本课题旨在原有工作基础上,为探索ECRG4在浸润性乳腺癌中是否为一新的候选抑癌基因开展系列研究：检测乳腺正常和乳腺癌细胞系之间、乳腺浸润性导管癌与相应癌旁乳腺组织之间ECRG4的mRNA和蛋白表达差异；检测ECRG4 基因启动子区CpG岛中各CpG位点的甲基化状态及该区域的启动子活性；并用去甲基化试剂处理ECRG4阴性表达的细胞系以观察是否能使其表达恢复，以期证实乳腺浸润性导管癌中ECRG4基因启动子区甲基化可使其转录沉默。用表达ECRG4的慢病毒感染乳腺癌细胞系，观察细胞恢复ECRG4表达后在细胞生长能力、凋亡率、细胞周期分布、细胞侵袭性等方面发生的功能变化；同时我们还将筛选的高表达ECRG4的乳腺癌细胞系导入BALB/c小鼠中观察成瘤性及肿瘤特性的变化。这将为证实ECRG4在乳腺癌中为一新的抑癌基因提供有力的证据，并有望成为乳腺癌早期诊断、预后判断的新指标。

To explore if ECRG4 is a new candidate tumor suppressor gene in invasive breast cancer, we will carry on a series of studies as follows. First, Expression of ECRG4 at mRNA and protein level will be detected in breast cell lines and invasive ductal carcinoma by RT-PCR,qRT-PCR，Western blot and immunohistochemistry. Second, to show the potential role of the methylation within CpG islands in promoter region of ECRG4 gene in silencing its transcription, we will assess the methylation status of each CpG site around the ECRG4 CpG islands in breast cancer cell lines and primary breast invasive ductal carcinoma tissues by means of bisulfate sequencing. To further establish the relationship between promoter methylation and downregulation of ECRG4 expression, we will test whether demethylation could reverse the suppression of ECRG4 in breast cancer cell lines. Third, the promoter activity of ECRG4 gene CpG islands region will be assessed using the Dual-Luciferase Reporter Assay System to further confirm the methylation silencing. Fouth, we will infect the breast cancer cell lines with the lentivirus expressing ECRG4, and the phenotype changes of infected cells with ECRG4 restored expression such as cell growth, apoptosis, cell cycle and the ability of migration and invasion will be studied. Finally, we will investigate the function of ECRG4 in mouse model.4T1 is a mouse mammary carcinoma cell lines with a high metastastic propensity. We will infect 4T1 cell lines with lentivirus expressing ECRG4 or control lentivirus without ECRG4 expression. After a period of selection with antibiotics, these two kinds of cells with or without ECRG4 expression will be injected separately into two groups of female BALB/c mice creating in vivo mammary carcinoma models. Ability of tumor-bearing, tumor volume and weight will be evaluated. All of the above studies will help to confirm that ECRG4 is a candidate tumor suppressor gene in breast invasive ductal carcinoma, and ECRG4 is supposed to be a new biomarker in ealry tumor diagnosis and analysis of prognosis.