Research ArticleOriginal Article

Identification of a potential tumor suppressor gene, UBL3, in non-small cell lung cancer

Xinchun Zhao, Zhou Yongchun, Hu Qian, Gao Sanhui, Liu Jie, Yu Hong, Zhang Yanfei, Wang Guizhen, Huang Yunchao and Zhou Guangbiao
Cancer Biology & Medicine February 2020, 17 (1) 76-87; DOI: https://doi.org/10.20892/j.issn.2095-3941.2019.0279

Article Figures & Data

Figures

  • Identification of UBL3 as a potential tumor suppressor in non-small cell lung cancer (NSCLC) cells. (A) Heat map showing 11 candidates with Z-scores ≥ 2 in both A549 and H1975 cells. (B, C) Heat map and scatter plot of the expression of the 11 candidates in 58 paired lung adenocarcinoma (LUAD) tissues and 50 paired lung squamous cell carcinoma (LUSC) tissues. The fold change indicates the tumor/normal ratio. (D–G) The association between the expression levels of the four genes and the prognosis of patients as determined by online survival analysis software. (H) The expression levels of the four genes in TCGA datasets. ***P
    Figure 1

    Identification of UBL3 as a potential tumor suppressor in non-small cell lung cancer (NSCLC) cells. (A) Heat map showing 11 candidates with Z-scores ≥ 2 in both A549 and H1975 cells. (B, C) Heat map and scatter plot of the expression of the 11 candidates in 58 paired lung adenocarcinoma (LUAD) tissues and 50 paired lung squamous cell carcinoma (LUSC) tissues. The fold change indicates the tumor/normal ratio. (D–G) The association between the expression levels of the four genes and the prognosis of patients as determined by online survival analysis software. (H) The expression levels of the four genes in TCGA datasets. ***P < 0.0001.

  • The expression of UBL3 in patients with NSCLC in the Oncomine and TCGA datasets. The expression of UBL3 in the Hou Lung (A), Selamat Lung (B), Su Lung (C), Stearman lung (D), Landi Lung (E), and Okayama lung (F) Oncomine datasets and the NSCLC (G), lung adenocarcinoma (LUAD) (H), and lung squamous cell carcinoma (LUSC) (I) of TCGA datasets is shown. (J, K) UBL3 expression levels in tumor tissues and the counterpart adjacent normal lung tissues from the TCGA LUAD (J) and LUSC (K) datasets. (L) Copy number loss analysis of 10 candidates in the TCGA database. (M–Q) The expression of UBL3 was tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (M), Western blot (N, O), and immunohistochemistry (P, Q) assays of lung cancer patients of our cohort. Size bar, 0.5 mm. The densitometry analysis of the Western blot results (O) and the immunoreactivity score of UBL3 were calculated (Q). P values, Student’s t-test. ***P
    Figure 2

    The expression of UBL3 in patients with NSCLC in the Oncomine and TCGA datasets. The expression of UBL3 in the Hou Lung (A), Selamat Lung (B), Su Lung (C), Stearman lung (D), Landi Lung (E), and Okayama lung (F) Oncomine datasets and the NSCLC (G), lung adenocarcinoma (LUAD) (H), and lung squamous cell carcinoma (LUSC) (I) of TCGA datasets is shown. (J, K) UBL3 expression levels in tumor tissues and the counterpart adjacent normal lung tissues from the TCGA LUAD (J) and LUSC (K) datasets. (L) Copy number loss analysis of 10 candidates in the TCGA database. (M–Q) The expression of UBL3 was tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (M), Western blot (N, O), and immunohistochemistry (P, Q) assays of lung cancer patients of our cohort. Size bar, 0.5 mm. The densitometry analysis of the Western blot results (O) and the immunoreactivity score of UBL3 were calculated (Q). P values, Student’s t-test. ***P < 0.0001.

  • The association between UBL3 expression and clinical characteristics. The expression of UBL3 in smoking and non-smoking patients with NSCLC in TCGA dataset (A) and Oncomine Lee Lung dataset (B). UBL3 expression and prognosis of smoking (C) and non-smoking (D) patients with NSCLC as determined by online survival analysis software. (E) The expression of UBL3 in patients with NSCLC at different stages. (F) Overall survival (OS) of patients with NSCLCs at different stages with high or low expression of UBL3. (G) Expression of UBL3 in female and male patients with NSCLC in TCGA (G) and the Oncomine datasets Kuner Lung (H) and Zhu Lung (I). The Kaplan-Meier survival curve of LUAD patients with high or low levels of UBL3 expression as determined by online survival analysis software (J) and TCGA database analysis (K). ***P
    Figure 3

    The association between UBL3 expression and clinical characteristics. The expression of UBL3 in smoking and non-smoking patients with NSCLC in TCGA dataset (A) and Oncomine Lee Lung dataset (B). UBL3 expression and prognosis of smoking (C) and non-smoking (D) patients with NSCLC as determined by online survival analysis software. (E) The expression of UBL3 in patients with NSCLC at different stages. (F) Overall survival (OS) of patients with NSCLCs at different stages with high or low expression of UBL3. (G) Expression of UBL3 in female and male patients with NSCLC in TCGA (G) and the Oncomine datasets Kuner Lung (H) and Zhu Lung (I). The Kaplan-Meier survival curve of LUAD patients with high or low levels of UBL3 expression as determined by online survival analysis software (J) and TCGA database analysis (K). ***P < 0.0001.

  • UBL3 inhibits NSCLC cell proliferation. (A) A549 and H1975 cells were transfected with siUBL3 (1# and 2#) and lysed 72-h later for the detection of UBL3 expression by Western blot assays (upper panel) and assessment of cell viability (lower panel). Error bars, SD. (B) A549 and H1975 cells were transfected with siUBL3-1, and cell proliferation was assessed by a Trypan Blue dye exclusion assays. (C, D) A549 and H460 cells were transfected with HA-tagged UBL3, and cell proliferation was assessed by a Trypan Blue exclusion assay. (E) Foci formation and soft agar assays of A549 cells transfected with siUBL3. Error bars, SD; P values, Student’s t-test. (F) A549 and H1975 cells were transfected with siUBL3 and the cell cycle distribution was assessed by propidium iodide staining of DNA followed by flow cytometric analysis. (G) A549 and H1975 cells were transfected with siUBL3, lysed 72 h later, and the lysates were subjected to Western blot assay using indicated antibodies.
    Figure 4

    UBL3 inhibits NSCLC cell proliferation. (A) A549 and H1975 cells were transfected with siUBL3 (1# and 2#) and lysed 72-h later for the detection of UBL3 expression by Western blot assays (upper panel) and assessment of cell viability (lower panel). Error bars, SD. (B) A549 and H1975 cells were transfected with siUBL3-1, and cell proliferation was assessed by a Trypan Blue dye exclusion assays. (C, D) A549 and H460 cells were transfected with HA-tagged UBL3, and cell proliferation was assessed by a Trypan Blue exclusion assay. (E) Foci formation and soft agar assays of A549 cells transfected with siUBL3. Error bars, SD; P values, Student’s t-test. (F) A549 and H1975 cells were transfected with siUBL3 and the cell cycle distribution was assessed by propidium iodide staining of DNA followed by flow cytometric analysis. (G) A549 and H1975 cells were transfected with siUBL3, lysed 72 h later, and the lysates were subjected to Western blot assay using indicated antibodies.

  • UBL3 suppresses lung cancer in vivo. (A) qRT-PCR analysis of UBL3 mRNA levels in pCDH-UBL3-expressing A549-luciferase cells. (B) A total of 5 × 105 pCDH-UBL3-expressing A549-luciferase cells were injected into SCID-beige mice and the relative luciferase intensity was determined by an IVIS Spectrum system at the indicated time point (B). Error bars, SD; P values, Student’s t-test. The Kaplan-Meier survival curve of the mice is shown (C). P value, log-rank test. Mice were sacrificed, and lung tissues were lysed for Western blot assays using the indicated antibodies (D) or subjected to hematoxylin-eosin (HE) or immunohistochemical staining (E). Scale bar, 1 mm for HE and 0.5 mm for IHC. (F, G) A549-luc cells with pCDH-vector or pCDH-UBL3 were assessed by propidium iodide staining of DNA followed by flow cytometric analysis (F), and cell cycle distribution was assessed (G).
    Figure 5

    UBL3 suppresses lung cancer in vivo. (A) qRT-PCR analysis of UBL3 mRNA levels in pCDH-UBL3-expressing A549-luciferase cells. (B) A total of 5 × 105 pCDH-UBL3-expressing A549-luciferase cells were injected into SCID-beige mice and the relative luciferase intensity was determined by an IVIS Spectrum system at the indicated time point (B). Error bars, SD; P values, Student’s t-test. The Kaplan-Meier survival curve of the mice is shown (C). P value, log-rank test. Mice were sacrificed, and lung tissues were lysed for Western blot assays using the indicated antibodies (D) or subjected to hematoxylin-eosin (HE) or immunohistochemical staining (E). Scale bar, 1 mm for HE and 0.5 mm for IHC. (F, G) A549-luc cells with pCDH-vector or pCDH-UBL3 were assessed by propidium iodide staining of DNA followed by flow cytometric analysis (F), and cell cycle distribution was assessed (G).

Tables

  • Table 1

    Summary of the baseline demographic characteristics of the 86 patients

    CharacteristicsCases, nUBL3-low, n (%)P
    Total8659 (68.6)
    Gender
     Male4836 (75)0.402
     Female2013 (65)
     Not recorded1810 (55.6)
    Age (years)
     < 654733 (70.2)0.691
     ≥ 652015 (75)
     Not recorded1911 (57.9)
    Smoking status
     Smoker3927 (69.2)0.547
     Non-smoker2922 (75.9)
     Not recorded1810 (55.6)
    Histology
     LUAD3929 (74.4)0.729
     LUSC2318 (78.3)
     LUAD + LUSC40 (0)
     SCLC22 (100)
     Not recorded1810 (55.6)
    TNM Stage
     I-II3623 (63.9)0.150
     III-IV3024 (80)
     Not recorded2012 (60)

    LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma. P values were calculated using a two-sided Fisher’s exact test. “UBL3-low” indicates that the levels of UBL3 were lower in tumor tissues than in normal tissues.

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