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. 2019 Apr 8:12:2553-2561.
doi: 10.2147/OTT.S196132. eCollection 2019.

Silencing UBE4B induces nasopharyngeal carcinoma apoptosis through the activation of caspase3 and p53

Chengyin Weng  1   2   3 , Yong Chen  1 , Yong Wu  2   3 , Xia Liu  2   3 , Haibo Mao  2   3 , Xisheng Fang  2   3 , Baoxiu Li  2   3 , Lina Wang  2   3 , Mingmei Guan  2   3 , Guolong Liu  2   3 , Lin Lu  2   3 , Yawei Yuan  1   4
Affiliations

Silencing UBE4B induces nasopharyngeal carcinoma apoptosis through the activation of caspase3 and p53

Chengyin Weng et al. Onco Targets Ther. .

Abstract

Aim: The human ubiquitination factor E4B (UBE4B) gene is frequently amplified in some solid cancers. However, the role of UBE4B in nasopharyngeal carcinoma (NPC) has not yet been investigated.

Methods: Firstly, we analyzed the expression of UBE4B in NPC samples using real-time quantitative PCR and Western blot analysis. After knocking down UBE4B using small interfering RNA technology, the functions of UBE4B on cell proliferation, apoptosis, and cell cycle, as well as underlying mechanism, were investigated.

Results: Compared with matched adjacent non-tumor tissues, both protein and mRNA levels of UBE4B were much higher in most NPC cancerous specimens. Deficiency of UBE4B could significantly inhibit tumor cell growth and induce cell apoptosis. Knocking down UBE4B could promote the expression of cleaved caspase3 and p53, and inhibition of caspase3 could prevent cell apoptosis induced by the deficiency of UBE4B.

Conclusion: These results indicate that expression of UBE4B was higher in most NPC tissues compared to adjacent non-tumoral tissues, and that knockdown of UBE4B inhibited the cell growth and induced apoptosis in NPC cells. This process was regulated by the activation of caspase3 and p53. Thus, UBE4B gene might act as a potential molecular target to develop novel strategy for NPC patients.

Keywords: UBE4B; apoptosis; caspase3; nasopharyngeal cancer; p53.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Characteristics of UBE4B expression in 24 clinical samples. (A) Real-time PCR was performed to detect UBE4B mRNA in nasopharyngeal carcinoma (NPC) cancerous tissues and their surrounding tissues. Data are shown as mean ± SD (n=3). (B) Protein levels of UBE4B in NPC cancerous tissues and surrounding tissues were examined by Western blotting (N, adjacent non-tumor tissues; T, NPC cancerous tissues). (C) Quantitative analysis of UBE4B protein in NPC cancerous tissues and their matched adjacent non-tumor tissues.
Figure 2
Figure 2
The impact of silencing UBE4B on the proliferation of nasopharyngeal carcinoma (NPC) cells. (A) Expression of UBE4B protein in NPC cell lines and normal nasopharyngeal epithelial cell line (NPEC). UBE4B protein was upregulated in HK1, CNE1, CNE2, SUNE1, and Hone1 cells (particularly in CNE2 and SUNE1 cells) compared with the normal NPEC. (B and C) Representative protein expression of UBE4B in (B) CNE2 or (C) SUNE1 cells treated with control siRNA (siNC) or UBE4B siRNA (siUBE4B#2) for 48 hours. GAPDH was used as a loading control. (D and E) Cell growth of (D) CNE2 or (E) SUNE1 cells treated with siNC or siUBE4B#2 for 1, 2, 3, 4, 5, 6, and 7 days. Data are shown as mean ± SD (n=3). *P<0.05, **P<0.01.
Figure 3
Figure 3
Knockdown of UBE4B induces cell apoptosis in nasopharyngeal carcinoma (NPC) cells; downregulation of UBE4B promotes cleaved caspase3 and p53 expression and decreases Bcl2 expression. (A and B) Knockdown of UBE4B expression with siUBE4B#2 induces cell apoptosis of (A) CNE2 or (B) SUNE1 cells. (C and D) Downregulation of UBE4B with siUBE4B#2 promotes protein levels of cleaved caspase3 in (C) CNE2 and (D) SUNE1 cells. Downregulation of UBE4B promotes p53 expression in (C) CNE2 and (D) SUNE1 cells. Downregulation of UBE4B decreases Bcl2 expression in (B) CNE2 and (C) SUNE1 cells.
Figure 4
Figure 4
Caspase3 inhibition could prevent cell apoptosis induced by the knockdown of UBE4B. (A and C) The cell apoptosis levels of CNE2-siNC, CNE2-siUBE4B#2, and SUNE1-siNC, SUNE1-siUBE4B#2 cells cultured in the presence of caspase3 inhibitor. FITC-labeled Annexin V-positive cells (upper right and lower right) were considered apoptotic cells: (A) CNE2 cell, (C) SUNE1 cell. (B and D) Quantitative analysis of apoptotic percentages of CNE2-siNC cells, CNE2-siUBE4B#2, and SUNE1-siNC, SUNE1-siUBE4B#2 cells cultured in the presence or absence of caspase3 inhibitor: (B) CNE2 cell, (D) SUNE1 cell. Abbreviation: FITC, fluorescein isothiocyanate.
Figure 5
Figure 5
Effect of UBE4B on the cell cycle in nasopharyngeal carcinoma (NPC) cells. Cell cycle distribution was not changed significantly between CNE2 (A and B) and SUNE1 (C and D) cells transfected with siNC and siUBE4B#2.
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