Downregulation of USP32 inhibits cell proliferation, migration and invasion in human small cell lung cancer - PubMed (original) (raw)
Downregulation of USP32 inhibits cell proliferation, migration and invasion in human small cell lung cancer
Wenyu Hu et al. Cell Prolif. 2017 Aug.
Abstract
Objectives: Ubiquitin specific protease 32 (USP32) is a highly conserved but uncharacterized gene, which has been reported to be associated with growth of breast cancer cells. However, the role of USP32 in human small cell lung cancer (SCLC) has not been uncovered. The aim of this study was to investigate and evaluate the clinical significance of USP32 in patients with SCLC.
Materials and methods: Expression of USP32 was firstly investigated using public online data sets and then determined in SCLC tissues and cell lines using quantitative real-time PCR, Western blotting and immunohistochemical staining. SCLC cells were transfected with a small-interfering RNA targeting USP32 mRNA and analysed for cell viability, proliferation ability, cell cycle distribution, apoptosis and invasion.
Results: USP32 was found to be overexpressed in SCLC tissues compared with normal tissues. High USP32 expression was significantly correlated with disease stage and invasion. In vitro experiments demonstrated that silencing of USP32 caused a significant decrease in the proliferation and migration rate of cells. Furthermore, USP32 silencing arrested cell cycle progression at G0/G1 phase via decreasing CDK4/Cyclin D1 complex and elevating p21. In addition, downregulation of USP32 significantly induced cell apoptosis by activating cleaved caspase-3 and cleaved PARP, as well as inhibiting cell invasiveness via altering epithelial mesenchymal transition expression.
Conclusions: Our results suggest for the first time that USP32 is important for SCLC progression and might be a potential target for molecular therapy of SCLC.
Keywords: USP32; cell apoptosis; cell cycle; cell proliferation; invasion; small cell lung cancer.
© 2017 John Wiley & Sons Ltd.
Conflict of interest statement
The authors declare no conflicts of interest.
Figures
Figure 1
USP32 expression is upregulated in lung cancer tissues by publicly available database. The
mRNA
expression of USP32 in Oncomine data sets including Su Lung data set (A), Landi Lung data set (B), Selamat Lung data set (C) and Okayama Lung data set (D); the
mRNA
expression of
USP
32 in
TCGA
database including lung adenocarcinoma (
LUAD
) data set (E),
TCGA BRCA
data set (F),
TCGA ESCC
(G),
TCGA STES
data set (H) and
TCGA STAD
data set (I)
Figure 2
USP
32 is frequently upregulated in small cell lung cancer (
SCLC
) tissues. The
mRNA
(A) and proteins (B) expression of
USP
32 in 10 pairs of paracancerous and cancer tissues from
SCLC
patients; N, paracarcinoma (normal) lung tissues. T,
SCLC
tissues; (C) representative images of immunohistochemistry staining of
USP
32 in
SCLC
tissues (−+, weak staining, + moderate staining, ++ strong staining); (D) Analysis of
USP
32 protein in tissues by immunohistochemistry.
USP
32 expression was significantly increased in
SCLC
tissues, when compared with normal lung tissues, P<.001
Figure 3
The expression of USP32 is efficiently silenced in two small cell lung cancer (
SCLC
) cell lines. (A)
USP
32 expression in six
SCLC
cell lines using Western blot analysis; (B) statistical analysis of the ratio of
USP
32/ β‐actin;
SCLC
, small cell lung cancer; ***P<.001; β‐actin was used as an internal control. The
mRNA
and proteins expression of
USP
32 was significantly reduced when
USP
32 was silenced by specific si
RNA
targeting USP32 (Si
USP
- compared with untreated (Mock) and scrambled si
RNA
(
NC
) in
GLC
4 (C) and H1688 (D) cells.
SCLC
, small cell lung cancer; all the results are represented as mean ±
SD
from three independent experiments. ***P<.001 as compared with
NC
cells; β‐actin was used as an internal control
Figure 4
USP32 silencing significantly suppressed small cell lung cancer (
SCLC
) cell proliferation. (A, B)
CCK
‐8 assay indicated that the cell viability of
GLC
4 and H1688 was decreased after USP32 silencing. (C, D) The number of colonies in
GLC
and H1688 cells was significantly suppressed after USP32 knockdown.
SCLC
, small cell lung cancer; Mock: untreated cells;
NC
: cells were treated with scrambled si
RNA
; Si
USP
32: cells were treated with si
RNA
targeting USP32; all the results are represented as mean ±
SD
from three independent experiments. ***P<.001 as compared with
NC
cells
Figure 5
USP32 silencing remarkably induced G0/G1 arrest in
GLC
4 cells. A, Representative images showed knockdown of USP32 altered cell cycle distribution profiles in
GLC
4 cells. B, The cell cycle was significantly arrested in G0/G1 phase in Si
USP
32 group. C, Western blot analysis of proteins associated with cell cycle regulation in
GLC
4 cells after USP32 knockdown. D, The percentage of cells in sub‐G1 phase was significantly elevated in Si
USP
32 group. Mock: untreated cells;
NC
: cells were treated with scrambled si
RNA
; Si
USP
32: cells were treated with si
RNA
targeting USP32; all the results are represented as mean ±
SD
from three independent experiments. **P<.01, ***P<.001 as compared with
NC
cells; β‐actin was used as an internal control
Figure 6
USP32 silencing remarkably promoted cell apoptosis, including early apoptosis and late apoptosis in
GLC
4 cells. A,
GLC
4 cells stained with Annexin V and 7‐
AAD
analysed using flow cytometry analysis. B, Quantification of the percentage of early apoptotic cells (Annexin V+/7‐
AAD
−) and late apoptotic cells (Annexin V+/7‐
AAD
+). C, Western blot analysis of proteins associated with cell apoptosis in
GLC
4 cells after USP32 knockdown. Mock: untreated cells;
NC
: cells were treated with scrambled si
RNA
; Si
USP
32: cells were treated with si
RNA
targeting USP32; all the results were represented as mean ±
SD
from three independent experiments. ***P<.001 as compared with
NC
cells; β‐actin was used as an internal control
Figure 7
Knockdown of USP32 impaired the invasive potential of H1688 cells. A, Crystal violet staining images of invasive H1688 infected with si
USP
32 or scrambled si
RNA
are shown. B, Quantification of the number of invaded cells are shown as mean ±
SD
. C, Western blot analysis of proteins associated with cell mobility in H1688 cells after USP32 knockdown. Mock: untreated cells;
NC
: cells were treated with scrambled si
RNA
; Si
USP
32: cells were treated with si
RNA
targeting USP32; ***P<.001 as compared with
NC
cells; β‐actin was used as an internal control
References
- Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359‐86. - PubMed
- Bordi P, Tiseo M, Barbieri F, et al. Gene mutations in small‐cell lung cancer (SCLC): results of a panel of 6 genes in a cohort of Italian patients. Lung Cancer. 2014;86:324‐8. - PubMed
- Bunn PA Jr. Worldwide overview of the current status of lung cancer diagnosis and treatment. Arch Pathol Lab Med. 2012;136:1478‐81. - PubMed
- Lally BE, Urbanic JJ, Blackstock AW, Miller AA, Perry MC. Small cell lung cancer: have we made any progress over the last 25 years? Oncologist. 2007;12:1096‐104. - PubMed
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