Detecting HIF-1α MRNA And Analyzing Its Regulation Mechanism In Crizotinib-induced Apoptosis Of Lung Adenocarcinoma Cell Line H2228

Objective:Hypoxia inducible factor1α（HIF-1α）is an important angiogenicregulators in non-small cell lung cance（rNSCLC）.To explore the role of HIF-1αmRNA in crizotinib-induced apoptosis of echinoderm microtubule associatedprotein like4-anaplastic lymphoma kinase （EML4-ALK）positive lungadenocarcinoma cell line H2228and analyze its mechanism, a tyrosine kinaseinhibitor crizotinib was employed for inducing apoptosis of EML4-ALKpositive lung adenocarcinoma cancer cell H2228in this study, and observed thegrowth and apoptosis of lung cancer cells, then detected mRNA expression levelof AKT/HIF-1α/VEGF pathway relevant indicators, further explored HIF-1αmRNA regulatory mechanism in crizotinib-induced apoptosis of lung cancercell.This study will provide theoretical basis and molecular biology reference tothe mechanism of tyrosine kinase inhibitor in the future. Methods:1、H2228cells were treated with10、30、90、270、810nM concentrationcrizotinib for48h, and using MTT method to measure the ablility ofproliferation. And then explore the role of crizotinib in cell proliferationinhibition.2、H2228were treated with100、200、300nM crizotinib for48h, the levelsof apoptosis were quantitated using Annexin V assay. And then explore the roleof crizotinib in inducing apoptosis.3、H2228were treated with50、100、200、400、800μm/L low oxygensimulation agent CoCl2for24h. Detecting the change of HIF-1α mRNAexpression level. H2228were treated with200μm/L CoCl2for0、3、6、12、24、48h. Detecting the change of HIF-1α mRNA expression level by reversetranscription-PCR(RT-PCR).4、H2228were treated with0、60、125、250、500、1000nM crizotinib for24h. Detecting the change of HIF-1α mRNA expression level by RT-PCR.5、Expression level of HIF-1α,and its upstream regulatory geneAkt,downstream target gene VEGF mRNA in hypoxia(hypoxic preconditionwith CoCl2) and normoxia groups were detected by RT-PCR after treated with500nM crizotinib for24h.Results：1、Cellular proliferation inhibition rate of H2228cell line was increasedalong with the increasing concentration of crizotinib, presented in a dose-dependent manner with IC50335nM.2、Apoptosis rate of H2228cell line was increased along with theincreasing concentration of crizotinib presented in a dose-dependent manner.3、When CoCl2effect time is6-48h or CoCl2concentration is more than100μ m/L，HIF-1α mRNA expression level decreased gradually with theincrease of CoCl2concentration and action time in a dose and time dependentmanner. In the200μ m/L concentration HIF-1α mRNA expression level had thegreatest reduction.4、HIF-1α mRNA expression level was increased gradually along with theincreasing concentration of crizotinib in H2228cells, then drop to a certainconcentration, but were higher than control group.5、Crizotinib can up regulate the expression of HIF-1α、Akt mRNA inH2228cells, but down regulate the expression of VEGF. Comparing tonormoxia group, the role of crizotinib up-regulating HIF-1α mRNA expressionwas more obvious in hypoxia group（P＜0.05）.Conclusion:1、Crizotinib inhibits proliferation and promotes apoptosis in lungadenocarcinoma cell line H2228with dose and time dependent manner.2、Low oxygen simulation agent CoCl2can down regulate HIF-1α mRNAexpression, which is consistent with previous prolong hypoxia study.3、Crizotinib can up regulate the expression of HIF-1α、Akt mRNA inH2228cells, but down regulate the expression of VEGF. The role of crizotinib up-regulating HIF-1α mRNA expression was more obvious in hypoxia group（P＜0.05）.4、The increase of HIF-1alpha mRNA expression plays an important rolein crizotinib-induced H2228cells apoptosis process. It is an vital transcriptionfactor.