The impacts of ubiquilin 1 (UBQLN1) knockdown on cells viability, proliferation, and apoptosis are mediated by p53 in A549 lung cancer cells - PubMed (original) (raw)

The impacts of ubiquilin 1 (UBQLN1) knockdown on cells viability, proliferation, and apoptosis are mediated by p53 in A549 lung cancer cells

Xinghua Zhang et al. J Thorac Dis. 2020 Oct.

Abstract

Background: Little is known about the relationship between ubiquilin 1 (UBQLN1) and p53, both of them have been implicated in the development and progression of non-small cell lung cancer (NSCLC). In this study, we aimed to explore the role of loss of UBQLN1 in cell viability and proliferation, and cell apoptosis in human lung adenocarcinoma A549 cells.

Methods: Cell viability, proliferation, and apoptosis were determined by MTT, BrdU, and TUNEL assays, respectively. Adenoviruses carrying cDNA or siRNA were used to overexpress or silence target protein. Dihydroethidium (DHE) staining was performed to measure the real-time formation of intracellular reactive oxygen species (ROS). The chymotrypsin-like activity of 20S proteasome core was determined by using synthetic fluorogenic peptide substrate.

Results: UBQLN1 silencing led to a reduction of p53 protein levels and overexpression of p53 reversed the effects of UBQLN1 knockdown (KD) on cell viability, proliferation, and apoptosis. Furthermore, deficiency of UBQLN1 activated autophagy activity but did not affect proteasome activity. Inhibition of autophagy restored p53 protein levels in UBQLN1-KD A549 cells. In addition, UBQLN1 KD markedly inhibited phosphorylation of mammalian target of rapamycin (mTOR) and its downstream ribosomal S6 kinase (S6K).

Conclusions: Our experiments suggested that the regulation of UBQLN1 on cell viability, proliferation, and apoptosis was mediated by mTOR/autophagy/p53 signaling pathway.

Keywords: Ubiquilin 1 (UBQLN1); autophagy; mammalian target of rapamycin (mTOR); non-small cell lung cancer (NSCLC); p53.

2020 Journal of Thoracic Disease. All rights reserved.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jtd-20-1362). The authors have no conflicts of interest to declare.

Figures

Figure 1

Figure 1

Effects of UBQLN1 knockdown (KD) on cell viability, proliferation, and apoptosis in A549 cells. (A) Cell viability determined by MTT assay; (B) cell proliferation determined by BrdU assay; (C) cell apoptosis determined by TUNEL assay. Left: representative images showing apoptotic cells; right: quantification of cell apoptosis; (D) representative Western blot images showing apoptosis markers; (E) quantification of cleaved caspase-3 in (D); (F) quantification of protein levels of Bcl-2 and Bax in (D); (G) quantification of Bcl-2/Bax in (D). n=4. **, P<0.01 and ***, P<0.001 vs. siRNA control group. UBQLN1, ubiquilin 1; KD, knockdown.

Figure 2

Figure 2

Effects of p53 overexpression on cell viability, proliferation, and apoptosis in UBQLN1-KD A549 cells. (A) Representative Western blot images showing p53 protein levels; (B) cell viability determined by MTT assay; (C) cell proliferation determined by BrdU assay; (D) cell apoptosis determined by TUNEL assay. Left: representative images showing apoptotic cells; right: quantification of cell apoptosis; (E) representative Western blot images showing apoptosis markers; (F) quantification of cleaved caspase-3 in (E); (G) quantification of Bcl-2/Bax in (E). n=4. ***, P<0.001 vs. siRNA control group; ##, P<0.01 and ###, P<0.001 vs. UBQLN1 KD group. OE, overexpression; UBQLN1, ubiquilin 1; KD, knockdown.

Figure 3

Figure 3

Effects of UBQLN1 knockdown (KD) on the activities of proteasome and autophagy in A549 cells. (A) Effects of UBQLN1 KD on proteasome activity; (B) representative Western blot images showing the effects of UBQLN1 KD on autophagic markers; (C) quantification of LC3-II/LC3-I in (B); (D) representative Western blot images showing the effects of autophagy inhibitor BFA on LC3-II expression; (E) quantification of autophagic flux based on (D); (F) representative Western blot images showing the effects of autophagy inhibitor BFA on p53 protein levels; (G) quantification of p53 protein levels in (F). n=4. **, P<0.01 and ***, P<0.001 vs. siRNA control group; ##, P<0.01 vs. UBQLN1 KD group. UBQLN1, ubiquilin 1; BFA, brefeldin A.

Figure 4

Figure 4

Effects of UBQLN1 knockdown (KD) on ER stress, ROS formation, and the mTOR signaling in A549 cells. (A) Representative Western blot images showing proteins markers of ER stress; (B) representative images showing real time formation of intracellular ROS (magnification ×400); (C) quantification of ROS formation in (B). (D) representative Western blot images showing phosphorylation of mTOR and its downstream S6K; (E) quantification of phosphorylation levels of mTOR and S6K in (C); (F) schematic diagram of UBQLN1 on cell viability, proliferation, and apoptosis in A549 cancer cells. UBQLN1 impairs mTORC1 activity leading to the activation of autophagy and subsequent degradation of p53. Loss of p53 will affect cell viability, proliferation, and apoptosis. n=4. **, P<0.01 and ***, P<0.001 _vs._ siRNA control group; #, P>0.05 vs. siRNA control group. UBQLN1, ubiquilin 1.

Figure S1

Figure S1

Effects of UBQLN1 knockdown (KD) on cell viability and proliferation in H358 cells and BEAS-2B cells. UBQLN1-silenced and its control cells were grown in RPMI medium containing 10% FBS for 24 h. (A) Cell viability determined by MTT assay; (B) cell proliferation determined by BrdU assay. n=4. **, P<0.01 and ***, P<0.001 vs. siRNA control group. UBQLN1, ubiquilin 1.

Figure S2

Figure S2

Effects of UBQLN1 knockdown (KD) on autophagy and p53 levels in H358 cells. UBQLN1-silenced and its control H358 cells were grown in RPMI medium containing 10% FBS, in the presence or absence of 20 nM BFA for 24 h. (A) Representative Western blot images showing the effects of UBQLN1 KD on autophagic markers; (B) quantification of LC3-II/LC3-I in (A); (C) representative Western blot images showing the effects of autophagy inhibitor BFA on p53 protein levels; (D) quantification of p53 protein levels in (C). n=4. **, P<0.01 and ***, P<0.001 vs. siRNA control group; ##, P<0.01 vs. UBQLN1 KD group. UBQLN1, ubiquilin 1; BFA, brefeldin A.

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