Effects of S100A6 gene silencing on the biological features of eutopic endometrial stromal cells and β‑catenin expression

Abstract

Protein expression levels of S100 calcium binding protein A6 (S100A6) are increased in various malignancies and are associated with tumor behavior; however, the association between S100A6 and endometriosis remains to be elucidated. In order to investigate the influence of S100A6 protein, recombinant lentivirus siS100A6 was used to transfect the eutopic endometrial stromal cells. CCK‑8 assay was performed to identify the proliferation ability of cell and the cell migration was detected by Transwell assay. Flow cytometry was performed to detect cell apoptosis, and western blotting and reverse transcription‑quantitative polymerase chain reaction were performed to identify the expression of β‑catenin. The present study investigated the role of S100A6 in endometriosis and its interaction with β‑catenin by transfecting eutopic endometrial stromal cells with a recombinant lentivirus containing S100A6‑specific small interfering RNA. Inhibition of S100A6 expression had a significant antiproliferative effect and reduced the migratory ability of eutopic endometrial stromal cells, and induced their apoptosis. In addition, inhibition of S100A6 expression suppressed β‑catenin expression. These results suggested that inhibition of S100A6 may represent a promising novel approach for the targeted therapy of endometriosis.

Figure 1.

Silencing of S100A6 downregulated the mRNA and protein expression levels of β-catenin in eutopic endometrial stromal cells, as determined by RT-PCR and western blot analysis. (A) Western blot analysis indicated decreased S100A6 and β-catenin protein levels in eutopic endometrial stromal cells following treatment with Lv-siS100A6 compared with Lv-control. Lane 1, untreated group; lane 2, Lv-control group; lane 3, Lv-siS100A6 group. The effect of siS100A6 on S100A6 (B) protein and (C) mRNA expression, and β-catenin (D) protein and (E) mRNA expression was evaluated 4 days post-transfection by western blotting and RT-PCR, respectively. Data are presented as the mean ± standard deviation. *P<0.05 vs. untreated and Lv-control groups. S100A6, S100 calcium binding protein A6; si, small interfering RNA; Lv-siS100A6, siS100A6 recombinant lentivirus vector; Lv-control, control recombinant lentivirus vector; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 2.

Silencing of S100A6 inhibited the proliferation and migration of eutopic endometrial stromal cells. (A) Proliferation of eutopic endometrial stromal cells was detected using a Cell Counting Kit-8 assay post-transfection with Lv-siS100A6. Negative group is Lv-control group. (B) Eutopic endometrial stromal cells transfected with Lv-siS100A6 or Lv-control, or untreated cells, were placed in serum-free medium and added to the upper chamber of a Transwell plate. Migratory capacity was assessed by counting the filtered cells. (C) Silencing of S100A6 markedly suppressed the number of cells that passed through the chamber (magnification, ×200). Data are presented as the mean ± standard deviation. *P<0.05 vs. untreated and Lv-control groups. S100A6, S100 calcium binding protein A6; si, small interfering RNA; Lv-siS100A6, siS100A6 recombinant lentivirus vector; Lv-control, control recombinant lentivirus vector.

Figure 3.

Eutopic endometrial stromal cell apoptotic rates were detected by flow cytometry following transfection with Lv-siS100A6. (A) Analysis of apoptosis was conducted 4 days post-transfection. Cells undergoing early apoptosis only bound to PE Annexin V, whereas cells that bound to PE Annexin V and 7-AAD were in the late stages, or had already undergone apoptosis. (B) Knockdown of endogenous S100A6 induced eutopic endometrial stromal cell apoptosis. The LR quadrants were used to determine apoptotic ratio. The results represent one of three experiments performed. Data are presented as the mean ± standard deviation. *P<0.05 vs. untreated and Lv-control groups. S100A6, S100 calcium binding protein A6; si, small interfering RNA; Lv-siS100A6, siS100A6 recombinant lentivirus vector; Lv-control, control recombinant lentivirus vector; PE, phycoerythrin; 7-AAD, 7-aminoactinomycin D; UL, upper left; UR, upper right; LL, lower left; LR, lower right.