Stanniocalcin 1 (STC1) is a glycoprotein hormone that is involved in calcium/phosphate homeostasis. Increasing evidence suggests that STC1 is involved in carcinogenesis; however, few studies have defined the mechanisms and functional roles of STC1 activity in prostate carcinogenesis. In the present study, MTT, flow cytometry and colony formation assays, and small interfering RNA (siRNA) and overexpression in multiple cell lines were used to investigate the function of STC1 in prostate carcinoma
Prostate carcinoma is one of the most prevalent malignant tumors in males. In 2012, 241,740 cases of prostate carcinoma were diagnosed in the US, and this was the most common type of newly diagnosed tumor among males, accounting for 29% of new diagnoses. It also accounted for ~28,170 mortalities, ranking as the second most common cause of cancer-related mortality in males (
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STC1 is a peptide hormone that was initially identified in teleost fish and is widely expressed in mammalian tissues (
LNCaP2 and DU145 prostate carcinoma and normal prostate RWPE-1 cells were cultured in Gibco RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) (both from Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cells were incubated at 37̊C in an atmosphere of 5% CO2.
mRNA was isolated from two prostate carcinoma and one normal prostate cell lines, and reverse transcribed and amplified using a One-Step RT-PCR System (Fermentas, Vilnius, Lithuania). The following primer sequences were used for PCR: GAPDH antisense, 5′-CCTGCTTCACCACCTTCTTG-3′ and sense, 5′-AATCCCATCACCATCTTCCA-3′; STC1 sense, 5′-TTCTGGTGCTGGTGATCAGTG-3′ and antisense, 5′-TTTGGGCACAGTGGTCTGTCT-3′. Samples were initially heated to 95̊C for 1 min, then subjected to 30 cycles (GAPDH, 28 cycles) of 95̊C for 30 sec, 56̊C for 30 sec and 72̊C for 90 sec; a final 10-min extension step at 72̊C was also included. All reaction products were purified on 1% agarose gels containing ethidium bromide. The relative expression levels of mRNA were analyzed by a Phosphor-Imager.
The sample cells were washed with cold phosphate-buffered saline (PBS), and then lysed in Laemmli buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol and 0.01% bromphenol blue] for 5 min at 98̊C. Cell lysate samples were separated by SDS-PAGE, and the proteins were electrophoretically transferred to polyvinylidene difluoride membranes. The blots were subsequently blocked for 1 h with non-fat milk and probed with the specific primary antibodies followed by a secondary detection step. The immunoreactive proteins were revealed by an enhanced chemiluminescence kit. The following antibodies were used in the western blotting: rabbit anti-STC1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-cyclin D1 (Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-cyclin E1 (Abcam, Cambridge, MA, USA), rabbit anti-cyclin-dependent kinase 4 (CDK4), and rabbit anti-CDK2 (both from Santa Cruz Biotechnology, Inc.).
To knock down STC1 expression, a pRNAT-U6.1/Neo vector encoding a small interfering RNA (siRNA) directed against the target gene, STC1, in prostate cells was utilized (si-STC1). The target sequence for STC1 was, 5′-TTAGTCCAGGAAGCAATAGTA-3′. An empty pRNAT-U6.1/Neo vector was used as a negative control (NC). For transfection, prostate carcinoma cells were cultured to 70% confluency, transfected with a recombinant plasmid, and harvested after 48 h for further experiments.
For the MTT assay, the cells were seeded in 96-well plates at a density of 103 cells/well (n=6) and cultured for 12, 24, 48 or 72 h. Subsequently, the cells were incubated with 10 µl MTT (50 µg/well; Sigma-Aldrich, St. Louis, MO, USA) for 4 h. The generated formazan was assessed at 490 nm to detect the cell viability.
Additionally, a colony formation assay was conducted as previously described (
Cells were cultured in RPMI-1640 medium containing 1% FBS for the first 24 h and 10% FBS for the subsequent 24 h (n=3). The cells were then harvested and resuspended in fixation fluid at a density of 106 cells/ml. Propidium iodide (PI) solution (1,500 µl) was then added, and the cell cycle was analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA).
To evaluate tumor growth
All experiments were repeated and data are expressed as the mean ± standard deviations. Differences among >2 groups were assessed by ANOVA, and differences between 2 groups were analyzed using a Student's t-test. Analyses were performed with GraphPad Prism software version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was indicated by P<0.05.
The mRNA and protein expression levels of STC1 were assessed by RT-PCR and western blotting in the normal prostate (RWPE-1) and prostate carcinoma (DU145 and LNCaP2) cell lines. The results revealed that STC1 mRNA and protein levels were markedly higher in the DU145 and LNCaP2 cells compared with the RWPE-1 cells (
To examine the biological function of STC1, STC1-knockdown DU145 and LNCaP2 cells were established. As shown in
The effect of STC1 knockdown on the proliferation of prostate cancer cells (DU145 and LNCaP2) was determined using MTT analysis. During a 6-day period, the proliferation results suggested that DU145/si-STC1 and LNCaP2/si-STC1 cells proliferated more slowly compared with DU145/NC and LNCaP2/NC cells (
Flow cytometric analysis was also used to assess cell proliferation. The results demonstrated that transfection with si-STC1 led to increased G1 phase cell cycle arrest in DU145 and LNCaP2 cells (
Given the aforementioned results, we hypothesized that STC1 overexpression may promote RWPE-1 cell proliferation. To confirm this, cells were transfected with a plasmid encoding STC1 (RWPE-1-STC1), and these cells exhibited increased levels of STC1 mRNA and protein compared with the control group (RWPE-1-C;
Furthermore, flow cytometric analysis demonstrated that in RWPE-1-STC1 cells a smaller proportion of cells were arrested in the G1 phase, while the percentage of cells in the S phase was increased compared with that in the RWPE-1-C cells (
To further determine the effects of STC1 on tumor growth and development
Previous studies revealed that cyclin D/CDK4 and cyclin E/CDK2 have vital roles in cell cycle progression and are often overexpressed in cancer cells (
Extensive evidence suggests that the STC1 participates in various types of carcinoma, including colorectal cancer (
Recent studies (
In the present study, a flow cytometric analysis was performed to assess cell cycle distribution. Cell proliferation is controlled by cell cycle progression, which is regulated by numerous cell proliferation signaling pathways (
In conclusion, to the best of our knowledge, the present study is the first to suggest STC1 as a potential biomarker associated with the development and metastasis of prostate carcinoma. The results indicate a novel mechanistic role for STC1 in the regulation of prostate carcinoma cell proliferation via cyclin E1/CDK2. This novel biomarker may aid in clinical treatment and prediction of prognosis in prostate carcinoma. Further research is necessary to explore the regulatory mechanism of STC1.
The present study was supported by the Natural Science Foundation of Hunan Province (14JJ7004), and the Independent Innovation Foundation of Central South University (2016zzts516) (Changsha, China).
Expression of STC1 mRNA and protein in DU145 and LNCaP2 prostate carcinoma and RWPE-1 normal prostate cell lines. (A) STC1 and GAPDH mRNA expression levels were examined by reverse transcription-polymerase chain reaction. (B) STC1 and β-actin protein expression levels were examined by western blotting. STC1, stanniocalcin 1.
Knockdown of STC1 inhibits the proliferation of prostate cancer cells. The effect of STC1 knockdown in (A) DU145 and (B) LNCaP2 prostate cancer cell lines was assessed by reverse transcription-polymerase chain reaction (left panels) and western blotting (right panels). MTT assays demonstrated the effect of STC1 knockdown (si-STC1) on the proliferation of (C) DU145 and (D) LNCaP2 compared with their respective NC group cells. Colony formation analysis revealed the effects of si-STC1 and NC transfection on (E and F) DU145 cells (**P<0.05 vs. DU145/NC) and (G and H) LNCaP2 cells (**P<0.01 vs. LNCaP2/NC). STC1, stanniocalcin 1; NC, negative control.
Flow cytometric analysis of cell cycle distribution in prostate cancer cells with or without STC1 knockdown. (A) Cell cycle distribution of DU145/NC and DU145/si-STC1 cells, and (B) the percentages of DU145/NC and DU145/si-STC1 cells in the G0/G1, G2/M and S phases. (C) Cell cycle distribution of LNCaP2/NC and LNCaP2/si-STC1 cells and (D) the percentages of LNCaP2/NC and LNCaP2/si-STC1 cells in the G0/G1, G2/M and S phases. STC1, stanniocalcin 1; NC, negative control; si-STC1, STC1 knockdown.
Overexpression of STC1 promotes cell proliferation in RWPE-1 normal prostate cells. (A) The overexpression of STC1 in transfected RWPE-1 cells was confirmed by reverse transcription-polymerase chain reaction (left panels) and western blotting (right panels). (B) An MTT assay demonstrated the effect of STC1 on the proliferation of RWPE-1-STC1 and control group cells. (C and D) Colony formation analysis was performed on RWPE-1-STC1 and RWPE-1-C cells (*P<0.01 vs. RWPE-1-C). (E) Cell cycle distributions of RWPE-1-STC1 and RWPE-1-C and (F) the percentages of RWPE-1-STC1 and RWPE-1-C cells in the G0/G1, G2/M and S phases. RWPE-1-C, control RWPE-1 cells; RWPE-1-STC1, RWPE-1 cells overexpressing STC1.
Tumor formation in nude mice. (A and B) The tumor volumes of nude mice were assessed every 5 days for a total of 40 days. *P<0.01 vs. LNCaP2/si-STC1, **P<0.01 vs. DU145/si-STC1. (C-F) After 40 days, the weights of the tumors were recorded. (G) The expression levels of STC1 in the xenograft tumors generated from STC1-knockdown and control cells (without STC-1 knockdown) were verified by reverse transcription-polymerase chain reaction and western blot analyses. STC1, stanniocalcin 1; si-STC1, STC1 knockdown; NC, negative control.
Expression levels of cyclin E1/CDK2 were verified by western blotting. (A and B) Cyclin E1 and CDK2 protein expression levels were detected in the prostate carcinoma cells with or without STC1 knockdown. Cyclin E1 and CDK2 protein expression levels were detected in (C) normal prostate cells with or without STC1 overexpression, and (D) xenograft tumors with or without STC1 overexpression. CDK2, cyclin-dependent kinase 2; STC1, stanniocalcin 1; NC, negative control; si-STC1, STC1 knockdown; RWPE-1-C, control RWPE-1 cells; RWPE-1/STC1, RWPE-1 cells overexpressing STC1.