Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jun 11:10:78.
doi: 10.1186/s13578-020-00437-9. eCollection 2020.

Identification of hub genes associated with RNAi-induced silencing of XIAP through targeted proteomics approach in MCF7 cells

Mehdi Agha Gholizadeh  1 , Fatemeh T Shamsabadi  1 , Ahad Yamchi  2 , Masoud Golalipour  1 , Gagan Deep Jhingan  3 , Majid Shahbazi  1   4
Affiliations

Identification of hub genes associated with RNAi-induced silencing of XIAP through targeted proteomics approach in MCF7 cells

Mehdi Agha Gholizadeh et al. Cell Biosci. .

Abstract

Background: The X-linked inhibitor of apoptosis protein (XIAP) is the most potent caspase inhibitor of the IAP family in apoptosis pathway. This study aims to identify the molecular targets of XIAP in human breast cancer cells exposed to XIAP siRNA by proteomics screening. The expression of XIAP was reduced in MCF-7 breast cancer cells by siRNA. Cell viability and the mRNA expression level of this gene were evaluated by MTS and quantitative real-time PCR procedures, respectively. Subsequently, the XIAP protein level was visualized by Western blotting and analyzed by two-dimensional (2D) electrophoresis and LC-ESI-MS/MS.

Results: Following XIAP silencing, cell proliferation was reduced in XIAP siRNA transfected cells. The mRNA transcription and protein expression of XIAP were decreased in cells exposed to XIAP siRNA than si-NEG. We identified 30 proteins that were regulated by XIAP, of which 27 down-regulated and 3 up-regulated. The most down-regulated proteins belonged to the Heat Shock Proteins family. They participate in cancer related processes including apoptosis and MAPK signaling pathway. Reduced expression of HSP90B1 was associated with apoptosis induction by androgen receptor and prostate specific antigen. Suppression of XIAP resulted in the enhancement of GDIB, ENO1, and CH60 proteins expression. The network analysis of XIAP-regulated proteins identified HSPA8, HSP90AA1, ENO1, and HSPA9 as key nodes in terms of degree and betweenness centrality methods.

Conclusions: These results suggested that XIAP may have a number of biological functions in a diverse set of non-apoptotic signaling pathways and may provide an insight into the biomedical significance of XIAP over-expression in MCF-7 cells.

Keywords: Apoptosis; Breast cancer; Molecular targets; Proteomics; RNA interference; XIAP.

PubMed Disclaimer

Conflict of interest statement

Competing interestsThe authors declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Expression of XIAP in response to XIAP silencing in MCF-7 cells. a Fold-change expression of XIAP gene. The expression level of XIAP mRNA was reduced in siRNA treated cells compared to the si-NEG after 24, 48 and 72 h post-transfection. ** illustrates the P < 0.001; *** represents P < 0.0001. b Protein expression level of XIAP in response to XIAP siRNA. Western blot shows that the highest down-regulation of protein occurred in 48 h post-transfection. c Densitometry of XIAP protein bands. The results of western blot were quantified by ImageJ software in order to measure the density of protein bands. The si-NEG was employed as a control group which exhibits the normal expression quantity of XIAP. The densitometry analysis revealed the low amount of XIAP protein at 48 h after transfection
Fig. 2
Fig. 2
Suppression of MCF-7 cell proliferation in post-transfection of siRNA XIAP. The highest reduction of cell viability was observed at 24 and 48 h post-transfection. Single star represent P < 0.05, ** shows P < 0.001, *** P < 0.0001
Fig. 3
Fig. 3
Two-dimensional gel electrophoresis of MCF-7 cells exposed to the XIAP siRNA. After silencing of XIAP, the expression of 30 proteins was differentiated in the XIAP siRNA-transfected cells compared to the si-NEG. The non-linear 18 cm, 3 to 10 pH range IPG strips in the first dimension and 12.5% polyacrylamide gel in the second dimension were used
Fig. 4
Fig. 4
Functional classification of DEPs in MCF-7 cells exposed to RNAi-induced silencing of XIAP. a A network of protein–protein interaction among the XIAP-regulated proteins was constructed. XIAP did not directly interact with the identified DEPs in MCF-7 cells. According to the textmining criteria, XIAP interacts with HSP90B1, HSPB1, and ALB. b The molecular function of the XIAP-regulated proteins are demonstrated. The precise mechanism of action of XIAP with the DEPs was not identified yet. Colors and shapes of line show binding (blue), reaction (black), catalysis (indigo), activation (light green), post-translational modification (fuchsia). The positive and negative effects were illustrated by arrowhead and bar, respectively. c Network of regulated proteins by XIAP was constructed according to the degree, betweenness centrality, and co-expression by CytoHubba application in Cytoscape software. The thickness of grey lines indicates the strength of co-expression. HSP90AA1 and HSPA6 were identified as the hub genes in this network. Although, it should not be ignored the impact of HSPA8 and ENO1 genes
Fig. 5
Fig. 5
GO analysis of the XIAP-regulated proteins. The number of genes involved in molecular function (a) and biological process (b) were demonstrated. Enrichment analysis revealed that most of regulated proteins by XIAP have binding and catalytic activities. Also, these proteins are implicated in cellular and metabolic processes
Fig. 6
Fig. 6
Proposed pathway for activation of apoptosis after XIAP silencing. Association of XIAP in the MAP-kinase signaling pathway is demonstrated in cancer
See this image and copyright information in PMC

References

    1. Hickman JA. Apoptosis and tumourigenesis. Curr Opin Genet Dev. 2002;12(1):67–72. - PubMed
    1. Uren AG, Coulson EJ, Vaux DL. Conservation of baculovirus inhibitor of apoptosis repeat proteins (BIRPs) in viruses, nematodes, vertebrates and yeasts. Trends Biochem Sci. 1998;23(5):159–162. - PubMed
    1. Takahashi R, Deveraux Q, Tamm I, Welsh K, Assa-Munt N, Salvesen GS, et al. A single BIR domain of XIAP sufficient for inhibiting caspases. J Biol Chem. 1998;273(14):7787–7790. - PubMed
    1. Li J, Feng Q, Kim J-M, Schneiderman D, Liston P, Li M, et al. Human Ovarian Cancer and Cisplatin Resistance: possible Role of Inhibitor of Apoptosis Proteins** This work was supported by grants from the Canadian Institutes of Health Research (MOP-15691), the University of Ottawa-Industry Grants Program, and the Ottawa Civic Hospital Foundation. Endocrinology. 2001;142(1):370–380. - PubMed
    1. Satoh K, Kaneko K, Hirota M, Masamune A, Satoh A, Shimosegawa T. Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors. Cancer. 2001;92(2):271–278. - PubMed