Effects of the ninein-like protein centrosomal protein on breast cancer cell invasion and migration

To investigate the effects of the centrosomal protein, ninein-like protein (Nlp), on the proliferation, invasion and metastasis of MCF-7 breast cancer cells, the present study established green fluorescent protein (GFP)-containing MCF7 plasmids with steady and overexpression of Nlp (MCG7-GFP-N1p) and blank plasmids (MCF7-GFP) using lentiviral transfection technology in MCF7 the breast cancer cell line. The expression of Nlp was determined by reverse transcription-quantitative polymerase chain reaction and western blott analysis. Differences in levels of proliferation, invasion and metastasis between the MCF7-GFP-Nlp group and MCF-GFP group were compared using MTT, plate colony formation and Transwell migration assays. The cell growth was more rapid and the colony forming rate was markedly increased in the MCF7-GFP-Nlp group (P<0.05) compared with the MCF7-GFP group. The number of cells in the MCF-GFP-Nlp and MCF7-GFP groups transferred across membranes were 878±18.22 and 398±8.02, respectively, in the migration assay. The invasive capacity was significantly increased in the MCF7-GFP-Nlp group (P<0.05) compared with the MCF7-GFP group. The western blotting results demonstrated high expression levels of C-X-C chemokine receptor type 4 in the MCF7-GFP-Nlp group. The increased expression of Nlp was associated with an increase in MCF7 cell proliferation, invasion and metastasis, which indicated that Nlp promoted breast tumorigenesis and may be used as a potent biological index to predict breast cancer metastasis and develop therapeutic regimes.


Introduction
Breast cancer is a malignant tumor, which severely affects female health, is life threatening and the incidence of which has increased gradually over recent years (1,2). With the prevalence of advanced diagnostic instruments and the development of standardized systematic therapy, the rate of early diagnosis in patients with recurrent-metastasis patients has increased and survival rates have improved, with mortality rates declining by 1-2% per year in China (1,2). The present study aimed to investigate the risk factors of breast cancer, recurrent-metastasis and intervention methods, which are important to decrease the breast cancer mortality rate. The human centrosomal ninein-like protein (Nlp) is a novel member of the γ-tubulin complex binding proteins (GTBPs) and is essential in the process of mitosis. The primary function of Nlp is to promote microtubule nucleation, which contributes to centrosomal maturation, spindle formation and chromosome segregation (3,4). The centrosome from almost all types of tumor exhibit abnormal structure, morphology and function. Previous studies have demonstrated that centrosome activity is important in cell division and in the transition from G1 phase to S phase (5,6). Abnormal centrosomes may lead to interruption of the cell cycle, including the polycaryon phenotype, which causes abnormal cell transformation, tumorigenesis and the development of malignancy (7)(8)(9). In the present study, the biological action of Nlp on metastatic capacity of breast cancer was investigated using advanced transfection technology.     data are expressed as the mean ± standard error of the mean. A least significant difference-t test was used for two sample comparisons from two groups. P<0.05 was considered to indicate a statistically significant difference.

Results
Overexpression of Nlp is established in the MCF-7 breast cancer cell line. The pEGFP-C1-Nlp plasmid or the pEGFP-C1 plasmid were transfected into MCF-7 cells to establish MCF7-GFP-NLP and MCF7-GFP cells. The mRNA and protein expression levels of Nlp were detected by RT-qPCR and western blotting (Figs. 1 and 2).

Effect of high expression of Nlp on the growth of MCF-7 cells detected using an MTT assay.
Under the same growth conditions, the growth rate was more rapid in the MCF7-GFP-NLP cells (P<0.05) compared with the MCF7-GFP cells, which indicated that Nlp promoted the growth of MCF-7 cells (Fig. 3).

Colony formation assay to detect cell proliferation ability.
The results demonstrated that colony numbers in MCF-GFP cells and MCF7-GFP-NLP cells were 49±3.45 and 206±14.35, respectively, under identical conditions. The colony formation rate was markedly increased in the MCF7-GFP-NLP cells (P<0.05) compared with the MCF7-GFP cells, which indicated that Nlp promoted MCF7 cell proliferation (Figs. 4 and 5).    Effect of high expression of Nlp on the migration ability in vitro. Under the same conditions, quantification of the cells migrated under the membrane in MCF7-GFP-Nlp cells and MCF7-GFP cells were 878±18.22 and 398±8.02, respectively. The migration ability was increased in the MCF7-GFP-Nlp cells compared with the MCF7-GFP cells and a significant difference was observed between two groups (P<0.05; Figs. 6 and 7).

Expression of CXCR4 was detected by western blotting in the MCF7-GFP-Nlp cells and MCF7-GFP cells.
The results of the western blotting revealed that the protein expression levels of CXCR4 were higher in the MCF7-GFP-Nlp cells compared with the MCF7-GFP cells (Fig. 8).

Discussion
Nlp is overexpressed in breast, lung and ovarian cancer, and head and neck squamous cell carcinoma. Notably, centrosomal Nlp causes spontaneous tumorigenesis in transgenic mice overexpressing Nlp (10)(11)(12). Previous studies have reported that centrosomal abnormalities occur in certain low-grade tumors and exhibit an demonstrate an increased trend in invasive tumors. In ovarian cancer tissues, the higher the pathological classification, the higher the number of centrosome abnormalities that are present. Notably, the presence of centrosome abnormalities are higher in malignant ovarian cancer (13)(14)(15)(16).
A previous study demonstrated that the overexpression of Nlp is observed in head and neck squamous cell carcinoma, which is associated with the clinicopathological characteristics (17). In addition, it has also been confirmed that the expression of Nlp significantly correlates with the tumor grade, and that the overexpression of Nlp is marginally associated with a decrease in overall survival rates (18). Furthermore, Nlp induces tumor development by interfering with the cell cycle, mitosis and cell apoptosis (19,20). However, the effect of Nlp on breast tumor metastasis remains to be elucidated. The MCF-7 breast cancer cell line retains several characteristics of differentiated mammary epithelium, including the ability to process estradiol via cytoplasmic estrogen receptors and the capability of forming domes. The present study established MCF7-GFP-Nlp and MCF7-GFP cells and observed, through growth curves that the MCF7-GFP-Nlp cells grew more rapidly compared with theMCF7-GFP cells. In addition, plate colony forming assays demonstrated that the MCF7-GFP-Nlp cells exhibited a increased colony formation capacity compared with the MCF7-GFP cells. These results indicated that Nlp promoted MCF-7 cell proliferation. Transwell chambers are considered as a permeability support, and usually, a Transwell chamber is put into culture plates and medium is added to the top and bottom chambers, with the cells were seeded into the top chamber. Since the membrane is permeable, cells can migrate to the lower chamber (21,22). The results of the present study revealed that the overexpression of Nlp promoted cell migration in the Transwell model in vitro.
Changes in tumor cell migration capacity is an important step affecting tumor invasion and metastasis. CXCR4 is encoded by 352 amino acids and is a seven-transmembrane G-protein chemokine receptor. In 1996, Feng et al identified that CXCR4 is a coreceptor for human immunodeficiency virus-1 entry, following which several studies have investigated CXCR4 (23). It has been demonstrated that CXCR4 is involved in the invasion and metastasis of several types of cancer, including breast carcinoma (24). Hiller and Chu (25,26) demonstrated that CXCR4 is important in several types of cancer, including breast cancer, and revealed that CXCR4 was highly expressed in areas common for breast cancer metastasis, including the axillary lymph nodes. Hernandez et al confirmed that CXCL12-CXCR4 is important in the process of breast tumor cell growth, angiogenesis, invasion and metastasis (27,28). A meta-analysis investigation based on thirteen eligible studies, consisting of 3,865 patients with breast cancer, demonstrated that the overexpression of CXCR4 was significantly associated with lymph node status and distant metastasis. In addition, the overexpression of CXCR4 indicated a poor overall and disease-free survival rates (29). The present study demonstrated that the expression of CXCR4 was higher in the MCF7-GFP-NLP cells compared with the MCF7-GFP control cells, which implied that Nlp improved the migration capacity of breast cancer cell lines through activated CXCL12 and CXCR4.
In conclusion, the results of the present study indicated that an increase in the expression of Nlp resulted in a malignant phenotype, which induced tumor cell proliferation and invasion. Furthermore, the results confirmed that Nlp exhibited certain biological characteristics, including promoting breast tumorigenesis and development, to provide a novel molecular index for breast cancer diagnosis. Therefore, Nlp may be an effective target of antitumor drugs for therapy against specific types of tumor.