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. 2013 Jun 20:6:667-77.
doi: 10.2147/OTT.S44177. Print 2013.

Cyclin D1 affects epithelial-mesenchymal transition in epithelial ovarian cancer stem cell-like cells

Jie Jiao  1 , Lu Huang, Feng Ye, Minfeng Shi, Xiaodong Cheng, Xinyu Wang, Dongxiao Hu, Xing Xie, Weiguo Lu
Affiliations

Cyclin D1 affects epithelial-mesenchymal transition in epithelial ovarian cancer stem cell-like cells

Jie Jiao et al. Onco Targets Ther. .

Abstract

Background: The association of cancer stem cells with epithelial-mesenchymal transition (EMT) is receiving attention. We found in our previous study that EMT existed from CD24- phenotype cells to their differentiated cells. It was shown that cyclin D1 functioned in sustaining self-renewal independent of CDK4/CDK6 activation, but its effect on the EMT mechanism in ovarian cancer stem cells is unclear.

Methods: The anchorage-independent spheroids from ovarian adenocarcinoma cell line 3AO were formed in a serum-free medium. CD24- and CD24+ cells were isolated by fluorescence-activated cell sorting. Cell morphology, viability, apoptosis, and migratory ability were observed. Stem-related molecule Bmi-1, Oct-4 and EMT-related marker E-cadherin, and vimentin expressions were analyzed. Cyclin D1 expression in CD24- phenotype enriched spheroids was knocked down with small interfering RNA, and its effects on cell proliferation, apoptosis, migration ability, and EMT-related phenotype after transfection were observed.

Results: In our study, CD24- cells presented stronger proliferative, anti-apoptosis capacity, and migratory ability, than CD24+ cells or parental cells. CD24- cells grew with a scattered spindle-shape within 3 days of culture and transformed into a cobblestone-like shape, identical to CD24+ cells or parental cells at 7 days of culture. CD24- cells or spheroids highly expressed cyclin D1, Bmi-1, and vimentin, and seldom expressed E-cadherin, while CD24+ or parental cells showed the opposite expression. Furthermore, cyclin D1-targeted small interfering RNA resulted in decreased vimentin expression in spheroids. Transfected cells also exhibited an obvious decrease in cell viability and migration, but an increase in cell apoptosis.

Conclusion: Cancer stem cell-like cells possess mesenchymal characteristics and EMT ability, and cyclin D1 involves in EMT mechanism, suggesting that EMT of cancer stem cell-like cells may play a key role in invasion and metastasis of ovarian cancer.

Keywords: cancer stem cell; cyclin D1; epithelial; mesenchymal transition; ovarian cancer.

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Figures

Figure1
Figure1
Cell viability, apoptosis, and stem-related genes expression in CD24− and CD24+ cells. Notes: (A) The proliferative rate of CD24+ cells was obviously lower than that of parental 3AO and CD24− cells. At 48 hours after culture, CD24+ cells showed lower proliferation than the other two kinds of cells. At 72 hours after culture, only CD24− cells still persisted in proliferation. (B: a) CD24− cells before dosing, (B: b) CD24− cells after dosing, (B: c) CD24+ cells before dosing, (B: d) CD24− cells after dosing. (C) Comparison of viable and nonviable apoptosis rates between CD24− and CD24+ cells. nonviable apoptosis rate of CD24+ cells was significantly increased beyond that of CD24− cells (Z = −3.363, P = 0.001). (D) Bmi-1 mRNA expression was significantly higher in CD24− cells than that in CD24+ cells (t = 4.761, P = 0.001), but gradually and significantly decreased during the differentiation cultivation (F = 11.584, P = 0.001); Oct-4 expression was not significantly different between CD24− and CD24+ (t = 0.296, P = 0.774), but gradually decreased during the differentiation cultivation in both cells (FCD24− = 6.016, PCD24− = 0.016; FCD24+ = 5.426, PCD24+ = 0.021).
Figure 2
Figure 2
Epithelial–mesenchymal transition (EMT) phenomenon in CD24− cells. Notes: (A) CD24− grew in the shape of scattered spindle, similar to fibroblast cells within 3 days of culture with complete medium, while CD24+ presented the typical cobblestone-like shape growth. Cell colonies grew towards the shape of parental 3AO cells as cultures were prolonged. (B) E-cadherin and vimentin expression in different cells. E-cadherin mRNA expression in CD24− cells was significantly lower than CD24+ cells (t = −4.095, P = 0.015). There was a clear upregulated trend of E-cadherin mRNA expression from fresh isolated, 3-day culture, to 7-day culture CD24− cells (F = 6.459, P = 0.012), but not among CD24+ cells with different culture times. Vimentin mRNA expression in CD24− cells was significantly higher than CD24+ cells (t = 5.767, P = 0.002). There was also a clear downregulated trend of vimentin mRNA expression from fresh isolated, 3-day cultures, to 7-day culture CD24− cells (F = 54.637, P = 0.001), but not among CD24+ cells with different culture times. (C and D) The wound-repair assay: spheroid-differentiated cells revealed significantly increased width closure than parental adherent cells at 4 hours (P = 0.068) and at 8 hours after scratch (P = 0.000).
Figure 3
Figure 3
The expression pattern of cyclin D1, N-cadherin, vimentin in parental cells and spheroids, and transfection efficacy of cyclin D1 siRNA. Notes: (A and B) The average value of cyclin D1 was higher in spheroids than that in parental cells with a significant difference (t = −3.351, P = 0.007). The value of N-cadherin was not statistically different between spheroids and parental cells (t = −1.322, P = 0.221). The average value of vimentin was also higher in spheroids than that in parental cells, with a significant difference (t = −2.471, P = 0.033). (C) Cyclin D1 mRNA was downregulated nearly 59% after siRNA transfection. (D) Cyclin D1 protein was down-regulated nearly 51% after siRNA transfection. Abbreviations: P1, parental cell sample 1; P2, parental cell sample 2; P1, spheroids sample 1; S2, spheroids sample 2; NT-siRNA, nontargeting small interfering RNA.
Figure 4
Figure 4
Cell viability, apoptosis induced by cyclin D1 silence. Notes: (A) At 0, 24, and 48 hours, cell viability was not significantly different between cyclin D1-siRNA and NT siRNA transfected cells, but a marked decrease of cell viability was observed at 72 hours post-transfection (Z = −2.241, P = 0.025). (B and C) At 30 hours after siRNA transfection, viable apoptosis was significantly increased in cyclin D1 siRNA-transfected cells compared with NT siRNA group (Z = −2.650, P = 0.008). Abbreviation: NT-siRNA, nontargeting small interfering RNA.
Figure 5
Figure 5
Cell migration and n-cadherin, vimentin expression induced by cyclin D1 silence. Notes: (A and B) Wound width of cyclin D1 siRNA-transfected cells was significantly decreased compared to that of control (t = −3.125, P = 0.004); (C and D) Knockdown of cyclin D1 resulted in significantly decreased expression of vimentin protein in spheroid-differentiated cells (t = 3.748, P = 0.002). N-cadherin expression was not significantly different (Z = −1.083, P = 0.279). Abbreviation: NT-siRNA, nontargeting small interfering RNA.
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