The Expression Of MiRNA-1 Regulated By Paclitaxel In EGFR-TKIs Resistant Lung Cancer

Objective and background:Non-small cell lung cancer(NSCLC)is one of the most common cancers.The incidence and mortality of NSCLC is top of the malignant tumors.EGFR-TKIs(epidermal growth factor receptor tyrosine kinase inhibitors)(erlotinib and gefitinib)are effective in the treatment of NSCLC,but the emergence of acquired drug-resistance to EGFR-TKIs limits their clinical application.Researches showed that EGFR-TKIs are effective for 50-80%of NSCLC patients with EGFR mutations.In Asia,including China,EGFR-TKIs are far more effective than traditional chemotherapy,with only little tolerant adverse side effects,and are widely used as the result.The IPASS subgroup analysis suggests that for patients with EGFR mutations,EGFR-TKI is better than standard platinum-based two drug treatment in Progress Free Survival(PFS).Then using specific molecular markers for screening,and blocking them to suppress tumor growth have become new ideas in lung cancer research.But in the course of the treatment,most of the patients initially sensitive to EGFR-TKIs become EGFR-TKIs resistant.PI3K/AKt signal continually activated by secondary C-MET gene amplification is one of the causes for EGFR-TKI acquired drug resistance.Thus,it is urgent to find out the gene to regulate MET.MicroRNA(miRNA)is small non-coding molecule RNA found in eukaryotic cells and regulates gene expression after transcription.Researches show that miRNA plays a role as oncogene or tumor-suppressor gene in cell proliferation,differentiation and apoptosis.It has important biological effects in the development of cancers,and it also may relate to drug resistance in lung cancer.It is possible that up-regulate tumor suppressor gene miRNA-l's expression may reverse EGFR-TKIs resistance.Apoptosis inhibitory factor Survivin not only participates in cell apoptosis and proliferation,but also induces drug resistance.The expression of Survivin is regulated by PI3K/Akt signal pathways.Thus we reckon that Survivin may also play an important role in EGFR-TKI resistance.Purpose:EGFR-TKIs are effective for the treatment of NSCLC,but their acquired drug resistance limits their clinical application.So far,there is no ideal drug to overcome the drug resistance in clinic.Paclitaxel is the only known drug to promote polymerization of tiny tubes and to stabilize polymerized tiny tubes.Phase II-III clinical researches show that paclitaxel is suitable to treat ovarian cancer and breast cancer.It is reported that paclitaxel is also effective for lung,gastric,esophageal,and head and neck cancers.But its ability to treat EGFR-TKIs-resistant cells and the molecular change has not been investigated yet.Our study was to found the influence of paclitaxel on EGFR-TKIs-resistant cells and its molecular change,and to discuss paclitaxel's potential therapeutic effect on EGFR-TKIs-resistant lung cancer.We also tested the microRNA-1 expression in an acquired-Gefitinib-resistant patient's plasma,who regains sensitivity to Gefitinib after the treatment of paclitaxel,including three periods of time:before Gefitinib treatment,before paclitaxel,and after paclitaxel.The microRNA-1 expression in plasma is in accordance with the in vitro study.And the expression of microRNA-1 in the plasma of control group(healthy people,esophageal cancer patients)was also detected.Methods:The cytotoxicity of paclitaxel was tested by CCK-8.Flow cytometry was used to measure the promotion of apoptosis.The expression of apoptosis associated protein MET,p-Akt and survivin was detected by Western blot.MicroRNA-1 expression in H1975,H820,A549 and PC-9 cells treated with or without paclitaxel was detected by RT-PCR.MicroRNA-1 expression in human plasma treated with or without paclitaxel was detected by RT-PCR.MicroRNA-I expression in human plasma in the NSCLC patient,healthy people,esophageal cancer patients was detected by RT-PCR.Results:Apoptosis rates of H1975,H820,A549 and PC-9 cells were all increased after the incubation of paclitaxel.The MET,p-Akt and survivin proteins were down-regulated by paclitaxel,while the Akt protein not affected.Paclitaxel could significantly increase the expression of microRNA-I in H1975,PC-9 and H820 cells,which was confirmed by real-time PCR.Correlation analysis found that miRNA-1 expression had a strong correlation with MET,p-Akt,survivin proteins.Real-time PCR also found that after treated by paclitaxel,microRNA-1 in the plasma was up-regulated.The expression of microRNA-1 in the plasma of the lung cancer patient was significantly higher than that of the control groups(esophageal cancer,healthy people).Conclusion:Paclitaxel induced the activation of microRNA-1,which may lead to the decreased levels of proteins of MET,p-Akt and survivin in H1975,PC-9 and H820 cell lines.Paclitaxel may increase the expression of miRNA-1 to make EGFR-TKIs-resistant patients restore sensitivity.MiRNA-1 may have the potential to be a new tumor marker for the prognosis and EGFR-TKIs' sensitivity of NSCLC patients.