The Mechanism Of LncRNA GAS5 Involved In Osimertinib Resistance In NSCLC

Objective: Non-coding RNAs play an important regulatory role in drug resistance of molecular targeted therapy in non-small cell lung cancer(NSCLC).This study aims to explore the long non-coding RNA GAS5(Growth arrest-specific transcript 5)involved in the possible mechanism of resistance to Epidermal-growth factor receptor tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer.Methods: H1975 cells and osimertinib-resistant H1975 OR cells were transfected by lentivirus;CCK-8 assay detected parental cell groups(H1975,H1975+Control,H1975+GAS5)and osimertinib-resistant cell groups(H1975OR,H1975OR-sh Control,H1975OR-sh GAS5).The osimertinib resistance index and IC50 were calculated;q RTPCR detection of LncRNA GAS5,PARP,Caspase-3,PI3K/AKT signaling pathway and apoptosis-related proteins in each group cells;flow cytometry was used to detect the apoptosis and cell cycle of each group cells;Western Blot experiment relatively quantitatively revealed the effect of LncRNA GAS5 on the relative expression of EMTrelated proteins.Results:1.The CCK-8 experiment showed that osimertinib had a proliferation inhibitory effect on parental cells and osimertinib-resistant cells after 24 and 48 hours of treatment,and the IC50 of both groups of cells decreased with the increase of dose and time.2.q RT-PCR results showed that the relative expression of LncRNA GAS5 in drugresistant cells was higher than that in parental cells.3.The results of flow cytometry experiments showed that:(1)The apoptosis rate of cells decreased after overexpression of LncRNA GAS5 in parental cells;the apoptosis rate of cells increased after LncRNA GAS5 was knocked out in osimertinib-resistant cells.The same results were observed after treatment with osimertinib,and the apoptosis rate was higher than that the corresponding unmedicated group,which proved overexpression of LncRNA GAS5 could not only inhibit the apoptosis rate of H1975 cells,but also resist the effect of osimertinib on promoting the apoptosis of H1975 cells.(2)The cell cycle results showed that after overexpression of LncRNA GAS5 in parental cells,the proportion of cells in G0/G1 phase decreased,while the proportion of cells in S phase and G2/M phase increased,and the proportion of cells in G0/G1 phase increased after adding osimertinib,and the proportion of cells in S phase increased.The proportion of cells in G0/G1 phase increased,and the proportion of cells in S phase and G2/M phase decreased.After adding osimertinib,the proportion of cells in G0/G1 phase increased more significantly,and the proportion of cells in S phase decreased more obviously,the difference in the proportion of cells in G2/M phase was not statistically significant.It is proved that the proliferation activity of parental cells is enhanced after overexpression of LncRNA GAS5,and knockout of LncRNA GAS5 can effectively reduce the proliferation activity of drug-resistant cells and arrest the cell cycle in G0/G1 phase.4.Western Blot results showed that: the expression of N-cadherin and ZEB-1 proteins in osimertinib-resistant cells was higher than that of parental cells,and the expression of Ecadherin protein was lower than that of parental cells.Moreover,the expression of cadherin and ZEB-1 protein increased,and the expression of E-cadherin protein decreased.After LncRNA GAS5 was knocked out in osimertinib-resistant cells,the expression of Ncadherin and ZEB-1 proteins decreased,and the expression of E-cadherin protein increased.These data indicates that overexpression of LncRNA GAS5 can induce EMT transformation in H1975 cells,and knockdown of LncRNA GAS5 can reverse this phenomenon.5.The results of the dual-luciferase reporter assay confirmed that LncRNA GAS5 has a binding site with mi R-196 a.By q RT-PCR,it was found that overexpression of LncRNA GAS5 in parental cells reduced mi R-196 a,which further verified that GAS5 can directly regulate mi R-196 a and inhibit its expression.Conclusion: Overexpression of LncRNA GAS5 can inhibit the apoptosis of human lung adenocarcinoma H1975 cells,enhance the proliferation of H1975 cells,and enhance the epithelial-mesenchymal transition phenotype transformation ability,while enhancing the resistance of H1975 cells to osimertinib;while knocking down LncRNA GAS5 in H1975 OR cells gave the opposite results,indicate long non-coding RNA GAS5 can promote the transformation of EMT and participate in osimertinib resistance in H1975 cells.