High-throughput Screening Lung Cancer-related Protein In Smokers

Background: Lung cancer remains one kind of the worldwide most serious malignanttumors carcinomas in recent years. In China, lung cancer patients increase by26%every year,and the death toll was more than600,000. The key facter for lung cancer is closely related totobacco smoking. Recently, our country contains1/3of the total worldwide smokers, with80%of male and19.3%of female lung cancer patients attributed to smoking. Therefore, toreduce the morbidity and mortality of lung cancer still is an urgent problem, it is so critical toimprove survival rate of patients with lung cancer through early detection and early diagnosisas well. In nowadays, a variety of tumor markers for early diagnosis of lung cancer have beenselected, but specific tumor markers for smoking lung cancer were still rare. The purpose ofthis study was to select some novel lung cancer associated phage clones in smokers by proteinmicroarray, so as to lay the foundation for building a diagnostic chip and screening tumormarkers in the future.Methods: The mRNA was isolated from total RNA extracted from the smoking relatedlung cancer tissues by RNeasy Mini Kit, and was used to synthesize double strained (ds)cDNA by the reverse transcription. Then not only the directional EcoRI/HindIII linkers wereligated into the ends of ds cDNA, but also the ds cDNA was further digested with EcoRI andHindIII, which resulted in ds cDNA with EcoRⅠand HindⅢ ends. The digested ds cDNAfragments longer than250bp in length were fractionated by Mini Column, then ligated intothe T7Select10-3vector with EcoRⅠand HindⅢ ends as well. After packaging in vitro,smoking lung cancer T7phage display cDNA library was constructed successfully. After that,the constructed phage library were biopanned, and the library was screened through the serumof smoking lung cancer patients, normal non-smokers and normal smokers on a large scale.With this, the data was standardized dealing with t test for smoking lung cancer group, normalnon-smokers group and normal smokers group. T test results with P <0.01indicated that thedifference was extremely significant. Combined with linear regression analysis, the phage clones were selected of high-expression in smokers with lung cancer group and normalsmokers group, but low-expression clones in normal non-smokers group.Results: It was indicated that the library contained1.35×106clones and the titer of theamplied library was4.4×1010pfu/ml. The PCR identification results of100clones picked atrandom suggested that97%clones were recombinant and91%of recombinant clonescontained cDNA fragments longer than250bp in length.158phage clones of high-expressionin smoking lung cancer group and162clones in normal smokers group were selected afteranalysing datas of smoking lung cancer and normal non-smokers group, normal smokers andnon-smokers group respectively when combining the phage library with the proteinmicroarray to screen phage clones. Last but not the least,82phage clones were selected,which were high expressed both in smoking lung cancer group and normal smokers group.Conclusions: A high quality T7phage display cDNA library was successfullyconstructed from smoking lung cancer.82lung cancer associated phage clones in smokerswere selected by protein microarray as well. Proven, these clones can be used to constructdiagnostic chips to screen lung cancer-related protein in smokers, and lay the foundation forearly stage lung cancer detection in smokers.