Fibulin-3 knockdown inhibits cervical cancer cell growth and metastasis in vitro and in vivo

Abstract

To explore the function of fibulin-3 in cervical carcinoma malignant cell growth and metastasis, fibulin-3 expression in normal cervical tissue, cervical intraepithelial neoplasia (CIN), and cervical carcinoma were evaluated by immunohistochemistry. Quantitative real-time-polymerase chain reaction, western blotting, and immunocytochemistry were performed to assess the expression of fibulin-3 at mRNA and protein levels in different invasive clone sublines. Fibulin-3 shRNA and fibulin-3 cDNA were used to transfect the strongly and weakly invasive clone sublines. Using in vitro and in vivo functional assays, we investigated the effects of down-regulating and up-regulating fibulin-3 expression on the proliferation and invasion of different clone sublines. Epithelial mesenchymal transition (EMT) and its signaling pathways PI3K/AKT and ERK were studied carefully in lentiviral transfection systems. Fibulin-3 was upregulated in cervical carcinoma, and its overexpression was significantly related with malignant phenotype and poor prognosis of cervical carcinoma. Fibulin-3 promoted cervical cancer cell invasive capabilities by eliciting EMT and activating the PI3K-Akt-mTOR signal transduction pathway. Fibulin-3 could facilitate the process of cervical cancer development. The results presented here will help develop novel prognostic factors and possible therapeutic options for patients with cervical cancer.

Figure 1

Expression of fibulin-3 in human cervical tissues. Fibulin-3 expression in (A) normal human cervical tissue, (B) cervical intraepithelial neoplasia (CIN), (C) stage I and stage II of cervical carcinoma, and (D) stage III and stage IV of cervical carcinoma were measured by IHC. (E) Cervical cancer patients with high fibulin-3 expression (blue line) had a worse prognosis than those with low fibulin-3 expression (green line). (Magnification x200).

Figure 2

Fibulin-3 expression in cervical cancer cell clone sublines with different invasive ability. Fibulin-3 expression in highly invasive clone sublines and low invasive clone sublines were measured by (A) western blotting (cropped blot), (B) real time q-RT-PCR, and (C) ICC staining. (Magnification x200). *P < 0.05.

Figure 3

Identification of downregulated and upregulated fibulin-3 expression in lentivirus transfection systems. Fibulin-3 expression in control shRNA-infected cells and control cDNA-infected cells, as well as fibulin-3 shRNA-infected cells and fibulin-3 cDNA-infected cells as measured by (A) ICC staining, (B) real time q-RT-PCR, and (C) western blotting (cropped blot) (Magnification x200). *P < 0.05.

Figure 4

Effects of fibulin-3 knockdown and overexpression on the proliferation and colony formation abilities of cervical cancer cells. (A) Downregulated fibulin-3 markedly inhibited cell proliferative abilities of the highly invasive clone subline cells, whereas upregulated fibulin-3 significantly promoted cell proliferative abilities of the low invasive clone subline cells. (B) The colony-forming abilities of the highly invasive clone subline cells were inhibited by fibulin-3 knockdown; at the same time, fibulin-3 upregulation promoted the colony forming abilities of the low invasive clone subline cells. (C) The colony images of control shRNA-infected cells and fibulin-3 shRNA-infected cells from a soft agar colony formation assay. (D) The colony images of control cDNA-infected cells and fibulin-3 cDNA-infected cells were examined from a soft agar colony formation assay (Magnification x200). *P < 0.05.

Figure 5

Effects of fibulin-3 knockdown and overexpression on cervical cancer cell migration and invasion abilities. (A) Cell migration images of control shRNA-infected cells and fibulin-3 shRNA-infected cells, as well as control cDNA-infected cells and fibulin-3 cDNA-infected cells were measured by cell migration assays using Boyden chambers without Matrigel. (B) Cell invasion images of control shRNA-infected cells and fibulin-3 shRNA-infected cells, as well as control cDNA-infected cells and fibulin-3 cDNA-infected cells were measured by cell invasion assays using Boyden chambers coated with Matrigel. (C) The average counts of migrating and invading fibulin-3 shRNA infected cells were much lower than those of controls; meanwhile, the average counts of migrating and invading fibulin-3 cDNA infected cells were much higher than those of controls (Magnification x200). *P < 0.05.

Figure 6

Effects of fibulin-3 knockdown and overexpression on tumor growth in vivo. (A) Knockdown of fibulin-3 inhibited tumor growth, while up-regulation of fibulin-3 promoted tumor growth. Images of xenografts in nude mice 2 months after subcutaneous inoculation of (B) control shRNA-infected cells and fibulin-3 shRNA-infected cells and (C) control cDNA-infected cells and fibulin-3 cDNA-infected cells. *P < 0.05.

Figure 7

Effects of fibulin-3 knockdown and overexpression on key EMT hallmarks. After lentivirus transfection, EMT markers including E-cadherin, N-cadherin, vimentin, Snail, Slug, Zeb2, and Twist were measured by (A) western blotting (cropped blot) and (B) real-time q-RT-PCR in the lentivirus transfection systems. *P < 0.05.

Figure 8

The correlations between fibulin-3 and the studied EMT markers E-cadherin, N-cadherin and Vimentin. E-cadherin (A), N-cadherin (B), and Vimentin (C) expression in normal human cervical tissues and cervical carcinoma tissues were measured by IHC. (D) Pearson’s product-moment correlation coefficient analysis showed that fibulin-3 was significantly positively correlated with N-cadherin and Vimentin, but negatively correlated with E-cadherin.

Figure 9

The correlations between fibulin-3 and the transcription factors closely related to EMT. Using transcriptomics data from the TCGA repositories, (A) fibulin-3 was significantly positively correlation with the transcription factors Snail 1 and Snail 2, (B) the transcription factors Twist1 and Twist2, and (C) the transcription factors Zeb1 and Zeb2.

Figure 10

Effects of fibulin-3 knockdown and overexpression on the PI3K/AKT/mTOR and ERK pathway as assessed by Boyden chambers. Fibulin-3 cDNA-infected cells, which were serum-starved and treated with DMSO or the indicated inhibitors PD98059 and LY294002 for 48 h, were measured by (A) cell migration assays without Matrigel and (B) cell invasion assays with Matrigel. Fibulin-3 shRNA-infected cells, which were serum-starved and treated with DMSO and the indicated inhibitors PD98059 and LY294002 for 48 h, were subjected to (C) cell migration assays without Matrigel and (D) cell invasion assays with Matrigel. (E) The average counts of migrating and invading fibulin-3 cDNA-infected cells were strongly decreased upon incubation with LY294002, but not with PD98059. (F) The average counts of migrating and invading fibulin-3 shRNA-infected cells were not significantly different among cells treated with DMSO, LY294002, and PD98059.

Figure 11

Effects of fibulin-3 knockdown and overexpression on the PI3K/AKT/mTOR and ERK pathway as determined by western blotting (cropped blot). (A) LY294002 treatment remarkably reduced the levels of PI3K, AKT, and mTOR phosphorylation, and accordingly changed the expression of EMT markers, but had no influence on fibulin-3 expression compared to the DMSO control. (B) PD98059 decreased ERK phosphorylation level but had no effect on fibulin-3 expression and could not block fibulin-3-mediated promotion of EMT. (C) Fibulin-3 knockdown reduced the PI3K, AKT, and mTOR phosphorylation levels to deactivate the PI3K/AKT/mTOR pathway but had no effect on the ERK phosphorylation levels; in contrast, fibulin-3 upregulation increased the PI3K, AKT, and mTOR phosphorylation levels to activate the PI3K/AKT/mTOR pathway, but also had no effect on the ERK phosphorylation level.

Figure 12

Expression of p-PI3K, p-AKT, and p-mTOR in human cervical tissues and the correlations between these proteins and fibulin-3. The expressions of p-PI3K (A), p-AKT (B), and p-mTOR (C) in normal cervical tissues and cervical carcinoma tissues were measured by IHC. (D) Pearson’s correlation coefficient analysis was used to separately analyze the correlations between p-PI3K, p-AKT, and p-mTOR and fibulin-3, and significant positive correlations were observed.