| Background:Lung cancer is the leading cancer death of the human disease, and it causes the patients deaths over 1 million worldwide every year, of which 80% to 90% of patients are non-small cell lung cancer (NSCLC),15% -25% are small cell lung cancer (SCLC). Because of the lowest degree of differentiation in various pathological types of lung cancer, the incidence of small cell lung cancer presents early hematogenous metastasis, and the prognosis is poor. If the patients are diagnosed as small cell lung cancer, chemotherapy is taken as the main treatment due to sensitivity to chemotherapy, and rarely surgical therapy even in the field of surgical treatment, but there will be earlier present drug resistance and Chemotherapy failure, resulting in tumor early progression, recurrence. The 5-year survival rate is less than 10%. Therefore, resistance to chemotherapy is the biggest problems of treatment to small cell lung cancer. How to transfer small-cell lung cancer resistance to chemotherapeutic drugs is the key problem faced by the researchers and clinical workers.Multidrug resistance is the most important acquired resistance of various resistant forms. It refers to the anticancer drugs that have been exposed in terms of function or structure uncorrelated to a variety of drugs exist drug resistance. Update the mechanism of tumor drug resistance include four forms:(1) pharmacological drug, it refers to that the drug resistance impacted by body, (2) micro-environment resistance,it is the tumor cell survival and growth depends on the organ micro-environment, micro-organs environment by different resistance genes to affect the tumor cells to chemotherapeutic drug sensitivity; (3) apoptosis resistance; Such as P53, BCL-2, C-myc Participate inhibition of apoptosis,(4) biochemical resistance;It refers to the tumor cells and genetic biochemical characteristics occur complex changes, the cell resistant to drugs present different channels, ABC superfamily of membrane transport proteins that pump drugs out is the most widely studied, such as P-glycoprotein (P-gp), multidrug-resistant Drug-related protein (MRP), lung resistance-related protein (LRP). Doyle LA, Yang W and others first detected breast cancer resistance protein (BCRP)in MCF-7/AdrVp in 1998. Then the works found that BCRP is ATP2binding cassette superfamily G member2 (ABCG2).This protein can pump chemotherapy drugs extracellular, which lead to drug decrease intracellular and failure of chemotherapy.We found that ABCG2 expression are significant difference between drug resistance small cell lung cancer cell lines and parental cell lines using cDNA microarray, So we speculated that ABCG2 played an important drug-related role in small cell lung cancer cells, We want to investigate the mechanism between ABCG2 and SCLC and to provide a new theoretical basis for clinical treatment.Purpose:1. This study choose H69 and H69AR as object to analyze the difference including morphology and sensitivity of chemotherapeutic drugs between H69 cell line and resistant cell line H69AR.2. To analyze the difference including expression of mRNA and protein levels of ABCG2 between H69 cell line and resistant cell line H69AR.3. To silent expression-of ABCG2 using RNAi technology and test the effect in H69AR, and to analyze drug resistant changes.Method:1. To observe the difference of morphology of H69 and H69AR by Optical microscope.. Application of CCK8 detect sensitive to etoposide, cisplatin of H69 and H69AR.2 Application of real-time fluorescence quantitative PCR and western-blot technique analyzes ABCG2 expression in two different cell lines.3.By real time quantitative PCR, western-blot technique confirmed H69AR levels of ABCG2 cells was significantly higher than H69 cells, the use of Lipofectamine2000 method ABCG2-siRNA are transfected into H69AR cells, real-time quantitative PCR and western-blot detect silencing effect.4.CCK8 detect ABCG2 silencing H69AR cell drug resistance changes.Results:1. Unlike H69 cells, multi-drug resistant cell line H69AR adherent grows. CCK8 indicated that H69AR to cisplatin, etoposide have cross-resistance phenomenon, respective 5.91 folds,11.99 folds than H69.2. The ABCG2 expression on mRNA level in H69AR were 95.5 times higher than H69 by real time quantitative PCR. Protein level also significantly increases notably too, show a significant difference (p<0.05).3. Three pairs of SiRNA to ABCG2, respectively, ABCG2-667, ABCG2-781, ABCG2-979 are designed by gene company. The expression levels of ABCG2 is successfully decreased in H69AR by gene silencing technique.Compared with the control group ABCG2-667 group, ABCG2-781 group and ABCG2-979 group, ABCG2 mRNA expresses respectively 0.525,0.875,0.620 times by the real time quantitative PCR.Compared with the control group, gray value of western-blot analysis showed that ABCG2-667 group, ABCG2-781 group, and ABCG2-979 group of ABCG2 protein level is 0.700,0.75,0.701 times, and show a significant difference(p<0.05).4. CCK8 assay showed that ABCG2-667 group is more sensitive to cisplatin and etoposide than other groups(p<0.05).Conclusion:1. Human small cell lung cancer cells lines H69AR are provided with multi-drug resistance;2. Compared with which of the parental small cell lung cancer cell lines H69, The ABCG2 expression levels of small cell lung cancer cells lines H69AR increases obviously, but after the ABCG2 level of H69AR being degrading by gene silencing techniques, the sensitive to cisplatin, etoposide of H69AR increases greatly.3. Multi-drug resistance of human small cell lung cancer cell line H69AR may be closely related to ABCG2 expression.4. ABCG2 may be an ideal target to reverse multi-drug resistance of small cell lung cancer. |
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