Decorin Gene Targeted Inhibiting A549Cells And Increasing Killing Efficiency Of Platinum Chemotherapeutic Drugs To Lung Cancer Cells In Vitro

Lung cancer is the primary cause of death of cancer, almost140thousandspatients succumb to lung cancer.Most patients were diagnosed with advanced lungcancer which cause the high death rate.In the classification of lung cancer. Genetargeted therapy of lung cancer is a newly rising therapeutic tool. It can kill the lungcancer cells with height selectivity, and not damage the function of normal lungtissue cells, however, just because the targeted therapy is designed for thespecificity-attack target molecule, we must find out the target point to develop thetherapeutic effect.Decorin is a major proteoglycan associated with the collagen fiber indwellconnective tissue,there is a variety of biological activity to regulate and control thetissue morphogenesis,cell differentiation,movement,proliferation, and collagenfiber formation process and so on. It is important to prevent the occurrence of tissueand organ fibrosis.Along with the research for the past few years, we can see that theabnormal expression of decorin in a variety of malignant tumors can make thegrowth factor binding especially and make the down-regulation of cell growth oftumor.Those above bring more and more attention to it in the tumorous developmentand treatment.The hSLPI (human secretory leukoprotease inhibitor),is usually overexpressedin lung cancer,which is produced by the epithelium mucosae of respiratory andgenital tract,especially in NSCLC. The hSLPI belongs to the tumor specificpromoter, can regulate the specifically expressed of decorin gene in lung cancer cells,targeted killing lung cancer cells without affecting normal lung tissue cells.The purpose of this study is to build the promoter hSLPI regulatory eukaryoticexpression vector of decorin gene, specifically expressed in lung cancer cells andtargeting inhibiting the cell, at last gene therapy improves the safety and theeffectiveness of the therapy of lung cancer. Research has made the followingprogress: 1．The application of recombinant technology was successfully constructed theregulation of decorin eukaryotic expression vector by the promoter of hSLPI.By genomic PCR,cloned from peripheral blood of hSLPI1250bp full–lengthpromoter sequence, the DNA sequencing analysis showed that the promotersequence with the Genebank in hSLPI upstream of the5′end of the transcriptionalregulatory region of the sequence was exactly concord; Separately clone both hSLPIpromoter sequence and decorin into eukaryotic expression vector pcDNA3.1(+),make sure that they are inserted to the correct position by the restriction enzyme andmolecular sequencing. Its molecular weight and DNA sequence fully consistent withGeneBank database to successfully construct pcDNA3.1-hSLPI-decorin eukaryoticexpression vector. In the end, we get the basis of this experiment by specificinhibiting the target cells through the implementation of the hSLPI regulates thedecorin gene.2．The successful implementation of the expression of decorin gene targetingspecific lung cancer cells which regulated by promoter hSLPIpcDNA3.1(+),pcDNA3.1-decorin,pcDNA3.1-hSLPI-decorin mediated byliposome stable transfect lung cancer A549cells and liver cancer HepG2cells.Theresult of RT-PCR,IHC,Westem blot and ELISA confirm that decorin generegulated by promoter hSLPI expressed only in lung cancer A549cells except forliver cancer HepG2cells. With the help of advanced vector,which is designed by ourreserch institute, the expression of decorin gene in lung cancer cells specificallyachieve the functions of targeting specificity expression of therapeutic gene, fromthe design level of molecular carriers, to provides the molecular structure basis ofside effects caused by the shield generally integrated gene into the host cell.3．The biofunction of decorin gene in targeting injurying lung cancer cells isconfirmedThe result of eukaryotic expression vector pcDNA3.1-hSLPI-decorin targetinginjury lung cancer cell in vitro showed that the lung cancer A549cell transfected bypcDNA3.1-hSLPI-decorin mediated by liposome,from the fourth day,lung cancerA549cell grows slowly,cell growth rate decrease obviously,the cell growth is inhibited obviously,compared with HepG2cell group transfected by same vector,theresults are statistics different, those prompt that the constructed pcDNA3.1-hSLPI-decorin eukaryotic expression vectors express efficiency and targeted inhibit lungcancer strongly. It provide an experimental basis for the realization of gene targetingtreatment of lung cancer.The analysis of eukaryotic expression vector pcDNA3.1-hSLPI-decorininhibited cell cycle in lung cancer growth, show that the pcDNA3.1-hSLPI-decorintranstected A549cell lines transit from G1phase to S phase was significantlyinhibited, it is obvious that the percentage of G1phase was higher compare with theempty vector transfected HepG2cells, suggesting that the eukaryotic expressionvector pcDNA3.1-hSLPI-decorin inhibited cancer growth by inducing G1phasearrest of cell cycle to inhibit cancer cell growth.4．A549cells transfected by eukaryotic expression vector pcDNA3.1-hSLPI-decorin obviously increased the sensitivity of chemotherapeutics.Different concentrations of the Cisplatin, Carboplatin separately use oneukaryotic expression vector pcDNA3.1-hSLPI-decorin, lung cancer A549cellstransfected by pcDNA3.1(+) and A549cells without transfecting. MTT assay resultsshowed that A549cells transfected by eukaryotic expression vector pcDNA3.1-hSLPI-decorin which Cisplatin and Carboplatin act on,its IC50significantly reducedby3times. The test of the A549cells, Bcl-2and ERCC-1mRNA detected byFluorescent real-time quantitative PCR technique indicated that, ERCC-1, Bcl-2mRNA expression level obviously depress in A549cells group transfected byeukaryotic expression vector pcDNA3.1-hSLPI-decorin. The probability of themechanism that decorin gene increase the A549cells' sensitivity to platinummetalsdrug by depressing ERCC-1,Bcl-2gene expression.In this research, by successfully constructing targeting-lung cancer cells'eukaryotic expression vector, the special expression of decorin gene and the specialinhibition of growth of lung cancer cells were come ture,. The eukaryotic expressionvector was transfected in A549cells,then the cells were more sensitive to platinum drugs. These results fully showed that after molecular optimized package of,thetargeting expressed vector pcDNA3.1-hSLPI-decorin could inhibit lung cancer cellsspecially,and it provided experimental basis for the treatment of lung cancer by usingof final targeting vecto.