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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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The expression of FN1BP1 induced G1 phase arrest.Synchronized by nocodazole for 20 h, Dox was added to half of the plates to induce the expression of FN1BP1 for 24 h. Following UV irradiation at 200 (×100 µJ/cm2) for 1 min, the cells were obtained after another 12-h incubation. Then the cells were collected and stained by propidium iodide (PI) for flow cytometry (FCM). (A–B) showed the representative figure of the result of FCM for DOX (−) and DOX (+), respectively. The statistical result is shown in (C). The statistical data demonstrate that more cells induced by Dox were arrested in the G1 phase compared with the non-Dox–induced cells. *P<0.05, n = 5.
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pone-0057574-g005: The expression of FN1BP1 induced G1 phase arrest.Synchronized by nocodazole for 20 h, Dox was added to half of the plates to induce the expression of FN1BP1 for 24 h. Following UV irradiation at 200 (×100 µJ/cm2) for 1 min, the cells were obtained after another 12-h incubation. Then the cells were collected and stained by propidium iodide (PI) for flow cytometry (FCM). (A–B) showed the representative figure of the result of FCM for DOX (−) and DOX (+), respectively. The statistical result is shown in (C). The statistical data demonstrate that more cells induced by Dox were arrested in the G1 phase compared with the non-Dox–induced cells. *P<0.05, n = 5.

Mentions: As indicated by Atlas cDNA microarray, some cell-cycle–arrest genes (p21cip1, p15, and cyclin E1) were up-regulated, while many repair genes (e.g., RAD, ERCC, DNA topoisomerase, polymerase, and ligase) were down-regulated when FN1BP1 expression was induced. Because these genes are involved in DNA repair after damage [17], [18] and cell cycle arrest [2], [19], FCM was performed to investigate the effect of FN1BP1 expression on cell cycle in Hep3B cells after the synchronization of nocodazole and UV irradiation. The result of FCM analysis showed that, compared with the non-induced cells, the Dox-induced FN1BP1 over-expression arrested 134±17% of Hep3B cells in the G1 phase (Fig. 5).


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)

The expression of FN1BP1 induced G1 phase arrest.Synchronized by nocodazole for 20 h, Dox was added to half of the plates to induce the expression of FN1BP1 for 24 h. Following UV irradiation at 200 (×100 µJ/cm2) for 1 min, the cells were obtained after another 12-h incubation. Then the cells were collected and stained by propidium iodide (PI) for flow cytometry (FCM). (A–B) showed the representative figure of the result of FCM for DOX (−) and DOX (+), respectively. The statistical result is shown in (C). The statistical data demonstrate that more cells induced by Dox were arrested in the G1 phase compared with the non-Dox–induced cells. *P<0.05, n = 5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585200&req=5

pone-0057574-g005: The expression of FN1BP1 induced G1 phase arrest.Synchronized by nocodazole for 20 h, Dox was added to half of the plates to induce the expression of FN1BP1 for 24 h. Following UV irradiation at 200 (×100 µJ/cm2) for 1 min, the cells were obtained after another 12-h incubation. Then the cells were collected and stained by propidium iodide (PI) for flow cytometry (FCM). (A–B) showed the representative figure of the result of FCM for DOX (−) and DOX (+), respectively. The statistical result is shown in (C). The statistical data demonstrate that more cells induced by Dox were arrested in the G1 phase compared with the non-Dox–induced cells. *P<0.05, n = 5.
Mentions: As indicated by Atlas cDNA microarray, some cell-cycle–arrest genes (p21cip1, p15, and cyclin E1) were up-regulated, while many repair genes (e.g., RAD, ERCC, DNA topoisomerase, polymerase, and ligase) were down-regulated when FN1BP1 expression was induced. Because these genes are involved in DNA repair after damage [17], [18] and cell cycle arrest [2], [19], FCM was performed to investigate the effect of FN1BP1 expression on cell cycle in Hep3B cells after the synchronization of nocodazole and UV irradiation. The result of FCM analysis showed that, compared with the non-induced cells, the Dox-induced FN1BP1 over-expression arrested 134±17% of Hep3B cells in the G1 phase (Fig. 5).

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