The Association Of DAL-1 Protein Expressi- On And Point Mutation (1810 C>T/575Cys> Tyr) With The Metastasis Of None Small Cell Lung Cancers

Lung cancer is one of the most common malignancies , among which 80%～85% are NSCLC. The direct cause of treatment failure and the vast majority of patient death are the invasion and metastasis of tumor. Thus, it becomes an urgent issue to seek out metastasis- related genes and its molecular targets of lung cancer at present.DAL-1 is a member of 4.1family, which expressed reducely or negatively in many type tumors such as NSCLC, breast cancer and meningiomas [1-4]. Reexpressed DAL-1 in NSCLC and breast cancer cells can inhibit them reproduction [1,5], suggest that protein DAL-1 may be a tumor suppressor.DAL-1 located at cell membrane by immunochemistry assay, and mainly focuses on the junction between cells, distributed as"honeycomb"[1]. Charboneau AL. et al. [6] find that DAL-1 expression enhances the attachment between breast cancer cells. Our pre-experiment in vitro show that silent the expression of DAL-1 can enhance migration and mobility of NSCLC cell [7]. We speculate that the loss of DAL-1 expression may weaken the connection and adhension between cells so that tumor cells detach and migrate easly. Therefore, we detected the protein expression of DAL-1 of human tissue, and further test the the association of it with the metastasis of NSCLC.Mechanisms of gene inactivation include genetic mechanisms such as gene deletion and mutation and epigenetic mechanisms such as DNA methylation and change in chromatin conformation. Our Preliminary studies suggest that, loss of DAL-1 expression is mainly caused by the methylation of promoter [7]. But part of tumors in which DAL-1 expressed negative hadn't the methylation of DAL-1 promoter. We suppose that there be some other causes that lead to the loss of DAL-1 expression. Point mutation is one of the common causes of gene loss. The current studies suggest that there are many point mutations in DAL-1 gene, including 1810 C>T/575Cys>Tyr, 2166C>T/704Thr>Thr, 2146G>T/716Ala>Ser, 2166 C>T/722Thr >Thr and so on. Among them 1810 C>T/575Cys>Tyr is missense mutation and have high frequency [8-10]. Therefore, we detected the point mutation of 1810 C>T/575Cys>Tyr of human tissues, and analyze the the association of it with DAL-1 protein expression and discus the causes that result in the loss of DAL-1 expression.Methods1. Detect DAL-1 protein expression of 167 cases NSCLC tissues (among them 149 cases have matching normal tissue adjacent to cancer) which were paraffin-embedded with immunohistochemistry (IHC). Analyze the association of it with the clinicopathological factors of NSCLC. Chi-square test was used to alanalysis data.2. Extract genome DNA of paraffin-embedded NSCLC tissues and matching normal lymph nodes of 204 cases, and fresh blood samples of 112 patients with NSCLC respectively. Detect the gene type of DAL-1 1810 C>T/575Cys>Tyr by PCR-RFLP. Analyze the association of DAL-1 1810 C>T point mutation with the protein expression of DAL-1 and the the metastasis of NSCLC. The genotypes frequeneies of DAL-1 1810 C>T/575Cys>Tyr were calculated by direct counting method. Chi-square test was used to alanalysis data.Results1. Among 167 cases human NSCLC tissues, DAL-1 protein expressed positively in 97 cases(58.1%). 70 cases (41.9%) expressed nigatively. Among 167 cases human NSCLC tissues, 149 cases have matching tissues which was corresponding normal tissues adjacent to cancer. DAL-1 protein expressed positively in 135 (90.6%) cases normal tissues adjacent to cancer. DAL-1 protein expressed negatively in 14 cases (9.4%) normal tissues adjacent to cancer. DAL-1 protein expressed positively in 87 cases (58.4%). DAL-1 protein expression of 62 cases (41.6%) showed negative. The positive expression rate of DAL-1 protein in NSCLC tissues was significantly lower than that in corresponding normal tissues adjacent to cancer (p<0.05).2. Among 167 cases human NSCLC tissues, the positive expression rate of DAL-1 protein in the group which tumor maximum diameter was≤3cm(47/66) was significantly higher than that in the group which tumor maximum diameter was>3cm (50/101) (p<0.010); which in the group NSCLC hasn't metastazed 82.05% (64/78) was significantly higher than that in the group NSCLC has metastazed 37.50% (33/88)(P<0.000), and which in the groupⅠstage,Ⅱstage (67/91)was significantly higher than that in the groupⅢstage,Ⅳstage(30/76) (P<0.000). The positive expression rate of DAL-1 protein in well differentiated group (63/93)was significantly higher than that in poorly differentiated group (27/55) (p<0.005).3. Detecting DAL-1 1810 C>T gene type of 112 cases blood specimens, 204 cases matching specimens of paraffin-embedded NSCLC tissues and normal node tissues. Distribution of DAL-1 1810 C>T gene types among 316 cases as follows: 293 cases were TT (92.7%), 20 cases were CT (6.3%), 3 cases were CC (0.9%). The mutation rate of DAL-1 1810 C>T is 4.1%. Each case of NSCLC tissue and the normal lymph node tissue have the same gene type, which suggest that cancer tissue has the same gene type of DAL-1 1810 C>T with normal tissues of corresponding patient.4. Among 167 cases NSCLC tissues which have been detected DAL-1 protein by IHC, 13 cases DAL-1 1810 C>T have mutated. Among mutated 13 cases, 5 cases of NSCLC tissues DAL-1 protein expressed positively and 8 cases of NSCLC tissues DAL-1 protein expressed negatively; 7cases of normal tissues adjacent to cancer DAL-1 protein expressed positively and 5 cases of normal tissues adjacent to cancer DAL-1 protein expressed negatively, 1 case has no matching normal tissues adjacent to cancer. All of 5 cases of NSCLC tissues which DAL-1 protein expressed positively have metastazed.5. Among 316 cases NSCLC, 23 cases DAL-1 1810 C>T are mutated type, 293 cases are wild type. Amang mutated type 23 cases, 17 cases have metastazed, but 6 cases have not metastazed. Amang wild type 293 cases, 149 cases have metastazed, but 144 cases have not metastazed (p<0.025).6. When we analyze the the association of Point mutation of DAL-11810 C>T in 167 cases NSCLC tissue with with the clinicopathological factors of NSCLC, we find that there is no significantly different in the distribution of DAL-1 1810 C>T gene types between the group which tumor maximum diameter was≤3cm and the group which tumor maximum diameter was>3cm, and between the groupⅠstage,Ⅱstage andⅢstage,Ⅳstage , but there is significantly different in the distribution of DAL-1 1810 C>T gene types between the group NSCLC hasn't metastazed and the group NSCLC has metastazed (p<0.025).Conclusions1. There is loss or reduction of DAL-1 protein expression in human NSCLC tissues.2. The positive expression rate of DAL-1 protein in the group which NSCLC has metastazed was significantly lower than that in the group NSCLC hasn't metastazed, which suggests that low expression of DAL-1 protein may associate with metastasis of NSCLC.3. The mutation rate of DAL-1 1810 C>T is 4.1% in human NSCLC tissue. 1810 C>T is not the main cause which result in the reduction or losss of DAL-1 protein expression; DAL-1 1810 C>T may associate with metastasis of NSCLC, which is needed to be confirmed further by directed mutagenesis.