Establishment Of Crizotinib-resistant Cell Line H3122 Of Non-small Cell Lung Cancer And Underlying Resistance Mechanisms

Non-small cell lung cancer(NSCLC)is one of the most common malignant tumors, about two-thirds of NSCLC patients cannot do operative treatment when they visit, and simple radiotherapy or chemotherapy effect is not ideal. However, individual target treatment is significantly more effective and specific, and has become a research focus in the NSCLC treatment. Unfortunately, resistance would be found after a period of target treatment, which can be a great threaten to clinical, so the research of drug resistance mechanisms in lung cancer is particularly important. As a new target, the study about echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase(EML4-ALK) in lung cancer is relatively fewer, not to the resistance mechanisms. In conclusion, this project aims to establish the crizotinib-resistant cell line of non-small cell lung cancer and explore the resistance mechanisms.H3122CR cell lines were established by continuous stepwise selection in increasing concentration of crizotinib. Cell morphological differences between parent cells and resistant cells were observed by optical microscope. CCK8 method was used to establish cell growth curves and detect the sensitivity of parent and resistant cells to crizotinib, and the resistance index was calculated. The differences of apoptosis and cell cycle distribution between lung cancer H3122 and H3122 CR cells were determined by flow cytometry. The mRNA levels of EML4-ALK and BCRP in lung cancer H3122 and H3122 CR cells were detected by real-time quantitative RT-PCR. To detect expressive changes of miRNA in lung cancer H3122 and H3122 CR cells by the gene sequencing and to further select differentially expressed microRNAs. The real-time quantitative RT-PCR was used to verify the selected miRNA. The miR-10a-5p mimics were transfected into H3122 CR cells with Lipofectamine 2000,and then changes of sensitivity to crizotinib and cell cycle distribution were detected.After six mouths inducement, the crizotinib-resistant lung cancer cells H3122 CR were established, whose volume increased, tentacles stretched and resistance index was 9.86. Comparing to H3122, the apoptosis rate of H3122 CR cells was significantly reduced(P<0.001) and the cell cycle proportion in S phase decreased(P<0.01) while that in G1 phase and G2 phase increased(P<0.05). The mRNA levels of EML4-ALK and BCRP were up-regulated obviously in resistant cells. The gene sequencing and RT-PCR revealed that the expression levels of hsa-mi R-30a-5p, hsa-miR-374 c, hsa-miR-143-3p, has-miR-148a-5p and hsa-mi R-125b-5p were up-regulated, and those of hsa-miR-31-5p, hsa-mi R-3182, hsa-mi R-7-5p, hsa-miR-148a-3p, hsa-miR-200c-3p and hsa-mi R-10a-5p were down-regulated. Transfecting miR-10a-5p mimics into H3122 CR cells discovered that the up-regulation of miR-10a-5p improved the sensitivity of resistant cells to crizotinib, to some degree, while the cell cycle proportion in G1 phase decreased.Above all, H3122 CR cells show some resist ant characteristics for crizotinib, which can be used as the model for resistance mechanisms as well as reversing the drug-resistant. The high expression of EML4-ALK and BCRP may participate in crizotinib resistance as well as some miRNAs.