Overcomes Osimertinib Resistance In Lung Adenocarcinoma Through EMT Transcription Factor Slug

Objective:Osimertinib is effective as a third-generation EGFR-TKI drug for the treatment of NSCLC.However,along with long-term and continuous medication,resistance to osimertinib is inevitable.EMT is one of the common resistance mechanisms of osimertinib,and there is a lack of effective countermeasures.Our group proposes to establish the EMT-associated osimertinib resistance cell line H1975/OE,compare the changes of EMT-related markers and analyze the resistance mechanism.We will add siRNA and DHA to compare and analyze the changes in m RNA and protein expression between parental cells and drug-resistant cells,to provide subsequent therapeutic strategies for patients with EMT-associated osimertinib resistance.Methods:In the first part of the experiment,the T790 M mutant human LUAD cell line NCI-H1975(H1975)was chosen as the parental cell.It was established by gradient concentration established of osimertinib and named H1975/OA,Transwell cell assay was used to compare the migration and invasion ability between parental and resistant cells.Flow cytometry was used to compare the apoptosis and cycle between parental and resistant cells.By comparing the morphological features and biological characteristics of H1975 and H1975/OA by magnification microscopy,the resistant cell lines with enhanced invasion ability were screened out in all H1975 / OA;that is,it was considered to be an occurrence of EMT,and it was named H1975 / OE.In the second part of the experiment,WB and qPCR assays were used to compare the difference in RNA levels and protein expression levels of EMT-related markers in H1975 and H1975/OE.The expression of Slug as a key EMT transcription molecule was knocked down in H1975/OE using si RNA to explore whether Slug could affect the expression of EMT-related markers in H1975/OE.To compare the difference in RNA levels and protein expression levels of EMT-related markers in H1975/OE and H1975/OE-Ss.To compare and contrast the biological properties of H1975/OE with H1975/OE-Ss.In the third part of the experiment,different concentrations of DHA were used to inhibit the expression of Slug and to explore whether DHA affects EMT-associated osimertinib resistance in LUAD.The cell viability of H1975 / OE + 1nmol / L osimertinib + different concentrations of the DHA model at different periods was detected.To compare the difference in RNA levels and protein expression levels of EMT-related markers in H1975,H1975/OE,and H1975/OE+ 40μmol/L DHA.To compare and contrast the biological properties of H1975/OE with H1975/OE+ 40μmol/L DHA.Comparison of changes in key proteins in the EMT tumor-associated signaling pathway in H1975/OE versus H1975/OE + 40 μmol/L DHA.RESULTS:In the first part of the experiment,the LUAD osimertinib-resistant cell lines H1975/OA was established by the gradient concentration induction method in our laboratory.The cell lines were cultured for 10 generations over 22 weeks.The IC50 was calculated to be approximately292.6nmol/L,and the resistance index was 298.Selection of EMT cell line H1975/OE from the total number of osimertinib-resistant cell lines by morphological features and biological characteristics.The total number of which was about 4% of all osimertinib-resistant cells were established in this experiment.H1975/OE cells showed large nuclei,poorly defined borders and loss of epithelial phenotype with 100 x microscopy.H1975/OE cells also showed G2/M cell cycle arrest,prolonged tumor doubling time,and increased migration and invasion ability compared with H1975 cells.In the second part of the experiment,the protein and mRNA expression levels of Slug as an invasion-promoting factor and N-cad as a tumor invasion promoter were both increased in H1975/OE compared to H1975,while the protein and m RNA expression levels of E-cad as an adhesion molecule between tumor cells were both down-regulated.After transfection by Slug-si RNA,in H1975/OE-Ss: Slug protein and m RNA expression levels were not significantly different from H1975;E-cad protein expression was up-regulated,higher than H1975/OE but lower than H1975,and significantly different from both H1975 and H1975/OE,and its m RNA levels were up-regulated,not significantly different from parental cells Both N-cad protein and m RNA expression levels were down-regulated,lower than H1975/OE and higher than H1975,and differed significantly from both H197 and H1975/OE.The invasive migration ability of H1975/OE-Ss was diminished compared to H1975/OE.In the third part of the experiment,there was no statistically significant difference in the inhibition of H1975/OE cells when the DHA concentrations were 10 and 20 μmol/L,respectively;when the DHA concentration was 40 μmol/L,the inhibition of H1975/OE was significant.Therefore,The experiment used 40 μmol/L DHA to act on H1975/OE.in H1975/OE-HDHA: the protein and m RNA expression levels of Slug were both down-regulated,lower than H1975/OE and higher than H1975,and the differences with both H1975 and H1975/OE were significant;the protein expression of E-cad was not significantly different from H1975/OE The protein expression of E-cad was not significantly different from that of H1975/OE,and its m RNA level was up-regulated,which was significantly different from that of H1975/OE and not significantly different from that of H1975;the protein and m RNA expression levels of N-cad were both down-regulated,which was significantly different from that of H1975/OE and not significantly different from that of H1975.The invasive migration ability of H1975/OE-HDHA was diminished compared to H1975/OE.Conclusion:The experiment has produced the process of osimertinib-resistant in LUAD in vitro.Then screened out EMT-resistant cell line H1975/OE and clarified the proportion of EMT in all osimertinib-resistant mechanisms.Finally,It can be confired confirmed that high concentrations of DHA could delay and reverse the process of EMT-associated osimertinib resistance.