| OBJECTIVE:Bronchial lung adenocarcinoma is one of the most common malignant tumors of the respiratory system. In recent years, due to the increasingly serious environmental pollution, the number of smokers and smoking frequencies,the incidence and mortality of lung cancer were gradually increased worldwide. The incidence of lung cancer in the world has reached approximately seven ten thousandths in recent years. The incidence of lung cancer has been in the first place of malignant tumors, which was consistent with the current situation in China. According to the data,more than half of the cancer patients died related to the presence of tumor drug resistance,whether directly or indirectly.Currently, platinum and cytotoxic drugs as the combined chemotherapy still was the first-line standard chemotherapy. Among the lung cancer and other tumors, cisplatin is one of the most widely used chemotherapy drugs, and was used in almost non-small cell lung cancer as the preferred chemotherapy. Compared with best supportive treatment, combined chemotherapy could significantly improve and control the clinical symptoms, prolong the median survival period, and improve the life quality. However, due to the primary or secondary drug resistance phenomenon, their clinical therapeutic effects were decreased. The mechanism of drug resistance exhibited in the lung cancer was a complex biological process with number of factors and causes. Therefore, it was necessary to research the mechanism of drug resistance in lung cancer. The two cells trains of cisplatin-resistant human lung adenocarcinoma cell line (A549/DDP) and human lung adenocarcinoma cell line (A549),were from the same parental origin, with the same genetic background, which had a primary difference on cisplatin sensitivity and resistance.As a result, this two cell lines were selected to study how to avoid the histological differences caused in the sampling, in order to find the cisplatin resistance gene in lung adenocarcinoma cells and the relationship between each gene. Therefore, it could basically identify candidate genes of cisplatin-resistant lung adenocarcinoma and in-depth explore the lung cancer resistance mechanisms.Nucleotide excision repair (NER) is the most important intracellular repair,which could repair DNA damage lead by external factors (UV, a variety of chemical substances). DNA damage repair ability is an important factor to cause tumor cell resistance. In recent years, studies of NER have made great progress, especially have many new understandings of the relationship with tumorigenesis and tumor resistance.Animal studies found that the XPA gene lack mice did not have the function of NER,and sensitivity to UV was significantly higher, and were more likely to be induced to a variety of tumors. Meanwhile,the sensitivity to UV in XP patients was elevated1000times, and XP patients were more easily vulnerable to skin cancer and other tumors. It follows that XPA have played a very important role either in cells or in tissues. Many studies have found that the expression of XPA in lung cancer cells was lower than normal lung tissue, and that the low expression of XPA was related with the occurrence of lung cancer. Recent studies have showed that XPA in non-small cell lung cancer and bladder transitional cell carcinoma was significantly lower expressed than that in normal lung tissue and bladder tissue. However, the sensitivity of lung cancer and bladder cancer to chemotherapy was not high.In the last few years, RNA interference is one of the newest techniques in the field of biological science research, which could make a specific post-transcriptional silence at certain sequence to generate a similar effect with gene knock-out. It has become an important approach in research of gene function. Genes with obvious up-regulating expressions are selected from the screening of differential gene expression in the drug resistance of lung cancer. RNA interference is used to seal off the expression of these specific genes in A549/DDP cells. It could prove that these specific genes do participate in the development of cisplatin-resistance in lung cancer cells in vitro.Several genes with differential expression were selected by the gene chip technology. In this study, we silenced the gene XPA by siRNA interference technique,which was observed for its effect on the bionomics of the cell strains A549/CDDP. The aim of this study was to investigate the influence of platinum-based chemotherapy sensitivity by silencing XPA gene expression in non-small cell lung cancer (NSCLC) drug resistance cell lines (A549/DDP).Further investigation on the effect of XPA in the development of cisplatin-resistance was necessary to be carried out, which would provide effective evidences for the development drug resistance in lung tumors with XPA as a target. The mechanism of drug resistance was discussed to provide clues for future researches and treatment for lung cancer.METHODS:We detected the expression of XPA in lung normal and tumor tissues by immunohistochemistry, quantitative Pol-ymerase Chain Reaction (qPCR) and western blot, we silenced XPA A549/DDP cells by XPA-shRNA transfection, and detected the expression of XPA by qPCR and Western blot analysis. The sensitivity to cisplatin and apoptosis of A549/DDP cells transfected with XPA-shRNA were determined by methyithiazolyl tetrazolium bromide (MTT).RESULTS:1.The expression of XPA in lung adenocarcinoma and squamous cell carcinoma tissueXPA was expressed in the cytoplasm as well as the nucleus of lung adenocarcinoma cells by immunohistochemical staining, but inthe lung squamous cell carcinoma, it was mainly expressed in the cytoplasm of lung cancer tissues. IOD analysis showed that, compared with adjacent tissues, the expression of XPA in lung adenocarcinoma and squamous cell carcinoma were significantly increased. 2. The expression of XPA-mRNA and protein in lung adenocarcinoma squamous cell carcinoma tissue and adjacent tissueThe expression of XPA-mRNA in lung adenocarcinoma and squamous cell carcinoma were significantly increased than the corresponding adjacent tissue by qPCR(P<0.01). Western blot analysis showed that compared with the adjacent tissues, in lung adenocarcinoma and squamous cell carcinoma, the expression of XPA was significantly increased.3. Detect the effect of silencing XPA gene in A549/DDP cell lineqPCR and Western blot analysis showed that expression of XPA-mRNA and XPA protein were dramaticlly decreased in A549/DDP cell line after transfected by XPA-shRNA plasmid compared with the untreated group.4. The effect of XPA gene expression levels on A549/DDP cells apoptosisThe sensitivity of A549/DDP cells to cisplatin was increased after transfected with XPA-shRNA plasmid by MTT analysis. Compared with the blank group and the control group,the results showed no significant difference; compared with the blank group, and the control group, the difference between transfected group and the two group was statistically significant.CONCLUSION:1. In lung adenocarcinoma and lung squamous cell carcinoma, XPA was higher expressed.2. Silencing of XPA gene could promote A549/DDP cells apoptosis,that indicated the relationship between XPA and A549/DDP cell proliferation and apoptosis, which would further prompt silencing the XPA gene could reverse the resistance to cisplatin of non-small cell lung cancer. |
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