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
. 2015 Nov 15;7(11):2397-411.
eCollection 2015.

Effects of siRNA-mediated silencing of myeloid cell leukelia-1 on the biological behaviors and drug resistance of gastric cancer cells

Bo-Pei Li  1 , Jin-Lu Liu  1 , Jun-Qiang Chen  1 , Zhen Wang  1 , Yuan-Tian Mao  1 , Ye-Yang Chen  1
Affiliations

Effects of siRNA-mediated silencing of myeloid cell leukelia-1 on the biological behaviors and drug resistance of gastric cancer cells

Bo-Pei Li et al. Am J Transl Res. .

Abstract

Objective: This study was to investigate the effects of siRNA mediated silencing of myeloid cell leukelia-1 (Mcl-1) on the biological behaviors and drug resistance of human drug-resistant gastric cancer (GC) cell lines, and to explore the potential mechanisms.

Methods: siRNA targeting Mcl-1 mRNA were designed and independently transfected into SGC-7901/VCR and SGC-7901/DDP. Cell proliferation and drug sensitivity were examined by MTT assay. Cell apoptosis and cell cycle were detected by flow cytometry. Cell Invasion and migration abilities were detected by transwell chamber assays. The expressions of drug-resistance-related genes and apoptosis-related proteins were detected by quantitative real-time PCR and Western blot assay, respectively.

Results: siRNA effectively inhibited the Mcl-1 expression, lowered the proliferation rate (P<0.05), raised the apoptosis rate (P<0.05), and arrested cells in S-phase (P<0.05). After inhibiting Mcl-1, the cell migration and invasion decreased (P<0.05), the resistance to VCR, DDP and 5-Fu was reversed to different extents (P<0.05), TS mRNA expression increased significantly (P<0.05), MDR1 remained unchanged (P>0.05), but DPD and TOP2A decreased significantly (P<0.05). Following Mcl-1 silencing, Bcl-2 was over-expressed in VCR-siRNA group, but the expressions of Fas and survivin reduced markedly (P<0.05); Bcl-2 and Fas expressions decreased significantly in DDP-siRNA group (P<0.05), but survivin expression remained unchanged.

Conclusion: Mcl-1 is implicated in the proliferation, invasion, apoptosis and drug resistance of GC cells, and may be a promising target for the therapy of GC.

Keywords: Myeloid cell leukelia-1 gene; drug resistance; gastric cancer; mechanism; siRNA.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Expression of Mcl-1 in human gastric cancer cell line SGC-7901 cells and human drug-resistant gastric cancer cell line SGC-7901/VCR cells and SGC-7901/DDP cells. A. mRNA expression of Mcl-1 significantly increased in SGC-7901/VCR cells and SGC-7901/DDP cell (*P<0.05 vs SGC-7901 cells). B, C. Protein expression of Mcl-1 markedly increased in SGC-7901/VCR cells and SGC-7901/DDP cells (*P<0.05 vs SGC-7901 cells).
Figure 2
Figure 2
Transfection efficiency of SGC-7901/VCR cells and SGC-7901/DDP cells (×100). A. SGC-7901/VCR cells under light microscope and fluorescence microscope; B. SGC-7901/DDP cells under light microscope and fluorescence microscope.
Figure 3
Figure 3
Relative Mcl-1 mRNA and protein expressions in SGC-7901/VCR cells and SGC-7901/DDP cells at different time points after transfection. GAPDH mRNA expression was significantly suppressed in SGC-7901/VCR cells (A) and SGC-7901/DDP cells (B) at 24 h, 48 h and 72 h after GAPDH-siRNA transfection (*P<0.05 vs CTRL). (C) Mcl-1 mRNA expression significantly decreased at different time points after Mcl-1-siRNAs transfection in SGC-7901/VCR cells and SGC-7901/DDP cells (*P<0.05 vs CTRL, Mock and NC groups), except at 72 h in SGC-7901/VCR-Mcl-1-siRNA2 group. (D, E) Mcl-1 protein expression significantly decreased at different time points after Mcl-1-siRNAs transfection in SGC-7901/VCR cells and SGC-7901/DDP cells, except at 72 h in SGC-7901/VCR-Mcl-1-siRNA2 group and SGC-7901/DDP-Mcl-1-siRNA1 group (*P<0.05 vs CTRL, Mock and NC groups).
Figure 4
Figure 4
Proliferation (MTT assay) and cell cycle distribution (flow cytometry) of gastric cancer cells after siRNA-Mcl-1 trasnfection. The proliferation rate in Mcl-1-siRNA3 group significantly decreased at each time point in SGC-7901/VCR cells (A) and SGC-7901/DDP cells (B) (*P<0.05 vs CTRL, Mock and NC groups). (C, E) In SGC-7901/VCR cells, the proportion of cells in S phase and G2/M phase after Mcl-1-siRNA3 transfection increased significantly, but that of cells in G0/G1 phase decreases markedly (*P<0.05 vs CTRL, Mock and NC groups). (D, F) In SGC-7901/DDP cells, the proportion of cells in S phase after Mcl-1-siRNA3 transfection increased significantly, but that of cells in G0/G1 phase and G2/M phase decreased markedly (*P<0.05 vs CTRL, Mock and NC groups).
Figure 5
Figure 5
Migration and invasion abilities of SGC-7901/VCR cells and SGC-7901/DDP cells in vitro after Mcl-1 silencing. A, B, E. Cell migration assay. The number of SGC-7901/VCR cells and SGC-7901/DDP cells crossing the basement membrane significantly decreased after Mcl-1-siRNA3 transfection (*P<0.05 vs CTRL, Mock and NC groups). C, D, F. Cell invasion assay. The number of SGC-7901/VCR cells and SGC-7901/DDP cells crossing the basement membrane markedly decreased after Mcl-1-siRNA3 transfection (*P<0.05 vs CTRL, Mock and NC groups).
Figure 6
Figure 6
Apoptosis and apoptosis related protein expressions in gastric cancers after Mcl-1 silencing in vitro. A-C. FCM showed the apoptosis rate of SGC-7901/VCR cells and SGC-7901/DDP cells increased significantly after Mcl-1-siRNA3 transfection (*P<0.05 vs CTRL, Mock and NC groups). D. Mcl-1 expression was evidently suppressed (*P<0.05 vs CTRL, Mock and NC groups), but the Bcl-2 expression increased and survivin and Fas expressions decreased markedly (*P<0.05 vs CTRL, Mock and NC groups) in SGC-7901/VCR cells after Mcl-1-siRNA3 transfection. E. Expressions of Bcl-2 and Fas decreased significantly (*P<0.05 vs CTRL, Mock and NC) but survivin expression remained unchanged (P>0.05) in SGC-7901/DDP cells after Mcl-1-siRNA3 transfection.
Figure 7
Figure 7
Drug resistance and expressions of drug resistance-related genes in SGC-7901/VCR cells and SGC-790/DDP after Mcl-1 silencing. A-C. As the drug concentrations of VCR, DDP and 5-Fu increased, the inhibition rate of SGC-790/VCR cells undergoing Mcl-1-siRNA3 transfection gradually increased (*P<0.05 vs CTRL, Mock and NC groups). D-F. As the drug concentrations of VCR, DDP and 5-Fu increased, the inhibition rate of SGC-790/DDP cells undergoing Mcl-1-siRNA3 transfection progressively increased (*P<0.05 vs CTRL, Mock and NC groups). G. In SGC-7901/VCR cells, Mcl-1 silencing significantly increased TS mRNA expression, but inhibited the DPD and TOP2A mRNA expressions (*P<0.05 vs CTRL, Mock and NC groups), while MDR1 mRNA expression remained unchanged (P>0.05). H. In SGC-7901/DDP cells, Mcl-1 silencing markedly increased TS mRNA expression, but inhibited the DPD and TOP2A mRNA expressions (*P<0.05 vs CTRL, Mock and NC groups), while MDR1 mRNA expression reduced significantly as compared to control group (ΔP<0.05).
See this image and copyright information in PMC

References

    1. Yecies D, Carlson NE, Deng J, Letai A. Acquired resistance to ABT-737 in lymphoma cells that up-regulate MCL-1 and BFL-1. Blood. 2010;115:3304–3313. - PMC - PubMed
    1. Li G, Zhang S, Fang H, Yan B, Zhao Y, Feng L, Ma X, Ye X. Aspirin overcomes Navitoclaxresistance in hepatocellular carcinoma cells through suppression of Mcl-1. Biochem Biophys Res Commun. 2013;434:809–814. - PubMed
    1. Akagi H, Higuchi H, Sumimoto H, Igarashi T, Kabashima A, Mizuguchi H, Izumiya M, Sakai G, Adachi M, Funakoshi S, Nakamura S, Hamamoto Y, Kanai T, Takaishi H, Kawakami Y, Hibi T. Suppression of myeloid cell leukemia-1 (Mcl-1) enhances chemotherapy-associated apoptosis in gastric cancer cells. Gastric Cancer. 2013;16:100–110. - PubMed
    1. Wurst M, Robles A, Po J, Luu VD, Brems S, Marentije M, Stoitsova S, Quijada L, Hoheisel J, Stewart M, Hartmann C, Clayton C. An RNAi screen of the RRM-domain proteins of Trypanosoma brucei. Mol Biochem Parasitol. 2009;163:61–65. - PubMed
    1. Davidson BL, McCray PB Jr. Current prospects for RNA interference-based therapies. Nat Rev Genet. 2011;12:329–340. - PMC - PubMed

LinkOut - more resources