Role Of Gene CA916798 In Cis-dichlorodiamine Platinum-induced Multidrug Resistance Of Pulmonary Carcinoma

Background:As a kind of severe diseases doing harm to human health, pulmonary cancer ranks the first place in aspects of morbidity and mortality, with mean 4-year survival rate of only 14%, saying that 75% of patients with pulmonary cancer have been at advanced stage on visiting doctors[1]. Chemotherapy is the main method for treating pulmonary cancer. Multidrug resistance (MDR) is one of clinical major causes for low survival rate of the patients and also a problem needing urgent management.MDR is a kind of phenomenon that tumor cells exert resistance against anticancer drugs with multiple structure and different mechanism after they contact with one sort of anticancer drugs. During chemotherapy for pulmonary cancer, cis-dichlorodiamine platinum (CDDP) is effective and widely-used drug, but the result is unsatisfactory because of drug resistance. Both domestic and overseas researches proved that the mechanism of pulmonary cancer cells resisting chemotherapeutics is rather complex event involving many genes. At present, there have been found typical drug resistance route mediated by transmembrane transporters including P-170 (P-glucoprotein), multidrug-associated protein and lung resistance-related protein and atypical drug resistance route mediated by other non-transmembrane mechanism like apoptosis, enzyme activity and pH change within cells. Nonetheless, the MDR mechanism of pulmonary cancer still remains unknown. Furthermore, the mechanism of different drugs inducing MDR of pulmonary cancer varies too[2-4]. Therefore, only by searching original drug resistance-related genes and deeper study on them, can MDR mechanism of pulmonary cancer be elucidated and drug resistance reversed.Gene CA916798 is obtained after MDR cell line of lung adenocarcinoma and its parental generation is screened by using suppression subtractive hybridization (SSH) technique, a new gene with high expression within drug resistance cell and has been logged into EST bank of GenBank (logging No.CA916798). It was found that the expression of gene CA916798 in drug resistance cell of pulmonary cancer was more significantly up-regulated than that in parental generation cells[5]. Currently, total length cDNA of gene CA916798 was cloned and verified by sequencing[5], which showed insignificant homology with identified drug resistance-related gene by sequence homological analysis. The author presumed that gene CA916798 may play a role in CDDP-induced MDR of pulmonary carcinoma. A deeper study on gene CA916798 and its codogenic proteins is important for elucidating molecular mechanism of CDDP conforming drug resistance as well as reversing drug resistance of CDDP.Objective:To understand the role of gene CA916798 in CDDP-induced MDR of pulmonary carcinoma and ascertain the relationship of gene CA916798 and its coded proteins with MDR of pulmonary so as to provide basis for further studying biological function of the gene and searching new reverse way of drug resistance.Methods:1.Construct protein expression vector of gene CA916798, express and purify gene CA916798-coded protein in colibacillus.2.Screen and prepare high titer polyclonal antibody of anti-CA916798, locate the intracellular expression of the protein by using fluorescence immuocytochemical method.3.By means of gene transfection, RNA interference, the effect of high and silent expressions of the gene on biological behaviors of pulmonary carcinoma cells was discussed from positive and negative aspects; meanwhile, the function of the gene was elucidated.Results:1.Clone open reading frame sequence of gene CA916798 and successfully construct prokaryotic expression recombinant plasmid pET42a/CA916798.2. CA916798 protein was mainly expressed highly in kytoplasm of colibacillus in form of insoluable cytorrhyctes, which met the requirements of antibody preparation.3.With CA916798 fusion protein immunizing animals, high titer anti-CA916798 polyclonal antibody was obtained and could discriminate the fusion protein and total protein of human lung adenocarcinoma NCI-A549, A549/CDDP, indicating that CA916798 fusion protein had sound immunogenicity and antigenicity. In the meantime, the prepared antibody provided basis for further study on function of CA916798.4 . Fluorescence immuocytochemical localization showed that the protein was expressed mainly in intracytoplasm, with high expression in drug resistance cell strain A549/CDDP and low expression in H446 cells (p<0.01).5.Eukaryotic expression vector of gene CA916798 was successfully constructed. Furthermore, after the eukaryotic expression vector was transferred into cell strain NCI-H446 of human small cell lung cancer, a human small cell lung cancer cell line NCI-H446/CA916798 of gene CA916798 was prepared, with good stability and high expression.6. After treatment with CDDP, cell NCI-H446/CA916798 grew and proliferated faster, with lower cell apoptosis rate (p<0.01) and better drug resistance against many antineoplastic agents, compared with cells in none-vector transfection group (p<0.05).7.We successfully constructed four recombinant plasmids that could express small interference RNA within cells to specifically degrade CA916798 mRNA and result in CA916798 gene silencing. Then, the plasmid were used to transfect drug resistance cell A549/CDDP expressing high level of CA916798 gene to establish cell strain A549/CDDP that could stably down-regulate and express CA916798.8.Posterior to stable transfection of 4 pieces of shRNA, either mRNA or protein expression of CA916798 was inhibited, with the most significant inhibition on expression of CA916798 in pRNAi-CA3 group and highest relative reverse rate of drug resistance against CDDP (p<0.01).9. After treatment with CDDP, cell pRNAi/CA916798-A549/CDDP grew and proliferated slowly, with higher cell apoptosis rate compared with cells in none-vector transfection group. Cycle was inhibited in G2 period.10.After being transfected by small hairpin interference RNA targeting CA916798 gene, cell strain pRNAi-CA3-A549/CDDP had weaker drug resistance against many chemotherapeutics and showed higher sensitivity (p<0.01).Conclusions:1.Construct protein expression vector of gene CA916798. CA916798 fusion protein is highly expressed in kytoplasm of colibacillus mainly in form of insoluable cytorrhyctes, which satisfies the requirements of antibody preparation, and sound immunogenicity and antigenicity. The prepared anti-CA916798 polyclonal antibody has high valence. The expression of the protein is expressed in intracytoplasm by means of fluorescence immuocytochemistry.2.Protein expression vector of the gene is successfully constructed. Small cell lung cancer cell strain NCI-H446 sensitive to CDDP and with low expression of gene CA916798 was transfected to screen positive cells and prepare cell stain with stable expression of CA916798; NCI-H446/CA916798 can stably express gene CA916798 and has stable resistance to multiple antineoplastic agents, which provides basis for deeper study on mechanism of CA916798-medicated MDR of carcinoma cells.3.Successfully construct eukaryotic expression plasmid of CA916798 siRNAs and prepare cell strain pRNAi/CA916798-A549/CDDP that can stably down-regulate expression of the gene. After CA916798 shRNA is transfected with cell strain A549/CDDP, pRNAi/CA916798-3 group has the highest relative reverse rate in four aspects of protein level, mRNA expression level, cell level and drug resistance reverse of CDDP, compared with that before transfection, which provides basis for deeper study on reverse of MDR of pulmonary cancer by targeting interference MDR gene.4. By means of gene transfection, RNA interference from positive and negative angles, gene CA916798 is proved to be new MDR-related gene of pulmonary carcinoma, which caters for breakthrough in overcoming drug resistance of chemotherapy for pulmonary carcinoma.