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HOXA11 hypermethylation is associated with progression of non-small cell lung cancer.

Hwang JA, Lee BB, Kim Y, Park SE, Heo K, Hong SH, Kim YH, Han J, Shim YM, Lee YS, Kim DH - Oncotarget (2013)

Bottom Line: HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs.HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively).HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival.

View Article: PubMed Central - PubMed

Affiliation: Cancer Genomics Branch, Research Institute, National Cancer Center, Goyang-si, Korea.

ABSTRACT
This study was aimed at understanding the functional significance of HOXA11 hypermethylation in non-small cell lung cancer (NSCLC). HOXA11 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using formalin-fixed paraffin-embedded tissues from 317 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA11 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA11 into H23 lung cancer cells resulted in the inhibition of cell migration and proliferation. HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs. HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively). HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival. Patients with pT2 and pT3 stages were 1.85 times (95% confidence interval [CI] = 1.04-3.29; P = 0.04) and 5.47 times (95% CI = 1.18-25.50; P = 0.01), respectively, more likely to show HOXA11 hypermethylation than those with pT1 stage, after adjusting for age, sex, and histology. In conclusion, the present study suggests that HOXA11 hypermethylation may contribute to the progression of NSCLC by promoting cell proliferation or migration.

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The effect of HOXA11 on cell migration and proliferation(A) To analyze the effect of HOXA11 on cell migration and proliferation, H23 lung cancer cells were transfected with pAcGFP-HOXA11 fusion construct, or empty vector. After transfection, the expression of HOXA11 was confirmed by western blot using primary antibody directed to GFP (sc-9996; Santa Cruz Biotechnology, CA, USA). (B) H23 cells transfected with pAcGFP-HOXA11 were re-seeded in 6.5 mm Transwell® with 8 μm pore size (Corning, USA). The cells were induced to migrate into 1% of FBS containing media in 24 well plate. After 48 hr each transwell insert was stained by 1% crystal violet and destained with 10% acetic acid. The absorbance was measured at 564 nm using VERSAmax microplate reader (Molecular devices). (C) H23 cells transfected with pAcGFP-HOXA11 were seeded in a 96 well plate, and cell proliferation activity was measured every 24hrs with MTT assay. Y axis indicates absorbance measured at 570 nm using VERSAmax microplate reader (Molecular devices), and the data are presented as mean ± standard error (SE) of eight experiments.
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Figure 3: The effect of HOXA11 on cell migration and proliferation(A) To analyze the effect of HOXA11 on cell migration and proliferation, H23 lung cancer cells were transfected with pAcGFP-HOXA11 fusion construct, or empty vector. After transfection, the expression of HOXA11 was confirmed by western blot using primary antibody directed to GFP (sc-9996; Santa Cruz Biotechnology, CA, USA). (B) H23 cells transfected with pAcGFP-HOXA11 were re-seeded in 6.5 mm Transwell® with 8 μm pore size (Corning, USA). The cells were induced to migrate into 1% of FBS containing media in 24 well plate. After 48 hr each transwell insert was stained by 1% crystal violet and destained with 10% acetic acid. The absorbance was measured at 564 nm using VERSAmax microplate reader (Molecular devices). (C) H23 cells transfected with pAcGFP-HOXA11 were seeded in a 96 well plate, and cell proliferation activity was measured every 24hrs with MTT assay. Y axis indicates absorbance measured at 570 nm using VERSAmax microplate reader (Molecular devices), and the data are presented as mean ± standard error (SE) of eight experiments.

Mentions: To investigate the function of HOXA11 in tumorigenesis, cell migration and cell proliferation was analyzed in H23 cells induced by transient transfection of GFP-tagged HOXA11. Anti-GFP antibody was used to detect the expression of HOXA11 (Fig. 3A) by western blot analysis. Cell migration was significantly reduced in H23 cells transfected with pAcGFP-HOXA11 construct (P = 0.01; Fig. 3B). Cell proliferation was also analyzed in the H23 cells after transfection of the GFP-fusion constructs. Cell proliferation was inhibited in H23 cells induced by pAcGFP-HOXA11 (Fig. 3C): pAcGFP-HOXA11 suppressed cell proliferation by 30% at 72 h after seeding the cells for MTT assay. Based on these observations, it is likely that HOXA11 may function as a tumor suppressor by inhibiting cell migration and cell proliferation in tumorigenesis of the lung.


HOXA11 hypermethylation is associated with progression of non-small cell lung cancer.

Hwang JA, Lee BB, Kim Y, Park SE, Heo K, Hong SH, Kim YH, Han J, Shim YM, Lee YS, Kim DH - Oncotarget (2013)

The effect of HOXA11 on cell migration and proliferation(A) To analyze the effect of HOXA11 on cell migration and proliferation, H23 lung cancer cells were transfected with pAcGFP-HOXA11 fusion construct, or empty vector. After transfection, the expression of HOXA11 was confirmed by western blot using primary antibody directed to GFP (sc-9996; Santa Cruz Biotechnology, CA, USA). (B) H23 cells transfected with pAcGFP-HOXA11 were re-seeded in 6.5 mm Transwell® with 8 μm pore size (Corning, USA). The cells were induced to migrate into 1% of FBS containing media in 24 well plate. After 48 hr each transwell insert was stained by 1% crystal violet and destained with 10% acetic acid. The absorbance was measured at 564 nm using VERSAmax microplate reader (Molecular devices). (C) H23 cells transfected with pAcGFP-HOXA11 were seeded in a 96 well plate, and cell proliferation activity was measured every 24hrs with MTT assay. Y axis indicates absorbance measured at 570 nm using VERSAmax microplate reader (Molecular devices), and the data are presented as mean ± standard error (SE) of eight experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926829&req=5

Figure 3: The effect of HOXA11 on cell migration and proliferation(A) To analyze the effect of HOXA11 on cell migration and proliferation, H23 lung cancer cells were transfected with pAcGFP-HOXA11 fusion construct, or empty vector. After transfection, the expression of HOXA11 was confirmed by western blot using primary antibody directed to GFP (sc-9996; Santa Cruz Biotechnology, CA, USA). (B) H23 cells transfected with pAcGFP-HOXA11 were re-seeded in 6.5 mm Transwell® with 8 μm pore size (Corning, USA). The cells were induced to migrate into 1% of FBS containing media in 24 well plate. After 48 hr each transwell insert was stained by 1% crystal violet and destained with 10% acetic acid. The absorbance was measured at 564 nm using VERSAmax microplate reader (Molecular devices). (C) H23 cells transfected with pAcGFP-HOXA11 were seeded in a 96 well plate, and cell proliferation activity was measured every 24hrs with MTT assay. Y axis indicates absorbance measured at 570 nm using VERSAmax microplate reader (Molecular devices), and the data are presented as mean ± standard error (SE) of eight experiments.
Mentions: To investigate the function of HOXA11 in tumorigenesis, cell migration and cell proliferation was analyzed in H23 cells induced by transient transfection of GFP-tagged HOXA11. Anti-GFP antibody was used to detect the expression of HOXA11 (Fig. 3A) by western blot analysis. Cell migration was significantly reduced in H23 cells transfected with pAcGFP-HOXA11 construct (P = 0.01; Fig. 3B). Cell proliferation was also analyzed in the H23 cells after transfection of the GFP-fusion constructs. Cell proliferation was inhibited in H23 cells induced by pAcGFP-HOXA11 (Fig. 3C): pAcGFP-HOXA11 suppressed cell proliferation by 30% at 72 h after seeding the cells for MTT assay. Based on these observations, it is likely that HOXA11 may function as a tumor suppressor by inhibiting cell migration and cell proliferation in tumorigenesis of the lung.

Bottom Line: HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs.HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively).HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival.

View Article: PubMed Central - PubMed

Affiliation: Cancer Genomics Branch, Research Institute, National Cancer Center, Goyang-si, Korea.

ABSTRACT
This study was aimed at understanding the functional significance of HOXA11 hypermethylation in non-small cell lung cancer (NSCLC). HOXA11 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using formalin-fixed paraffin-embedded tissues from 317 NSCLC patients, and Ki-67 expression was analyzed using immunohistochemistry. The promoter region of HOXA11 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). Transient transfection of HOXA11 into H23 lung cancer cells resulted in the inhibition of cell migration and proliferation. HOXA11 hypermethylation was found in 218 (69%) of 317 primary NSCLCs. HOXA11 hypermethylation was found at a higher prevalence in squamous cell carcinoma than in adenocarcinoma (74% vs. 63%, respectively). HOXA11 hypermethylation was associated with Ki-67 proliferation index (P = 0.03) and pT stage (P = 0.002), but not with patient survival. Patients with pT2 and pT3 stages were 1.85 times (95% confidence interval [CI] = 1.04-3.29; P = 0.04) and 5.47 times (95% CI = 1.18-25.50; P = 0.01), respectively, more likely to show HOXA11 hypermethylation than those with pT1 stage, after adjusting for age, sex, and histology. In conclusion, the present study suggests that HOXA11 hypermethylation may contribute to the progression of NSCLC by promoting cell proliferation or migration.

Show MeSH
Related in: MedlinePlus