Stanniocalcin1 (STC1) Inhibits Cell Proliferation and Invasion of Cervical Cancer Cells

Abstract

STC1 is a glycoprotein hormone involved in calcium/phosphate (Pi) homeostasis. There is mounting evidence that STC1 is tightly associated with the development of cancer. But the function of STC1 in cancer is not fully understood. Here, we found that STC1 is down-regulated in Clinical tissues of cervical cancer compared to the adjacent normal cervical tissues (15 cases). Subsequently, the expression of STC1 was knocked down by RNA interference in cervical cancer CaSki cells and the low expression promoted cell growth, migration and invasion. We also found that STC1 overexpression inhibited cell proliferation and invasion of cervical cancer cells. Moreover, STC1 overexpression sensitized CaSki cells to drugs. Further, we showed that NF-κB p65 protein directly bound to STC1 promoter and activated the expression of STC1 in cervical cancer cells. Thus, these results provided evidence that STC1 inhibited cell proliferation and invasion through NF-κB p65 activation in cervical cancer.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. The expression of STC1 was down-regulated in cervical cancer tissues (15 cases).

(A) The expression level of STC1 mRNA in cancerous (T) and adjacent normal (N) matched cervical tissues was examined by RT-PCR. GAPDH served as a loading control. (B) Box plot graph showed the quantitative results of STC1 mRNA expression in all pairs of samples (p<0.05). (C) Protein level of STC1 in tumor and normal tissues were examined by immunohistochemistry analysis. Bar size: 100 µm. (D) Quantity analysis of protein level of STC1 expression. The intensity of reactivity was scored using a three-tier system (p<0.05). Data was expressed as mean ± SEM of three separated experiments. A value of P<0.05 was considered as statistical significance.

Figure 2. Down-regulation of STC1 promoted CaSki cells growth and invasion.

(A) Knock down of STC1 in CaSki cells. CaSki cells were transfected by STC1 targeting siRNA, and knockdown efficiency was shown by RT-PCR and western blotting. (B) MTT assays showed that the effect of decreased STC1 on CaSki cell growth. Following a 7-day period, the growth of CaSki/siRNA cells was much faster than CaSki/NC cells (*p<0.05). (C) Colony formation assay demonstrated the large number of cell colonies from CaSki/siRNA cells compared to CaSki/NC cells (p<0.05). (D) Wound healing assays showed the effect of STC1 on the migration of CaSki cells. CaSki/siRNA cells migrated faster compared to CaSki/NC cells (left panel). The relative migration distance of CaSki cells was calculated (right panel) (p<0.05). Bar size: 100 µm. (E) Matrigel invasion assays showed the effect of STC1 on the invasion of CaSki cells. The number of CaSki/siRNA cells on the filter surface was larger than CaSki/NC cells (left panel) (p<0.05). The mean value of invaded cells was shown in right panel. Bar size: 100 µm. (F) The growth curves of the xenografts were determined by tumor volume (left panel) (*p<0.05). The growth rates of the xenografts were valuated by tumor volume/days (right panel) (*p<0.05). CaSki/siRNA or CaSki/NC cells were injected subcutaneously into nude mice. (G) At end of experimental period, the final xenograft tumors were shown. (H) RT-PCR analyzed the expression of STC1 in representative xenograft tumors. (I) Representative images of histological inspection of xenograft tumors. The sections of xenograft tumors were stained with H&E. Bar size: 20 µm. Data was expressed as mean ± SEM of three separated experiments. Data was expressed as mean ± SEM of three separated experiments. A value of P<0.05 was considered as statistical significance.

Figure 3. Overexpression of STC1 inhibited cell proliferation and invasion of CaSki cells.

(A) Overexpression of STC1 in CaSki cells. STC1 expression vector was transfected into CaSki cells, and increased expression of STC1 was shown by RT-PCR and western blotting. (B) MTT assays showed that the growth of CaSki/STC1 cells was much slower than CaSki/NC cells (*p<0.05). (C) Colony formation assay showed that a small amount of cell colonies from CaSki/STC1 cells demonstrated a low activity (p<0.05). (D) Matrigel invasion assay revealed that up-regulation of STC1 mitigated the invasion of cells in vitro. Bar size: 100 µm. (E) STC1 overexpressed tumors emerged later and slowly grew compared to control tumors (*p<0.05). (F) At end of experimental period, the final weights of STC1 overexpressed tumors were found to be lower than controls. (G) RT-PCR of STC1 in xenograft tumors indicated that increased STC1 expression had been maintained throughout experimental time course. (H) H&E staining of STC1 overexpressed tumors showed a low nuclear/cytoplasmic ratio, and limited to the cancer nests compared to control tumors. Bar size: 20 µm. Data was expressed as mean ± SEM of three separated experiments. A value of P<0.05 was considered as statistical significance.

Figure 4. STC1 sensitized CaSki cells to drugs.

(A) Effect of cisplatin on CaSki cells growth. CaSki cells were treated with with or without cisplatin (0, 1, 2, and 3 mg/L) for 96 h, removing aliquots every 24 h to evaluate cell viability. (B) Effect of thapsigargin on CaSki cells growth. CaSki cells were treated with with or without thapsigargin (0, 1, 3, 6, and 9 µM) for 72 h, removing aliquots every 24 h to evaluate cell viability. (C) Effect of rapamycin on CaSki cells growth. CaSki cells were treated with with or without rapamycin (0, 0.01, 0.1, 0.5, and 1 mg/L) for 72 h, removing aliquots every 24 h to evaluate cell viability. (D) STC1 sensitized CaSki cells to cisplatin. CaSki/STC1 or CaSki/NC cells were treated with cisplatin (2 mg/L) for 96 h. MTT assays detected the cell growth of CaSki/STC1 or CaSki/NC cells in the face of cisplatin (*p<0.05). (E) STC1 sensitized CaSki cells to thapsigargin. CaSki/STC1 or CaSki/NC cells were treated with thapsigargin (3 µM) for 72 h. MTT assays detected the cell growth of CaSki/STC1 or CaSki/NC cells in the face of thapsigargin (*p<0.05). (F) STC1 sensitized CaSki cells to rapamycin. CaSki/STC1 or CaSki/NC cells were treated with rapamycin (0.5 mg/L) for 72 h. MTT assays detected the cell growth of CaSki/STC1 or CaSki/NC cells in the face of rapamycin (*p<0.05). Data was expressed as mean ± SEM of three separated experiments. A value of P<0.05 was considered as statistical significance.

Figure 5. Direct binding of NF-κB p65 protein to STC1 promoter and regulated the expression of STC1.

(A) The binding sites of STC1 were tested in CaSki cells by chromatin coimmunoprecipitation. The site was found to bind to NFκB p65. (B) Western blotting detected the activity of PARP, caspase-3, STC1, and NF-κB p65 in CaSki cells. CaSki cells were treated with thapsigargin (3 µM) for 12 h. (C) Western blotting detected the activity of p65, STC1, and caspase-3 in CaSki cells. CaSki cells were treated with increasing time of TNFα (10 mg/L) for 2.5 h. (D) Western blotting detected the activity of p65 and STC1 in CaSki cells. CaSki cells were treated with PDTC (10 µM) for 60 min. (E) Subcellular activity of p65 and STC1 in CaSki cells was analyzed by Western blotting. CaSki cells were treated with siRNA knockdown of p65 for 72 h. (F) The expression of p65 and STC1 in CaSki was detected by RT-PCR at mRNA level. CaSki cells were treated with siRNA knockdown of p65 for 48 h. (G) Schematic representation of some findings in this work.