Overexpression of TROP2 promotes proliferation and invasion of ovarian cancer cells

Abstract

Human trophoblastic cell-surface marker, tumor-associated calcium signal transducer 2 (TROP2), is a newly identified marker that has a vital role in the proliferation and invasion of various tumors. However, its specific function in ovarian cancer has not been researched. The purpose of the present study was to investigate the role of TROP2 in the formation of ovarian cancer and its possible mechanism. TROP2 was knocked down by small interfering (si)RNA in ovarian cancer cell line, A2780. The expression of TROP2 protein following transfection was detected by western blot analysis. Cell viability was determined using a Cell Counting kit-8. Cancer cell migration and invasion were examined by wound healing and cell invasion assays, respectively. Apoptosis-related proteins, such as B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax), were measured by western blotting. Results demonstrated that the expression levels of TROP2 were markedly downregulated by siRNA in A2780 cells compared with the control groups, which led to strong inhibition of proliferation and invasion. Furthermore, TROP2 downregulation also reduced cell migratory ability. Additionally, in the TROP2-knockout group, Bcl-2 was downregulated and Bax was upregulated compared with the control. The present study suggested that the expression of TROP2 was related to cellular proliferation, migration and invasion. TROP2 may disrupt the balance in the Bax family to participate in apoptosis regulation in A2780 cells. Therefore, the overexpression of TROP2 may have a crucial role in tumorigenesis and tumor progression by disturbing the Bax/Bcl-2 balance in ovarian cancer.

Keywords: migration; ovarian cancer; proliferation; small interfering RNA; tumor-associated calcium signal transducer 2.

Figure 1.

The expression of TROP2 in ovarian cancer cell lines. Immunofluorescence assay demonstrated a layer of red fluorescent staining on the membrane of SK-OV-3, HO8910 and A2780 cells, and the staining on A2780 cells was markedly brighter than the other two cell lines. Western blot analysis demonstrated the same result. TROP2, tumor-associated calcium signal transducer 2; DAPI, 4, 6-diamidino-2-phenylindole.

Figure 2.

A2780 cells were observed and photographed by a fluorescence microscope 48 h after transfection with TROP2-homo-550 and TROP2-homo-1100 small interfering RNA sequences. The bright green stain for the expression of GFP protein implied successful transfection. TROP2, tumor-associated calcium signal transducer 2.

Figure 3.

(A) Western blot analysis measured the effects of siRNA-mediated knockdown of TROP2. GAPDH was used as an internal control. Data are presented as the mean + standard deviation. Cells were transfected with (B) TROP2-homo-550 and (C) TROP2-homo-1100 siRNA and the viability was assessed using Cell counting kit-8 assay at five time points (24, 48, 72, 96 and 120 h). Transfected cells demonstrated a reduction in cell viability, and differences were significant when cells were cultured for 72 h (group 550) and 48 h (group 1100). Data are presented as the mean ± standard deviation. *P<0.05 vs. the control groups, ^#P<0.05 vs. group 550. TROP2, tumor-associated calcium signal transducer 2; siRNA, small interfering RNA; C, control; NC, negative control.

Figure 4.

The effect of TROP2 expression on cell migration. Cells with TROP2-knockdown demonstrated slower wound recovery compared with the control groups at 24 and 48 h after wounding. Data are presented as the mean + standard deviation. *P<0.05 vs. the control groups at the corresponding time points. TROP2, tumor-associated calcium signal transducer 2.

Figure 5.

The influence of TROP2 expression on the invasive capability of A2780 cells. Downregulation of TROP2 inhibited cell invasion. Data are presented as the mean + standard deviation. *P<0.05 vs. the control groups. TROP2, tumor-associated calcium signal transducer 2.

Figure 6.

Western blot analysis of expression of Bcl-2 and Bax proteins. GAPDH was used as an internal control. Following the knockdown of TROP2, the expression of Bax was enhanced, whereas Bcl-2 protein expression levels were decreased compared with the NC. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; NC, negative control.