The Constructin Of ShRNA Plasmid For YAP Gene And The Effects Of Gene Silencing On Lung Adenocarcinoma A549 Cell

BackgroundLung cancer is one of the most common malignant tumor, the incidence and mortality of lung cancer increased gradually in recent years. According to the reports, it has been ranked the first of those malignant tumor in most developed countries, which is relavant to the development of industry, the pollution of the environment and the aging population. In nowdays, early detection and treatment of lung cancer have become global concern. According to the report of WHO Cancer Institute released in 2010: the incidence cases was about 1.71 million in 2009, 1.4 million patients died of this cancer. In our country, the incidence and mortality rate of lung cancer ranks the first and second respectively in the malignant tumors. The majority of patients are older than forty. Meanwhile, the incidence of female patients has been increasing in recent years, the incidence ratio of male to female has been increased from 4:1 to 1.5:1. The reason for the incidence of lung cancer are complicated, what is the most important reasons are smoking, environment pollution and the occupation factors according to the current research. These factors may damage the gene and result in gene mutation. It has been identified in lung cancer with multiple tumor suppressor gene inactivation or mutation in cancer genes. Mutation of cancer genes and inactivation of these tumor suppressor genes lead to stronger colony formation ability in the malignant tumors than normal cells. In addition, while proliferation of malignant cells increased and extended to the surrounding, the phenomenon of cell contact inhibition can eliminate, which result in the formation of deposits in further. Now the main method of treatment for lung cancer include operation, chemotherapy, radiotherapy and biotherapy etc. Although the operation method for lung cancer remains the most effective, most of the patients without any symptoms in the early stage, patients have entered the mid and late while the symptoms presentation, which lost the best period of operation treatment. The malignant tumor is a disease which may be caused in gene level, so the targeting therapy has become a research hotspot nowadays. Hippo signal transduction pathway was initially named the Drosophila Ste20 like kinase Hippo. There are several main biological effects of Hippo pathway: regulate the size of organ, keep the balance of cell proliferation and apoptosis, and maintain homeostasis, participate in the regulation of cell contact inhibition, and involve in tumor generation. In human, the Hippo pathway has negative regulation to the cell proliferation, and YAP is the key transcription activation factors after the phosphorylation. Recent studies have demonstrated that YAP is an oncogene, which can induce normal cells to malignant tumor cell, for example, the expression of YAP m RNA and protein in pancreatic cancer cells, liver cells, and colon cancer cells are obviously up-regulation. As the downstream signal molecule of Hippo pathway, YAP may promote cell proliferation, inhibit apoptosis, result in the loss function of cell contact inhibition and promote cell differentiation to malignant. The main role of YAP acts as a cancer promoting factor, but it can also promote the apoptosis. In response to DNA damage, PML recruits the YAP and p73 protein to the nucleus as a transcriptional coactivator, YAP enhanced transcription of p73 related apoptosis gene. In addition, some scientists found that the expression of YAP in breast cancer was significantly down-regulated or even missing, and breast cancer cells which were lacking YAP showed stronger ability of invasion and metastasis. Therefore, some researchers think that the definition of YAP as oncogene needs to be repositioning. Our study intends to learn the expression of YAP protein in the normal lung tissue and lung cancer tissues by immunohistochemistry, interference the expression of YAP and detect the proliferation and invasion ability changes of the lung adenocarcinoma cells. And in further to explore the related mechanism of YAP and find a new target for the treatment of lung adenocarcinoma. ObjectiveTo construct small hairpin YAP gene RNA(sh RNA), and transfected into human lung cancer cell line A549, and to find out the optimal jamming effect of sh RNA. Find the new target for treatment of adenocarcinoma. Method1.Detect the expression of YAP protein in lung adenocarcinoma tissues and normal tissues by by immunohistochemistry.2.based on information retrieval, 4 sh RNA were constructed(sh RNA1, sh RNA2, sh RNA3, sh RNA4) plasmid expression vector and a negative control expression vector(NCRNA), and then were transfected into human lung cancer cell line A549 by inverted fluorescence microscope, fluorescence quantitative PCR and Western-blot were detected in m RNA level and protein level of inhibitory effect.3.Test the proliferation and invasive ability changes of the interference cells by CCK8 and transwell. ResultThere is high expression in the lung adenocarcinoma tissue, but low expression and no expression in the normal lung tissue. After transfected with YAP-sh RNA, the expression level of YAP m RNA and protein were down-regulated, the proliferation and invasion of A549 cell line was inhibited in the YAP-sh RNA transfection group. ConclusionThe expression of YAP protein in lung adenocarcinoma tissue are higher than in normal lung tissues. The construction of YAP+sh RNA interference plasmid reduce the expression of YAP protein in lung adenocarcinoma cell line A549,and weaken the proliferation and invasive ability of lung adenocarcinoma A549 cell line.