| Background：Lung cancer has been the greatest threat to the health and life among all kinds of themalignant tumors. In the last50years, reports from many countries have pointed out thatthe incidence and mortality of lung cancer significantly increased. Among all kinds ofcancers,both the incidence and mortality of lung cancer topped the list in man while rankedsecond in women. However, its etiology was still not completely clear. Chemotherapypresents a main and common treatment for lung cancer[1], more than90%of lung cancerpatients need chemotherapy treatment. The curative effect on the patients with small celllung cancer either at early stage or at late stage is positive whereas the remission rate onthese patients with non-small cell lung cancer is40%~50%. In any case, chemotherapycan prolong survival and improve the quality of life of the tumor patients[2].Multidrug resistance(MDR) is defined that tumor cells selectively resistant against anatural product, such as doxorubicin, are often cross-resistant to a range of other drugs withvery different chemical structures or cellular targets. For lung cancer, cisplatin(Cis-dichlorodiamine, platinum, CDDP) is the most commomly used drug due to its broadanticancer spectrum, strong activity, synergistic effect and non-cross resistance. CDDPbelongs to cell cycle non-specific drugs, which exerts cytotoxicity effect via inhibitingDNA replication process of cancer cell. However, its clinical efficacy is not satisfyingbecause of MDR. So far, the mechanism of multidrug resistance remians unclear, butstudies have shown that resistance of lung cancer cells to chemotherapeutic treatment is aprocess involved multiple genes, such as P-170(P-gp) glycoprotein mediated classicalresistance pathway and non-classical mechanism of resistance like lung resistance related protein (LRP), multidrug resistance associated protein (MRP), apoptosis, enzyme activities,intracellular pH changes etc. Importantly, the mechanisms of resistance induced bydifferent chemotherapy drugs are not same[1][2][3]. Therefore, furtherly exploring multipledrug resistance associated genes and investigating the exact mechanism can be an effectiveway to solve MDR phenomenon, and also lay foundation for obtaining[4]more effectivefirst-line chemotherapy in clinical.In our previous study, we first discovered the CA916798gene from human MDR lungadenocarcinoma cell line SPC-A-1/CDDP by suppression subtractive hybridization (SSH).CA916798, locating on human chromosome19, with a full-length cDNA of1692bp,contains five exons.The fifth exon encodes a protein of117amino acids. Thesequence homology analysis showed that CA916798gene has no obvious homology withthe original resistance-related genes. Further studies showed that expression of CA916798gene was significantly up-regulated in lung cancer tissue than in noncancerous tissues[5].The transfection of CA916798into small cell lung cancer cell line H446, the geneexpression level was very low and sensitive to cisplatin, could increase its resistance tocisplatin and apoptosis decreased. While by using RNA interference to inhibit CA916798gene expression, it could reverse the resistance of A549/CDDP to cisplatin and cellapoptosis, in which CA916798gene has a high expression level and resistance to cisplatin.The above results strongly suggested that CA916798was a new gene related to MDRinduced by cisplatin.ScanProsite results showed that the protein coded by CA916798may contain1sulfatetyrosine sites,3protein kinase C phosphorylation sites and3casein kinase IIphosphorylation sites, suggesting that the expression of the protein may be affected by aseries of signal transduction and involved in the multidrug resistance of cancer cells tocisplatin.But CA916798gene promoter region is not clear. The mechanism on how cisplatinparticipate in transcriptional regulation of CA916798and drug resistance remains unclear.So, study on the regulation of CA916798gene expression will be conducive tofurther clarify the mechanism of MDR and provide new therapeutic targets for clinicalreversal of lung cancer MDR.Objective：This research focuses on the localization of CA916798gene promoter region. The coregene promoter region of CA916798will be found through the promoter prediction software,which lay the foundation for follow-up study of mechanism.Method：1. Prediction of CA916798gene promoter sequence through bioinformatics.2. Construction of the vectors of CA916798gene promoter sequence using molecularbiology techniques.3. Screening of gene promoter sequence through the inspection report for dualluciferase activity.Result：1. Promoter site of CA916798gene may located in1.5kb and5.2kb on the fourth exonby using bioinformatics method and Promoter2.0software prediction.2. The fragments of promoter region were amplified and10luciferase expressionvectors were successfully constructed. These vectors were used to further analysis.3. After COS7cells were transfected with above vectors, we screened the promoterregion of CA916798gene through detecting dual luciferase activity report gene with orwithout cisplatin inducing. The core gene promoter region of CA916798was confirmed tolocate in upstream~179bp, and the length was568bp. Furthermore, cisplatin can act onthe promoter sequence and induce gene expression.4.9H sequence region contains many potential transcription factor binding sites, suchas hypoxia inducible factor HIF-1, apoptotic factor NIP, transcription factor SP1and NF-κB, c-Rel etc.Conclusions：1.Bioinformatics Method and Promoter2.0software prediction strongly suggestedthat human CA916798gene proximal promoter might located in1.5kb and5.2kb on thefourth exon. 2. The core gene promoter region of CA916798was confirmed to locate in upstream~179bp, and the length was568bp.3. The bioinformation analysis showed that the9H sequence region contained manypotential transcription factor binding sites, such as hypoxia inducible factor HIF-1,apoptotic factor NIP, transcription factor SP1and NF-κ B, c-Rel etc.. CA916798gene isvery likely to be regulated by several signal transduction pathways. The drug resistance oftumor cells should be affected by inhibiting the apoptosis of tumor cell. This analysis laidthe foundation for further investigating on the molecular mechanism of CA916798generelated MDR. |
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