Relevant Research On Effect Of Centrosome Protein Nlp In Breast Cancer Potential Metastasis

Purpose and background: Breast cancer had become one of the major serious threaten toglobal women’s lives and health. Its incidence presented a very clear upward trend in theannual growth rate2-3%, especially this phenomenon more pronounced in developingcountries. According to the American Cancer Society, in2012the United States diagnosedwith226,870new cases of breast cancer cases,39510patients died of breast cancer patients.Recurrence and metastasis of breast cancer was the leading cause of death in patients. Inrecent years, due to the advanced diagnostic equipment and standardization of systemsdevelopment and popularization of treatment, early diagnosis and recurrence rate, patientswith prolonged survival, decreased risk of death. Therefore, risk factors and interventions toinvestigate breast cancer recurrence and metastasis is one of the important measures to reducebreast cancer mortality.Centrosome protein Nlp （ninein-like protein） was γ-tubulin binding protein （GTBPs）,which was essential ingredients of during mitosis. The main function of Nlp was to promotemicrotubule nucleation, thereby promoting centrosome maturation, spindle formation andchromosome segregation. Almost all of the centrosomal tumors and tumor-derived cells wereabnormal appeared on the morphology, structure and function. Centrosome abnormalitiescould lead to interruption of the cell cycle, chromosome separation and included polynuclearphenotype, which may result in abnormal cell transformation, tumor formation anddevelopment of malignancy. Almost all of the tumors and tumor-derived cell centrosomes inthe presence of abnormal morphology and composition of the amplification phenomenon, and had been reported that centrosome abnormalities existed in lower level tumors, and graduallyincreased in invasive cancers. In recurrence of malignant and invasive ability of tumor cells,the centrosome had significant abnormalities. Thus, centrosome protein Nlp may play animportant role in the development and metastasis of breast cancer invasion process.This study appled the lentiviral transfection, Western blotting detection techniques（Western blotting）, MTT method, cloning, and gene profiling to explore the role ofcentrosome protein Nlp in the growth of breast cancer proliferation and invasion andmetastasis impact.Methods: Application PUBMED to retrieve NLP gene sequences, NM₀25176.Artificial construct pEGFP-C1-NLP plasmid, while the empty plasmid pEGFP-C1served ascontrol, used lentiviral transfection technique to stably transfect pEGFP-C1-NLP plasmid andthe empty plasmid pEGFP-C1into MCF-7breast cancer cell lines, the MCF7-GFP-NLP cellsoverexpressed NLP and MCF-7-GFP cells expressed empty plasmid detected by RT-PCR andWestern blotting analysis to identify mRNA and protein expression levels. Flow cytometry todetect changes in cell cycle after transfection. By MTT assay, colony formation assay, andtranswall invasion chamber method to compare MCF7-GFP-Nlp cells and MCF7-GFP cells inthe proliferation, invasion and metastasis potential. And by Western blotting analysis toestimate the expression level of metastastic associated protein CXCR4（chemokine receptor4）in MCF7-GFP-NLP cells and MCF-7-GFP cells. MCF7-GFP-Nlp cells and MCF7-GFP cellswere detected by gene expression profiling sequencing, and screened differentially expressedgenes.Results: Compared with MCF7-GFP cells, the growth of MCF7-GFP-Nlp cells wasmore accelerate （P<0.05）, the number of colony-forming cells in MCF7-GFP-Nlp andMCF7-GFP cells were respectively:206±14.35,49±3.45, colony formation rate increasedsignificantly （P<0.05）. The number of MCF7-GFP-Nlp and MCF7-GFP migrated to themembrane were respectively:878±18.22,398±8.02, compared with MCF7-GFP cells,themigrated cells of MCF7-GFP-Nlp was significantly increased （P<0.05）. Western blottinganalysis showed that the expression of CXCR4was higher in MCF7-GFP-Nlp cells.Differences in gene expression profiles sequencing results showed that in accordance with theselection criteria were selected out of206differentially expressed genes: compared with MCF7-GFP cells, MCF7-GFP-Nlp cells upregulated genes46, and160down-regulatedgenes.Conclusion: The overexpression of Nlp in breast cancer cells MCF-7was associatedwith increased proliferation, invasion and metastasis, which suggested that Nlp mightpromote the progress of breast cancer, and would be the potential biological markers topredict breast cancer and guide the treatment of breast cancer.