| Backguound and purpose:Lung cancer is the most common malignant tumor in the world, whose incidence and mortality showed a clear upward trend in the world. At present the main treatments for lung cancer is still surgery, chemotherapy and radiation therapy, the overall effect is not as intention. Especially for some advanced cancer patients, they often need large doses chemotherapy to prolong survival, and chemotherapy significant side effects also make patients rather painful. As a new molecular target drug, Gefitinib can restrain the activity of the epidermal growth factor receptor tyrosine kinase. No matter used single or with the chemotherapy drug, it showed a strong role in clinical application, prolonged the patient's survival time and improved the quality of survival. But its long-term efficacy was limited by the drug resistance in almost all of the patients. The occurrence of Gefitinib resistance may be a result of joint action of various mechanisms, it was showed that microRNA may be involved in the acquired Gefitinib resistance in tumor.miRNA was small, 20-22 nucleotide-long, highly conserved non-coding RNA and widely expressed in organisms. They regulated target genes which were involved in development, apoptosis, metabolism and human diseases. More and more studies have indicated that miRNA played important roles driing cancer development. Tumourgenesis was closely related to abnormal cellular proliferation, differentiation and apoptosis. some miRNA associated with cancer was discovered by the pathway related to tumor,such as miR-126,miR-14, etc. in the studies of miRNA regulating resistant genes, we found microRNAs miR-27a and miR-451 are involved in activating the expression of P-glycoprotein, the MDR1 gene product, result in drug resistance in tumor cells. It was shown that microRNA-128b is a regulator of EGFR in non-small-cell lung cancer (NSCLC) cell lines. MicroRNA-128b loss of heterozygosity was frequent in tumor samples and correlated significantly with clinical response and survival following gefitinib. The overexpression of let-7a, miR-126 and miR-45 in lung cancer cell can regulate the activity of AKT and ERK, and enhance the sensibility to gefitinib. Post-transfection miR-126 can make the resistance index increased six times in lung cancer cell, the studies about the role of miRNA in acquired gefitinib-resi stance mechanism of tumor were still vety few both at home and abroad, so the further studies were of significance for choosing individualized therapy and avoiding and overcoming the acquired gefitinib-resistance.On the basis of analysis and identification of the difference between gefitinib-sensitivity and -resistance in lung adenocarcinoma cell, this study found the miRNA difference profile of gefitinib-sensitivity and -resistance in lung adenocarcinoma cell, and screened the miRNA that was closely related to the gefitinib-resistance in lung adenocarcinoma cell, and then laid the foundation for further studying the gefitinib-sensitivity and -resistance biomarker,guiding lung cancer individualized therapy.Methods:PC-9 cell line is gefitinib sensitive human lung adenocarcinoma cell line. PC-9/Ab11, PC-9/AB2, PC-9/BB4 cell lines are all PC-9 derived gefitinib acquired resistance cell lines. And all of the four cell lines were used for this experiment. The cells morphous were observed by inverted microscope. The cell growth curve was diagraphed by counting cells and calculated cell doubling time. Gefitinib IC50 value in each cell line was measured by MTT method. The cell cycle of the fore cell lines was detected by flow cytometry. Then, miRNA microarray were applied for screening the Gefitinib resistance-associated miRNAs of four cell lines. Constructed miRNA expression vector and control vector, and transfected the miRNA.Results:1 Compared with the drug-sensitivity cell PC9 cell, drug-resistance cell PC9/AB11, PC9/AB2 had no obvious change in morphous, PC9/BB4 cell is longer. compared with the drug-sensitivity cell PC9 cell, the cell cycle of drug-resistance cell line changed signigicantly, G0/G1 phase cell percentage of PC9/AB11 increased signigicantly and of PC9/AB2 and PC9/BB4 decreased signigicantly; S phase cell percentage of PC9/AB11 decreased signigicantly and of PC9/AB2 and PC9/BB4 increased signigicantly. The doubling time of all the drug-resistance cells prolonged signigicantly.2 The IC50 value of PC9 cell line was (0.022±0.01)μmol/L, the IC50 value of PC9/AB11 cell line was (2.07±0.11)μmol/L, The IC50 value of PC9/BB4 cell line was (0.973±0.176)μmol/L, The IC50 value of PC9/AB2 cell line was (12.383±1.352)μmol/L, the IC50 value of all of the drug-resistance cell lines were higher signigicantly.3 A significant difference of miRNA differential expression profiles was existed among the four cell lines. Compared with PC9 cell,19 differentially expressed miRNA upregulated and 11 differentially expressed miRNA down-regulated in PC9/AB2 cell line; 4 differentially expressed miRNA upregulated and 9 differentially expressed miRNA down-regulated in PC9/AB11 cell line; 18 differentially expressed miRNA upregulated and 7 differentially expressed miRNA down-regulated in PC9/BB4 cell line.4 Constructed pcDNA3.1(+)/Hygro-mir-503, the expression vector of miR-503, and transfected it into PC9/AB11 cell and PC9/AB2 cell, the cell lines' IC50 decreased significantly compared with that be transfected with control (P<0.05).5 Constructed pcDNA3.1(+)/Hygro-mir-100, the expression vector of miR-100, and transfected it into PC9/AB11 cell and PC9/AB2 cell, the cell lines' IC50 had no significantly change compared with that be transfected with control (P>0.05).6 Constructed pcDNA3.1(+)/Hygro-mir-138, the expression vector of miR-138, and transfected it into PC9/AB2 cell, the cell line's IC50 had no significantly change compared with that be transfected with control(P>0.05). Transfected it into PC9/AB11 cell, the cell line's IC50 decreased significantly compared with the control(P<0.05).7 Transfected the inhibitor of miR-21 and its control into PC9/BB4 cell, the IC50 had no significant difference between them(P<0.05).Conclusions:The difference of the IC50 value between the drug- sensitivity and-resistance lung cancer cell lines is signigicantly, the IC50 value of acquired drug-resistance was signigicantly higher compared with the drug- sensitivity cell. The difference of microRNA profile between the the drug- sensitivity and -resistance lung cancer cell lines is signigicantly. MiR-503 and miR-138 which were low expression in drug-resistance cell lines may play a role in acquired gefitinib-resistance. |
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