The Study On Survivin Antisense Oligonucleotides(Survivin-ASODN) Induced Apoptosis Of Xunwei Lung Adenocarcinoma Cell And Its Susceptibility To Chemotherapy

Objective and background:Lung cancer owns the highest morbidity and fatality worldwide. Yunnan Xuanwei is famous for its high incidence of lung cancer in China and even this world. The regional specificity is obvious. High morbidity of lung cancer in Xunwei has close relationship with indoor coal contamination. Recent study shows us that CDK2, RNA composition of lung cancer telomerase,antigen of Ki-67,mutation of P53and its mutational site also play critical role in the incidence ofXuanwei lung cancer in female patients. All these mentioned above are statistically significant when being compared with that in other region.This also reminds us the special characteristics of Xunwei lung cancer existing. Lung adenocarcinoma takes up a great deal in histological type in Xuanwei patients. Early diagnosis can be very hard so it is very sorry that most of the victims are diagnosed in advanced stage,which means they lose important opportunity to perform radical surgery. Chemotherapy is critical in treatment.Chemotherapeutic drug resistance is related to survival rate and prognosis. Current molecular biology and gene engineering technology has proved lung cancer is a kind of polygenic disease as other tumor.According to that, scientists has tried to cure cancer and prevent its occurence through interference of key gene and protein. Antisense technology is a kind of gene treatment which will regulate the replication, transcription, translation of target gene or shear target gene on molecular level so that transition of genetic information from gene to protein will be affected. Sequentially, we can restrain, seal or destroy target gene. Antisense technology is based on the principle of base complementation and hybridization of nucleotide. Artificially synthesized nucleotide section and natural nucleotide section,which will complement the target gene(single-chained or double-chained DNA)or specific mRNA section, will be utilized in this technology.Will the expression of drug resistance protein in Xuanwei lung adenocarcinoma be different from that in other regions? Can we boost apoptosis of lung cancer cell by antisense oligonucleotides so that its susceptivity to chemotherapy may be increased?We designed experiments to solve these questions. Four parts has been designed according for different purposes. Firstly,we would test the expression of drug resistance protein Survivin,RRMl,ERCCl,BRCA1in specimen we collected from Xunwei patients and patients from other region respectively in order to study relevant protein expression in different places.Also, the relationship between age, gender, and clinical stage would be researched in the first part. Second, Survivin-ASODN would be composed and in order to study its role in cell cycle and apoptosis, XWLC-05lung adenocarcinoma cell would be transfected. In the third part, we would study the role of Survivin-ASODN during the expression of SurvinnmRNA and Survivin protein in XWLC-05lung adenocarcinoma cell and whether it would increase susceptivity of cancer cell to docetaxel.In the forth part,we would further investigate if Survivin-ASODN can inhabit tumor growth in vivo and its combinational function with docetaxel in nude mice tumor model. We hope our experiment contribute to Survivin-ASODN gene remedy in clinics and experimental basis for further study in the future.[Method]1. Patients who had been treated with surgery in Clinical Study of Lung Cancer Institute in Yunnan Provincial Tumor Hospital were enrolled and pathological section of these patients were collected. Immunohistological technology were used to test the expression of drug resistance protein Survivin,RRMl,ERCCl,BRCAl in these section.We studied possible difference in protein expression between Xunwei and other places. And we studied the relationship between age,gender and clinical stages at the same time.2. Specially designed ASODN and SODN was composed according to the sequence of SurvivinmRNA232－251bp.Lung adenocarcinoma cell XWLC-05was transfected by Survivin-ASODN with liposome.MTT was used to find out the biological effects of Survivin-ASODN on XWLC-05and we tested apoptosis rate of lung adenocarcinoma cell XWLC-05,which was transfeeted with Survivin-ASODN, by flow cytometer.3. We investigated the expression of SurvivinmRNA by RT-PCR and Western blot tested the expression of Survivin protein in lung adenocarcinoma cell XWLC-05. We studied its dose-effect relationship and time-effect relationship with Survivin-ASODN.4. Effect of the combination of both Survivin-ASODN and docetaxel on cancer cell was tested by MTT. TUNEL analysis was used to invesitigate the apoptosis effect in cancer cell in order to research if synergy exists when both Survivin-ASODN and docetaxel were used at the same time.5. After modeling nude mice of XWLC-05,we performed multi-point injection of different drugs in mice hypodermically according to different group. After a specific period,we studied the growth and pathological changes of nude mice according to groups.[Results]1. The expressional rate of protein Survivin,RRMl,ERCCl,BRCA1was respectively57.55%(61/106),76.41%(81/106),54.72%(58/106) and45.28%(48/106)in Xunwei patients. Also, expression was not so closed related with age, gender, clinical stage.2. Survivin and ERCC1expressed significantly higher in Xunwei patients when being compared with that of patients from other regions. The difference of RRMK BRCAl expression between Xunwei patients and that of other patients was not statistically significant.3. After transfected by Survivin-ASODN, survival rate of XWLC-05cell decreased within the same period (48h)when being compared with other group and this effect was enhanced with the increase of Survivin-ASODN density. Giving the same Survivin-ASODN density(600nmol/L),inhibitory effect increased as time passed by. All that mentioned above was significantly different when compared with group Control, Lip and Survivin-SODN4. G2/M blocking happened in XWLC-05cell after treating with different density Survivin-ASODN and apoptosis rate increased gradually. Statistically meaningful dose-effect relationship showed(P<0.01). We increased Survivin-ASODN density from600nmol/L to800nmol/L.However, apoptosis rate did not increase(P>0.05) proportionally and this was not statistically meaningful when being compared with group Control,Lip and Survivin-SODN.5. After being processed by Survivin-SODN,the testing result of RT-PCR and Western blot of cancer cell showed significantly low expression of SurvivinmRNA and Survivin protein when being compared with group Control,Lip and Survivin-SODN. Time-effect relationship, which means intensity of expression declined as time passed, showed in our experiment.6. The result of MTT showed Survivin-ASODN,docetaxel Survivin-ASODN+docetaxel could inhabit the growth of cancer cell and enhanced its apoptosis. Inhibition ratio and apoptosis rate of group Survivin-ASODN+docetaxel was as high as72.25%,42.36±2.53%respectively. It was higher than that of group Survivin-ASODN(32.67%,25.27±1.43%)and group docetaxel(42.19%33.32±1.94%).It was statistically meaningful.7. In nude mice model study,we found out that tumor size shrinked in group Survivin-ASODN, docetaxel and group Survivin-ASODN+docetaxel. Time-effect relationship showed and it was more obvious in group Survivin-ASODN+docetaxel.8. We conducted pathological diagnosis in nude mice. We found spotted and focal necrosis of tumor in group Survivin-SODN and group docetaxel. More serious necrosis happened in group Survivin-ASODN+docetaxel and we saw a great deal of patchy necrosis with non-structure red substance in the section. [Conclusion]1. Obvious relationship between the protein expression such as Survivin,RRMl,ERCCl,BRCAl and age,gender,and clinical stage was not established. Expression of Survivn and ERCC1in Xuanwei lung adenocarcinoma patients was higher than that in patients from other places in Yunnan.2. Liposome-mediated Survivin-ASODN transfection could inhabit the growth of lung adenocarcinoma cancer cell XWLC-05and enhance apoptosis of cancer cell. Time-effect relationship and dose-effect relationship both showed.3. Survivin-ASODN could inhabit the expression of SurvivinmRNA and Survivin protein in XWLC-05and time-effect relationship showed.4. Survivin-ASODN could increase the susceptibility of cancer cell to docetaxel and inhabit the growth of cancer cell and enhanced its necrosis.5. Injecting Survivin-ASODN or Survivin-ASODN+docetaxel could restrain enlargement of tumor size on nude mice and this was safe without side effect in our experiment.