The Role Of Neogenin In Breast Cancer And Its Related Molecular Mechanism

Breast cancer is one of the most common malignant tumors in women, which affects women’s health seriously and may even be life-threatening. Although recent substantial progress has been achieved in early diagnosis and treatment of breast cancer, it is still now the leading cause of cancer-related death in females worldwide. Early detection of breast cancer is of great importance for improving patients’ survival rate. So studies of relevant genes and markers which are involved in breast cancer are of great value, which could be conducive to the improvement of early diagnosis and personalized treatment.Neogenin, a member of DCC family and immunoglobulin superfamily, encodes a 1461 amino acid identity. Neogenin protein belongs to transmembrane glycoprotein which is widely and conservedly expressed in most tissues of vertebrates. Neogenin could bind to its ligands and deliver signals into cells to modulate development of embryo and adult tissues. While its ligands being absent, Neogenin could also function as a dependent receptor inducing cell apoptosis. Neogenin m RNA and protein are widely expressed in both embryo and adult tissues during development. Evidence is now accumulating that Neogenin is a multi-functional receptor regulating many diverse developmental processes, including signal transduction, cell migration, cell apoptosis, tissue development and differentiation, angiogenesis, neural tube formation. Current evidence indicates members of DCC family could act as tumor inhibiting factors. Also abnormal expression of neogenin has been observed in a variety of human cancers, such as gliomas, gastric cancer, colon cancer,esophageal squamous cell carcinoma(ESCC),ovarian cancer, prostate cancer and lung cancer. It has been implicated that Neogenin may play an important role in tumorigenesis and progression of these cancers. Another study has observed that Neogenin expression may be inversely correlated to the tumorigenicity in human breast cancer cells which were immortalized derivatives from the same type I human breast epithelial cell lines with different tumorigenicity. It is suggested that Neogenin may act as tumor inhibiting factor and prognostic factor in breast cancer. However, there is few relevant study and report on Neogenin expression level and its function in breast cancer. The clinical significance of Neogenin in breast cancer diagnosis, treatment and prognostic evaluation is not clear.In this study, we firstly detected the expression level of Neogenin in one breast epithelial and three breast cancer cell lines. Secondly, we up-regulated Neogenin expression in MDA-MB-231 cells by plasmid transfection, and investigated the change in cell proliferation, migration and apoptosis. Thirdly, we explored the mechanisms of Neogenin in inhibiting malignant activities of MDA-MB-231 cell. Then we measured the expression level of Neogenin in paired normal and cancer tissues from 54 breast cancer patients by immunohistochemical staining and Real-time PCR. We also analyzed the correlationship between Neogenin expression and other breast cancer indicators to estimate the potential prognostic value of Neogenin. Methods1. The expression profile of Neogenin m RNA and protein was firstly characterized in one human mammary epithelial cell(MCF-10A) and three human breast cancer cells(MDA-MB-231, MCF-7 and T47D) by using Real-time PCR and Western blot.2. MDA-MB-231 cells were transfected with pc DNA3.1-e GFP-Neogenin or the control pc DNA3.1-e GFP plasmid using Lipofectamine 2000 respectively. The transfection efficiency was observed by fluorescence microscopy. The expression level of Neogenin in MDA-MB-231 cells after transfection was examined by Real-time PCR and Western blot.3. After up-reguating Neogenin expression in MDA-MB-231 cells, proliferation and apoptosis tests of transfected cells were detected by using CCK-8 assay, propidine iodide assay and TUNEL staining. m RNA expression levels of genes related to proliferation and apoptosis were also detected by Real-time PCR to analyze this mechanism.4. After up-reguating Neogenin expression in MDA-MB-231 cells, migration and invasiveness tests of transfected cells were detected by using scratch wound model and Boyden Chamber assay. m RNA expression levels of genes related to migration and invasiveness were also detected by Real-time PCR to analyze this mechanism.5. The expression levels of Neogenin protein and m RNA in paired normal and cancer tissues from 54 breast cancer patients were detected by immunohistochemical staining and Real-time PCR. We also compared the correlationship between Neogenin expression and clinical features, pathological and prognostic indicator. Results1. As a result of Real-time PCR and Westernblot analysis, the data showed that the expression levels of Neogenin m RNA and protein in three breast cancer cell lines(MCF-7 、 BT-549 and MDA-MB-231) were lower than that in normal mammary epithelial cell line(MCF-10A). The expression level of Neogenin showed nagatively correlated to metastasis potency in three breast cancer cell lines.2. The results of fluorescence microscope observation, Real-time PCR and Westernblot analysis verified the successful and high-efficient transfection of MDA-MB-231 cells with pc DNA3.1-e GFP-Neogenin. The expression levels of Neogenin m RNA and protein in MDA-MB-231 cells were up-regulated after transfection.3. Neogenin overexpression resulted in a reduction of cell proliferation, an increase of cell apoptosis rate and the G1�'S cell cycle arrest of MDA- MB- 231 cells. Real-time PCR showed that the m RNA levels of Bcl-2 and c-Myc were decresed after Neogenin overexpression, while the expression levels of CTGF, Med19, RPL13, Jun B and Suvivin had no obvious change.4. Neogenin overexpression resulted in a reduction of cell migration and invasion. Real-time PCR showed that the m RNA levels of Vimentin, MMP-2 and MMP-9 were decresed after Neogenin overexpression, while the expression levels of TIMP3 was increased.5. The expression and correlationship of Neogenin with clinical features, pathological and prognostic indicator was described as follows.5.1 IHC staining results showed that: 1 Neogenin was moderately expressed in cytoplasm of 54 para-carcinoma tissues; 2 24.1%(13/54)of breast cancer tissues had no expression of Neogenin; 64.8%(35/54) had weak expression of Neogenin; Only 11.1%(6/54) had moderate expression of Neogenin(p<0.05).5.2 Real-time PCR results showd that the level of Neogenin m RNA was significantly lower in breast cancer tissues than in the matched para-carcinoma tissues(51/54, 94.4%). The relative quantitative average of Neogenin expressed in breast cancer tissues was 0.264±0.0330 while value of each matched para-carcinoma tissue was normalized to 1.5.3 The relative quantitative average of Neogenin expressed in beast cancers of pathological histology Grade II was 0.330±0.034, while that of Grade III was 0.192±0.031. The expression level of Neogenin in breast cancer tissues is inversely associated with pathological histology grade(p<0.05).5.4 By Real-time PCR the expression level of Neogenin m RNA in breast cancer was lower in groups of ER negative(0.221±0.029 vs. 0.309±0.036), PR negative(0.256 ±0.034 vs. 0.279±0.031), HER-2 positive(0.239±0.029 vs. 0.281±0.035), Ki67 positive(0.256±0.033 vs. 0.287±0.035) and P53 negative(0.237± 0.030 vs. 0.308±0.037).5.5 By Real-time PCR the expression level of Neogenin m RNA in breast cancer was lower in groups of Senile breast cancer(0.042±0.003 vs. 0.270±0.032), diameter >20 mm(0.237±0.028 vs. 0.298±0.037), no lymph node metastasis(0.164±0.025), TNM Grade I(0.206±0.3), vascular nerve infiltration(0.247±0.026 vs. 0.272±0.03) and H subtype breast cancer(Lumianl A: 0.407±0.400, Luminal B: 0.217±0.314: TNBC:0.244±0.356). Conclusion1. The expression level of Neogenin was reduced in breast cancer cell lines, which was negatively related to the metastatic potential of breast cancer cells. The results suggested that the Neogenin could be a predictor for metastasis of breast cancer.2. Overexpression of Neogenin in breast cancer cells inhibited the proliferation, migration and invasion ability and induced G1�'S cell cycle arrest in cells. The results indicated that the Neogenin up-regulation may have inhibitory effect on breast cancer, and Neogenin may play a role of suppressor in breast cancer.3. The negative effect on the cell proliferation and the positive effect on the cell apoptosis of Neogenin overexpression may be caused by inhibiting of expression of Bcl-2 and c-Myc genes. And the influence on the breast cancer cell migration and invasion may due to down-regulated expression MMP-2, MMP-2, and Vimentin genes and up-regulated expression of TIMP-3 gene caused. However, the specific mechanism remains to be further clarified.4. The expression level of Neogenin m RNA was significantly lower in breast cancer tissues than in para-carcinoma tissues, and the level is inversely associated with pathological histology grade. Neogenin was lower in groups of ER negative, HER-2 positive, Ki67 positive, P53 negative, senile breast cancer, diameter >20 mm, no lymph node metastasis, TNM Grade I, vascular nerve infiltration and H subtype breast cancer. These results indicated that Neogenin may be involved in tumorigenesis and invasion process of breast cancer and it could be used as a potential prognostic predictor. Key words...