Silencing of Wnt10B reduces viability of heptocellular carcinoma HepG2 cells - PubMed (original) (raw)
. 2015 May 15;5(6):1911-20.
eCollection 2015.
Affiliations
- PMID: 26269753
- PMCID: PMC4529613
Silencing of Wnt10B reduces viability of heptocellular carcinoma HepG2 cells
Guohui Wu et al. Am J Cancer Res. 2015.
Abstract
Dysregulation of Wnt-mediated β-catenin signaling is associated with carcinogenesis and progression of hepatocellular carcinoma (HCC). Our previous studies showed that the Wnt10B gene, a member of Wnt gene family, over-activated in HCC tissues and cells. Here we demonstrate that stable silencing of Wnt10B reduces the viability of HCC cells in culture. HepG2, a human HCC cell line, was cultured in vitro and Wnt10B gene in the cells stably silenced, as showed in Western blotting analysis, by the shRNA interference with lentivirus plasmid transfection. Compared to the control (HepG2 cells without Wnt10B silencing), the Wnt10B-silencing cells showed significant reductions in proliferation, colony formation, migration and invasion. Furthermore, serum deprivation-induced apoptotic death, assessed by Hoechst 33342 staining and fluorescent microscopy, increased significantly in the Wnt10B-silencing cells. FACScan analysis indicated an arrest of the cell cycle in the Wnt10B-silencing HCC cells, with significant increases in the number of cells in G0-G1 and S phases. Thus, we hypothesize that Wnt10B plays an oncogenic role in HCC and is a potential therapeutic target.
Keywords: Wnt10B; apoptosis; hepatocellular carcinoma cells; invasion; proliferation.
Figures
Figure 1
Fluorescent staining analysis of HepG2 cells in culture with lentivirus vector transfection. HepG2 cells infected by lentivirus vectors pLVTHM (control), pLV-shwnt10b1 (shWnt10b#1) or pLV-shwnt10b2 (shWnt10b#2) were under fluorescent staining and microscopy (photos), with a relatively high efficiency of the transfection (the rate of infection) achieved (bar graph, *P<0.05, n=3).
Figure 2
Expression of Wnt10B protein in HepG2 cells in culture. Western blotting analysis of protein expression of Wnt10B in HepG2 cells transfected with pLVTHM (control), pLV-shwnt10b1 (shWnt10b#1), or pLV-shwnt10b2 (shWnt10b#2). Photos: Protein signals of Wnt10B (bands at ~42-55 kDa) presented on Western blots in a representative result from three independent experiments. Bar graph: relative levels of Wnt10B proteins are showed as mean ± SD (n=3); *P<0.01, vs. control; #P<0.05, vs. shWnt10b#1.
Figure 3
Silencing of Wnt10B attenuates the proliferation in HepG2 cells in culture. HepG2 cells transfected with pLVTHM (control), pLV-shwnt10b1 (shWnt10b#1), or pLV-shwnt10b2 (shWnt10b#2) were cultured for 2 to 92 hours and at different time points assessed with a CCK-assay to determine cell proliferation rate (absorbance at 450 nm). Results are expressed as the mean ± SD (n=3); *P<0.05, **P<0.01, vs. control; #P<0.05, vs. shWnt10b#1.
Figure 4
Silencing of Wnt10B delays the colony formation in HepG2 cells in culture. HepG2 cells transfected with pLVTHM (control), pLV-shwnt10b1 (shWnt10b#1), or pLV-shwnt10b2 (shWnt10b#2) were cultured for 2-3 weeks, then cell growth and colony formation determined. Photos: a representative result from three independent experiments. Bar graph: data are shown as mean ± SD (n=3). *P<0.05, **P<0.01 vs. control; #P<0.05, vs. shWnt10b#1.
Figure 5
Silencing of Wnt10B obstructs migration in HepG2 cells in culture. HepG2 cells transfected with pLVTHM (control), pLV-shwnt10b1 (shWnt10b#1), or pLV-shwnt10b2 (shWnt10b#2) were cultured to reach to confluence, scratched and cultured continually for 48-96 hours, then the cell migration and wound healing assessed by microscopy at different time points. Photos: a representative result from three independent experiments. Bar graph: data are shown as mean ± SD (n=3). *P<0.05, **P<0.01 vs. control; #P<0.05, vs. shWnt10b#1.
Figure 6
Silencing of Wnt10B reduces invasion of HepG2 cells in culture. HepG2 cells transfected with pLVTHM (control), pLV-shwnt10b1 (shWnt10b#1), or pLV-shwnt10b2 (shWnt10b#2) were cultured in Matrigel chamber for 48-96 hours and analyzed at different time points as described in Materials and Methods. The rate of invasion in control cells was as 100% and the inhibitory rate of invasion in Wnt10B silencing cells compared to the control. Data are shown as mean ± SD (n=3). *P<0.05, **P<0.01 vs. control; #P<0.05, vs. shWnt10b#1.
Figure 7
Silencing of Wnt10B promotes apoptosis in HepG2 cells in culture. HepG2 cells transfected with pLVTHM (control), pLV-shwnt10b1 (shWnt10b#1), or pLV-shwnt10b2 (shWnt10b#2) were cultured under serum-free DMEM for 48-96 hours, stained with Hoechst 33342, and analyzed by fluorescence microscopy. Photos: representative images of cells cultured for 72 hours from three independent experiments. Bar graph: data are showed as mean ± SD (n=3), *P<0.05, **P<0.01, vs. control; #P<0.05, vs. shWnt10b#1.
Figure 8
Silencing of Wnt10B in cultured HepG2 cells decreases cells at G0-G1 phase, increases S phase cells, and does not changed the number of G2-M cells. HepG2 cells transfected with pLVTHM (control), pLV-shwnt10b1 (shWnt10b#1), or pLV-shwnt10b2 (shWnt10b#2) were analyzed with FACS as described in Materials and Methods. A representative result from three independent experiments presented here indicates the percentage of cells in the different phases.
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