were obtained from the Hep3B-Tet-on cells transfected with pTRE2hyg-FN1BP1. (A) The 11th sense clone of Hep3B Tet-On-FN1BP1 (marked as Hep3B-Tet-On-FN1BP1/S11) showed a positive band identified by either anti-HA tag antibody (a) or anti-FN1BP1 antibody (b) when the cells were induced by Dox. S10 and S18 represent the 10th and 18th sense clones of Hep3B Tet-On-FN1BP1. AS3 represents the third antisense clone of Hep3B Tet-On-FN1BP1, respectively. (B) The cell proliferation activity curve of 6 days shows that expression of FN1BP1 suppressed the Hep3B cell proliferation activity significantly from day 3 to day 6. The data are represented as mean ± SD, * P<0.05 vs non-induced S11 cells, n = 4. (C) The data indicate that the expression of FN1BP1 induced by Dox distinctly decreased the cell colony formation compared to non-induced Hep3B cells, *P<0.05, n = 4. The upper panel shows the representative figure, while the statistical result is shown in the lower panel.
Mentions: After it was screened in the medium containing hygromycin for more than 8 wk and identified by western blotting, the 11th clone (named the Hep3B Tet-On FN1BP1/S11 cells) showed a positive band while the cells were induced by Dox using either HA tag antibody or FN1BP1 polyclonal antibody (Fig. 3A). The cell proliferation activity of Hep3B Tet-On FN1BP1/S11 cells with or without Dox treatment is shown in Fig. 3B, the data demonstrate that the over-expression of FN1BP1 suppressed Hep3B cell growth. A marked decline in growth began on the third day of culture. In the colony formation assay, the number of colonies in the group of Hep3B Tet-On FN1BP1/S11 cells with Dox induction was greatly decreased (Fig. 3C) compared to the non–Dox-induced Hep3B cells. These results prove that FN1BP1 can repress cell colony formation.

Bottom Line: Some interesting genes (p21cip1, ID2, GMSF, ERCC5, and RPA1), which changed in the cDNA microarray analysis, were confirmed by semi-qRT-PCR, and similar changes in expression were observed.FCM cell-cycle analysis indicated that FN1BP1 over-expression could result in G1 phase arrest.These results indicate the potential role of FN1BP1 as a treatment target for hepatocellular carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Jingsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, Jiangsu, China.

ABSTRACT
A novel human gene, FN1BP1 (fibronectin 1 binding protein 1), was identified using the human placenta cDNA library. Northern blotting showed a transcript of ∼2.8 kb in human placenta, liver, and skeletal muscle tissues. This mRNA transcript length was similar to the full FN1BP1 sequence obtained previously. We established a conditionally induced stable cell line of Hep3B-Tet-on-FN1BP1 to investigate the preliminary function and mechanism of the secretory FN1BP1 protein. Cell-proliferation and colony-conformation assays demonstrated that FN1BP1 protein suppressed Hep3B cell growth and colonization in vitro. Analysis of Atlas human cDNA expression indicated that after FN1BP1 Dox-inducing expression for 24 h, 19 genes were up-regulated and 22 genes were down-regulated more than two-fold. Most of these gene changes were related to cell-cycle-arrest proteins (p21cip1, p15, and cyclin E1), transcription factors (general transcription factors, zinc finger proteins, transcriptional enhancer factors), SWI/SNF (SWItch/Sucrose NonFermentable) complex units, early-response proteins, and nerve growth or neurotrophic factors. Down-regulated genes were subject to colony-stimulating factors (e.g., GMSFs), and many repair genes were involved in DNA damage (RAD, ERCC, DNA topoisomerase, polymerase, and ligase). Some interesting genes (p21cip1, ID2, GMSF, ERCC5, and RPA1), which changed in the cDNA microarray analysis, were confirmed by semi-qRT-PCR, and similar changes in expression were observed. FCM cell-cycle analysis indicated that FN1BP1 over-expression could result in G1 phase arrest. FN1BP1 might inhibit cell growth and/or colony conformation through G1 phase arrest of the Hep3B cell cycle. These results indicate the potential role of FN1BP1 as a treatment target for hepatocellular carcinoma.

Show MeSH
Related in: MedlinePlus