Silencing BRIT1 Facilitates the Abilities of Invasiveness and Migration in Trophoblast Cells

Abstract

BACKGROUND The improper invasion of trophoblast cells (TC) can cause various diseases. BRCT-repeat inhibitor of hTERT expression (BRIT1) is involved in the invasion of tumors. Here, we analyzed the effects of BRIT1 on the invasion of TC. MATERIAL AND METHODS The expression of BRIT1 in JEG-3, B6Tert, and HTR8/SVneo cells was evaluated by transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting. The viability, invasion, and migration of HTR8/SVneo cells were measured using cell counting kit-8 (CCK-8) and Transwell assays. The activities of pro-matrix metalloproteinase-2 (pro-MMP-2) and pro-MMP-9 were tested by gelatin zymography assay. The levels of invasion- and Wnt/β-catenin pathway-related factors were assessed by RT-qPCR and Western blotting. RESULTS Levels of BRIT1 in HTR8/SVneo cells were higher than that of JEG-3 and B6Tert cells. The transfection efficiency of BRIT1 siRNA-2 was better than BRIT1 siRNA-1 in HTR8/SVneo cells. BRIT1 siRNA-2 did not change cell viability, whereas it promoted cell invasion and migration. BRIT1 siRNA-2 enhanced the activities of pro-MMP-2 and pro-MMP-9, as well MMP-2 and MMP-9 levels, and reduced tissue inhibitor of metalloproteinases-1 (TIMP-1) and TIMP-2 expression. Moreover, BRIT1 siRNA-2 significantly increased the levels of Wnt2, Wnt3, and β-catenin. CONCLUSIONS BRIT1 silencing accelerated the invasion and migration of TC and activated the Wnt/β-catenin pathway. Our results may provide new insights for finding new molecular targets to cure disease caused by insufficient invasion of TC.

Figure 1

Expression of BRIT1 in different trophoblast cells. (A) The mRNA level of BRIT1 in human choriocarcinoma cell line (JEG-3 cells) and normal trophoblast cell lines (B6Tert and HTR8/SVneo cells) was detected by RT-qPCR. (B) The proteins levels of BRIT1 were measured by Western blotting. * P<0.05, ** P<0.01, versus JEG-3.

Figure 2

Effect of BRIT1 siRNA-2 on the viability of HTR8/SVneo cells. HTR8/SVneo cells were transfected with PBS (control), unspecific scrambled siRNA (control siRNA), BRIT1 siRNA-1, and BRIT1 siRNA-2 plasmids. (A) RT-qPCR was performed to test the mRNA level of BRIT1. (B) Western blotting was used to assess the protein level of BRIT1. (C) Cell viability was analyzed by CCK-8 analysis. * P<0.05, ** P<0.01, versus control; ^# P<0.05, ^## P<0.01, versus control si-RNA.

Figure 3

Effect of BRIT1 siRNA-2 on the invasion and migration of HTR8/SVneo cells. (A, B) Transwell assay was carried out to examine the invasion (A) and migration (B) of HTR8/SVneo cells. * P<0.05, ** P<0.01, versus control; ^# P<0.05, ^## P<0.01, versus control siRNA. Bar=50 μm.

Figure 4

Effect of BRIT1 siRNA-2 on invasion-related factors in HTR8/SVneo cells. (A) The activities of pro-MMP-2 and pro-MMP-9 were investigated by gelatin zymography assay. (B–E) The mRNA levels of MMP-2 (B), MMP-9 (C), TIMP-1 (D), and TIMP-2 (E) were surveyed using RT-qPCR. (F, G) The proteins levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were identified by Western blotting. * P<0.05, ** P<0.01, versus control; ^# P<0.05, ^## P<0.01, versus control siRNA.

Figure 5

Effect of BRIT1 siRNA-2 on Wnt/β-catenin pathway in HTR8/SVneo cells. (A–D) Western blotting was performed to test the protein levels of Wnt2 (B), Wnt3 (C), and β-catenin (D). * P<0.05, ** P<0.01, versus control; ^# P<0.05, ^## P<0.01, versus control siRNA.