The Expression Of Ku70 In Lung Adenocarcinoma A459 And A549DDP Cells

Ku protein is a DNA end-binding protein, including two subunits ,that is Ku70 and Ku80 (Advanced Bio-86), which constitute hetero-dimers. DNA double breaks (DSBs) can be caused by a variety of exogenous or endogenous damages , and they can cause genetic mutation, inactivation, transcription function losing, leading to the occurrence of cancer, even the death of the body. In mammals non-homologous end joining (NHEJ) of DNA has the dominant position of DSBs repair. DNA-dependent protein kinase (DNA-PK) is the DNA activated nucleus protein kinase, which is constituted of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and two DNA-binding protein regulation subunits Ku70 and Ku80. When DSBs occurs , Ku protein as DNA-PK regulatory subunit congtact on the DNA binding and repair it. Ku protein plays an important role in the NHEJ repair pathway of the DSBS as a DNA repair proteins.In addition, Ku protein also has a variety of biological functions. V (D) J re-activation of genes includes the restructuring and re-activation of a gene encoding a product of two reactions catalyzed by DNA division, as well as through cell DSBs repair mechanisms to connect to the end of the reaction. As mentioned above, Ku plays an important role in the repair of DSBs,which thereby demonstrates the Ku70 involves in immunoglobulin and T cell surface antigen receptor V (D) J re-activation. Ku protein can congtact with a variety of replication initiation and replication-related factors, indicating that the proteins involved in the regulation of gene replication. In vivo, transcription is the first stage of gene expression, and also the main phase of gene regulation. Ku70 connection with the sequence-specific DNA activity, alone or with other proteins can be combined with certain cis-acting element connectivity and thus play a role in regulating transcription process. Telomeres are located at the end of eukaryotic linear chromosomes of a DNA protein complex, whose main function is to stabilize chromosome ends, preventing the latter occurrence fusion, degradation, rearrangement and loss of such changes in genes on the chromosome structure protection. It in founded that,even without DNA-PKcs and telomere DNA Ku can also congtact with telomerase. And in vitro experiments, even though there is no subunit of human telomerase RNA or telomeric DNA, Ku can also occur with human telomerase reverse transcriptase, the length of telomere. Apoptosis influenced by the waterfall activation process wich contraled with endogenous genes, enzymes and signal transduction pathways. Ku protein combine with Bax in the cytoplasm, involed in Apoptosis , in apoptotic cells the expression of Ku protein decreased, but in lung tissues, Survivin gene's expression were increased, suggesting that Ku protein may play an important role in the process. Ku protein helps maintain normal biological functions of cells, but the abnormal content can induce tumorigenesis: Ku protein's overexpression may enhance cell proliferation and induce int-apoptosis and tumorigenesis; the lack of Ku protein expression or low expression will lead to telomere shortening, genomic instability can lead to tumor formation.Both varies endogenous factor and extrinsic factor could trigger DNA injury, alteration, result in therioma. DSBs repairing mechanism and structures of telomere's stability play an important role in development and evolution of neoplasm inhibition. Ku protein take the important position in DSBs resoring, And is inextricably linked to the structural stability of telomeres. So Ku protein could be a tumor suppressor protein.Furthermore, because of the core fuction of Ku protein and DNA-PKcs in DSBs restoring, Ku proteins can cause abnormal cells on the same resistance to cancer treatment.Expression quantity of Ku has obvious relativity with occurrence, development and prognosis of neoplasm. In current, the research of Ku is gradually increased and it may direct new direction for cancer's diagnosis, treatment and prognosis.The research provided the basement for the work of all.Lung cancer is one of the most common malignancy,. The incidence is still increasing in most countries. Chinese scholars have found that in several type of primary Lung cancer, the proportion of adenocarcinoma in the last 30 years have a rising trend of growing,which gradually rise to major attention of scholars.Cis-platinum complexes is the most common chemotherapeutics, and one of the perfect medicine, but multiple resistance placed restrictions on clinical practice. Known that Ku protein o have a significant impact on the tumor sensitivity to chemotherapy treatments, this experiment is intended to explore the sensitivity of Ku70 on the impact of lung cancer chemotherapy,and laid the theoretical foundation for further clinical and basic researchExperimental Purpose:Using RT-PCR, immunoblot assay experiment, the research observed sensitive lung cancer cell,A549 and cis-platinum complexes; lung cancer cell, mRNA expression of Ku70 in A549DDP. And then clarify Ku70 expression may have clinical significance on sensity of lung cancer chemotherapy; Apply of RNAi to silence Ku70 gene in mouse cancer cells,and then observe the effect of cisplatin on the growth of lung cancer befor and after Ku70 silence. and taking it as the target to research the basic theory of lung gene treatment, providing a new concept of the research on reversing neoplasm chemo and radiation tolerance mechanism, giving the support to the selection and individuation on anti-neoplasm treatment.Experimental method: using RT-PCR detecte Ku70mRNA expression in A549 and A549DDP; using Western bloting detecte Ku70 protein expression quantity in above cell. Apply of SiRNA to silence Ku70 gene in mouse cancer cells,and then observe the effect of cisplatin on the growth of lung cancer befor and after Ku70 silence.Experimental result:(1)Ku70mRNA expression in A549DDP upper A549 the differences has obvious significance. P<0.05.(2) Ku70 protein expression in A549DDP upper A549, the differences has obvious significance P<0.05. (3)Compare with the control group (SiRNA-control group), cisplatin can inhibite the growth of tumor which group Ku70 silenced (SiRNA-Ku70 group) obviously.Experimental conclusion: Ku70mRNA in lung cancer sensitive A549 and Ku70 protein expression are obviously below DDP tolerance in lung cancer cell A549DDP; Cisplatin can inhibite the growth of lung cancer which Ku70 silenced obviously.Showing that Ku expression may have the clinical effect on sensity of lung cancer chemotherapy, helping to select clinical chemotherapy scheme, and taking it as the target to research the basic theory of lung gene treatment.