Research On The Role Of NRP2 In Breast Cancer And Its Correlation With Wnt/β-catenin Signaling Pathway

【Objective】To analyze the expression of NRP2 in breast cancer(BC)and its correlation with clinicopathological features and investigate the biological function and molecular mechanism of NRP2 gene in the malignant progression of BC,which could provide new biomarkers and molecular targets for the clinical diagnosis and treatment of BC.【Methods】Part Ⅰ: 1.The expression of NRP2 in BC tissues and adjacent tissues was detected by qRT-PCR assay and immunohistochemical technique.2.Statistical analysis of the correlation between NRP2 and BC clinicopathological features in BC tissues.Part Ⅱ: 1.Using qRT-PCR assay and Western blot assay to detect the expression of NRP2 in 5 different BC cell lines and with the highest expression level were selected as the research object.2.Construction of NRP2 small molecule interfering RNA(siRNA)and transiently transfected into BC cell line.The inhibitory rate of NRP2 expression was detected by qRT-PCR assay and Western blot assay.3.MTT assay and cell colony formation assay were used to detect the BC cell proliferation activity changes after inhibited the expression of NRP2.4.After inhibited the expression of NRP2 in BC cell line,flow cytometry technology was used to detect the apoptosis rate.5.Scratch test and Transwell assay were used to detect the BC cell invasion and metastasis changes after inhibited the expression of NRP2.Part Ⅲ: 1.The expression of β-catenin in Wnt/β-catenin signaling pathway was detected by qRT-PCR assay and Western blot assay after reduced the expression of NRP2 in BC cell line.2.Immunohistochemical technique was used to detect the differential expression of β-catenin in BC tissue and adjacent tissues,and to analyze the correlation between NRP2 and β-catenin expression in BC tissues.3.qRT-PCR assay and Western blot assay were used to detect the expression of CyclinD1,c-myc,MMP2,E-Cadherin and vimentin of β-catenin important downstream effector molecules after reduced the expression of NRP2 in BC cell.4.Immunohistochemical technique was used to detect the expression level of each effector molecule in the downstream of β-catenin,and the correlation between the expression of NRP2 and the expression of each molecule was statistically analyzed.【Results】Part Ⅰ: 1.There was a significant difference in the expression of NRP2 in BC tissues and adjacent tissues.Compared with paracancerous tissue,the expression level of NRP2 in BC tissue was significantly higher 2.There was a significant difference between NRP2 and lymph node metastasis,TNM stage(p<0.05),but not with age,diameter of tumor,differentiation degree,ER,PR,and HER-2 status(p>0.05).Part Ⅱ: 1.NRP2 was expressed in five kinds of BC cell lines,and the expression level was the highest in MDA-MB-231 cell with the highest metastatic potential.2.The expression level of NRP2 was significantly inhibited after transfection of NRP2 siRNA in MDA-MB-231 cell line.3.NRP2 siRNA could significantly decrease the proliferation activity of MDA-MB-231 cell.4.There was no significant change in the rate of apoptosis after inhibited the expression of NRP2 in MDA-MB-231 cell line.5.Scratch test results showed that it significantly suppressed the migration of MDA-MB-231 cell after inhibited the expression of NRP2 in MDA-MB-231 cell line,and the sclerosis healed slowly.The results of Transwell assay showed that NRP2 siRNA could markedly reduce the number of transmembrane cells,the invasion and metastasis of MDA-MB-231 cell line.Part Ⅲ: 1.It significantly reduced the expression of β-catenin in the Wnt/β-catenin signaling pathway after inhibited the expression of NRP2 in MDA-MB-231 cell line.2.β-catenin in the BC tissues with 71.698% of the high-intensity expression and correlated with NRP2(p<0.05).3.The expression of CyclinD1,c-myc and MMP2 transfected with NRP2 siRNA decreased to a certain extent,and E-Cadherin increased,while the expression of vimentin did not change.4.The expression of c-myc,MMP2 and E-Cadherin in BC tissues was statistically different from that of NRP2(p<0.05).【Conclusions】1.NRP2 was highly expressed in BC tissues and correlated with lymph node metastasis and TNM staging.2.The proliferation,invasion and metastasis of BC cells were reduced by siRNA targeting interfering with NRP2.3.The expression of NRP2 in BC tissues was correlated with the expression of β-catenin and its downstream c-myc,MMP2 and E-cadherin.NRP2 abnormal activation of Wnt/β-catenin signaling pathway and up-regulates the expression of β-catenin,a key signal molecule in the Wnt/β-catenin signaling pathway,which causes the aberrant expression of c-myc,MMP2 and E-cadherin in the downstream of β-catenin,and eventually contributing to the malignant progress of BC.