The effect of sortilin silencing on ovarian carcinoma cells

Abstract

Background: Our preliminary data on the protein expression of SORT1 in ovarian carcinoma tissues showed that sortilin was overexpressed in ovarian carcinoma patients and cell lines, while non-malignant ovaries expressed comparably lower amount of this protein. In spite of diverse ligands and also different putative functions of sortilin (NTR3), the function of overexpressed sortilin in ovarian carcinoma cells is an intriguing subject of inquiry. The aim of this study was, therefore, to investigate the functional role of sortilin in survival of ovarian carcinoma cell line.

Methods: Expression of sortilin was knocked down using RNAi technology in the ovarian carcinoma cell line, Caov-4. Silencing of SORT1 expression was assessed using real-time qPCR and Western blot analyses. Apoptosis induction was evaluated using flow cytometry by considering annexin-V FITC binding. [(3)H]-thymidine incorporation assay was also used to evaluate cell proliferation capacity.

Results: Real-time qPCR and Western blot analyses showed that expression of sortilin was reduced by nearly 70-80% in the siRNA transfected cells. Knocking down of sortilin expression resulted in increased apoptosis (27.5±0.48%) in siRNA-treated ovarian carcinoma cell line. Sortilin silencing led to significant inhibition of proliferation (40.1%) in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart (p < 0.05).

Conclusion: As it was suspected from overexpression of sortilin in ovarian tumor cells, a cell survival role for sortilin can be deduced from these results. In conclusion, the potency of apoptosis induction via silencing of sortilin expression in tumor cells may introduce sortilin as a potential candidate for developing a novel targeted therapy in patients with ovarian carcinoma.

Keywords: Apoptosis; Cancer; Ovary; Silencing; Sortilin; siRNA.

Figure 1

Western blot analysis of sortilin expression in ovarian cancer and non-malignant tissues. Seven ovarian carcinoma tissues (Table 1) as well as five ovarian carcinoma cell lines that overexpressed sortilin were compared with five non-malignant ovarian tissues (A and B). The lower band in non-malignant ovarian tissues is likely to be related to the second variant of sortilin with a molecular weight of 80-85 kDa. The level of β-actin as an internal protein loading control was detected in each sample. OC: ovarian carcinoma tissue, N: non-malignant ovarian tissue

Figure 2

Real-time quantitative PCR analysis of SORT1 expression in Caov-4 cells following siRNA treatment. Results revealed 6.1 fold and 4.2 fold reduction in SORT1 expression in siRNA-transfected cells as compared to mock control-transfected cells 24 and 48 hr post-transfection, respectively (p < 0.001). Values are presented as mean±SEM in 3 separate experiments

Figure 3

Western blot analysis of sortilin protein levels in siRNA-transfected cells. Densitometric analysis showed that the level of sortilin was markedly reduced by 72, 69 and 61% in siRNA-treated cells as compared to mock control-treated cells at 48, 72 and 96 hr after transfection, respectively. The level of β-actin as an internal protein loading control was detected in each sample

Figure 4

Analysis of apoptosis following down regulation of sortilin expression. A) Caov-4 cells were treated with sortilin siRNA or mock control and the levels of apoptosis were then evaluated by annexin V FACS analysis 48, 72 and 96 hr post-transfection. The picture shows one of the three experiments. B) Numerical results from three independent experiments of FACS analysis of annexin V staining. Values are presented as mean±SEM in 3 separate experiments

Figure 5

Assessment of cell proliferation following sortilin down regulation. Cell proliferation was inhibited (40.1%) in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart 48 hr post-transfection (p < 0.05). Data is represented as mean±SEM in 2 independent experiments with 6 replicates