Clone And Initial Identification Of Differentially Expressed Genes Of Human Small-cell Lung Cancer Multi-drug Resistance Cell Line

Multidrug resistance(MDR) is a major impediment to successful chemotherapy for SCLC and therefore has become the focus of current tumour research and medical studies.Mechanisms of lung caner MDR are as follows:1.Exocytosis is enhanced by transport protein in cell membrane such as P-gp,MRP,BCRP,and LRP;2.The tumour cell increases its resistance to the anti-tumour drug(e.g.GST);3.Alternation in target enzyme(e.g.TopoⅡ) leads to a decrease in the drug susceptibility;4.DNA repair function is enhanced;5. Apoptosis-related gene is abnormally expressed;6.The correlation factor in signal transduction is abnormally expressed.However,these mechanisms cannot fully explain the formation of SCLC MDR,which suggests that some other MDR mechanisms exist.With the development of molecular biological technology,gene cloning strategy keeps being improved.The improved gene-cloning stage may help exploring new SCLC MDR gene and its function.Objectives:This research aims to build the subtracted cDNA library of differentially expressed genes of SCLC MDR cell and to detect the expression of some differentially expressed cDNA fragments in SCLC MDR cell and various other tumour cells,using a cell line created in the preliminary research.Methods:1.The tester is SCLC MDR cell H446/CDDP cDNA;the driver is SCLC cell H446 cDNA;SHH and T/A cloning technology were done to build the subtracted cDNA library of differentially expressed genes of SCLC MDR cell in this research.2.The dot blot hybridization and sequencing and homology analysis were used to obtain differentially expressed cDNA fragments.3.Semi-quantitative RT-PCR and Northern blot were used to detect the expressions of PDE2A and I-2^PP2A in seven tumour cells including SCLC cell H446,SCLC MDR cell H446/CDDP,human lung adenocarcinoma cell A549, human lung Adenocarcinoma MDR cell A549/CDDP,human hepatoma carcinoma cell SK-HEP-1,human B lymphoma cell Namalwa and human gastric adenocarcinoma cell SGC7901.Results:1.The study successfully built the subtracted cDNA library of differentially expressed genes of SCLC MDR cell H446/CDDP and obtained 21 differentially expressed cDNA fgragments of cell H446/CDDP.2.Sequencing and homology analysis shows that the 21 fragments respectively represents 6 known genes(96%-100%homology).3. Semi-quantitative RT-PCR and Northern blot shows that PDE2A only has expression in H446/CDDP;I-2^PP2A has expression in H446,H446/CDDP,and Namalwa.4.Further image analysis and statistical treatment show that I-2^PP2A expression in H446/CDDP is significantly higher than that in H446(P＜0.01) and that I-2^PP2A expression in H446/CDDP is not significantly different from that in Namalwa(P＞0.05).Conclusion:SSH is an effective method for screening new function genes;gene PDE2A and gene I-2^PP2A have differential expression in SCLC MDR cell H446/CDDP;the two genes might participate in the formation of the MDR in H446/CDDP.Further studies are needed to determine how the two genes function in SCLC MDR mechanism.