Study On The Mechanism Of YTHDF3 Regulating Drug Sensitivity In Lung Cancer A549 Cells

Lung cancer is the leading cause of cancer death and is one of the most threatening malignant tumors to human health and life.In the clinical treatment of cancer,some tumor cells will show insensitive characteristics to drugs,so that the treatment cannot produce effective and sustained effects,which seriously threatens the life and health of patients.Although various approaches have emerged with the progress of research,the effectiveness of the new approaches has been limited as they have been applied in clinical practice.the problem of differential sensitivity of tumor cells to these methods also occurs.There is an urgent need for new solutions to the problem of differential drug sensitivity of tumor cells.RNA methylation is a key link in epigenetics,with m6A modification being the most extensive.Due to its high plasticity and regulatory flexibility,m6A plays an important role in multiple cellular life including cell growth,metabolism,secretion,death,and immune processes.Moreover,studies have shown that m6A modification is involved in the process of cellular stress response.When cells are treated with chemotherapy drugs,they will also trigger a stress response to deal with the damage of drugs to tumor cells.The ability of cells to respond to external stimuli,especially chemotherapy drugs,determines the sensitivity of cells to this drug.Therefore,it is worth exploring what role m6A modification plays in the change of drug sensitivity of tumor cells,especially lung cancer cells.In addition,the comprehensive score calculated by multiple m6A-related proteins in lung cancer tissues as evaluation factors can be used as key indicators to evaluate the prognosis and survival rate of cancer patients.It has great potential to use m6A modification as a breakthrough to solve the problem of drug sensitivity differences.RNA methylation-binding protein is a bridge between m6A modification and cell life activities.YTHDF3,a member of the RNA methylation-binding protein family,plays an important role in the function of m6A.However,whether YTHDF3 plays a role in the difference of drug sensitivity in lung cancer cells and the underlying mechanisms have not been reported.Here,we report the correlation between the dynamic changes of YTHDF3 expression level and the therapeutic effect of drugs on A549 cells.Our results showed that the YTHDF3 expression level was increased gradually from the time of drug stimulation to the beginning of cell death,but the YTHDF3 protein level was decreased rapidly with the aggravation of cell death.We speculate that the elevation of YTHDF3 prior to cell death is a stress response triggered by the stimulation of cells to cope with the stress caused by this stimulus.We constructed a YTHDF3 knockdown cell line to test our hypothesis.The results showed that inhibiting the increase of YTHDF3 significantly enhanced the efficacy of SPC and pirarubicin.Overexpression of YTHDF3 attenuated the effect of SPC and pirarubicin.We confirmed this result by rescue experiments and demonstrated that YTHDF3 knockdown enhanced the therapeutic effect of pirarubicin by subcutaneous injection of YTHDF3 knockdown tumor cells in nude mice.In addition to this,we also conducted a preliminary study on the mechanism of the dynamic changes of YTHDF3,the increase of YTHDF3 protein level is due to the increase of its mRNA level,and the decrease of YTHDF3 protein level is due to the enhancement of the proteasomal degradation pathway.As an RNA methylation-binding protein,YTHDF3 is required to bind to its target mRNA.By sequencing and RIP-seq,we identified the target of YTHDF3,c-JUN,which is one of the most active subunits of AP-1.Studies have shown that AP-1 plays a role in drug resistance of various tumor cells,and c-JUN is also involved in the regulation of cellular stress response.We verified the direct binding and regulatory relationship between YTHDF3 and c-JUN.We also predicted and verified the specific binding sites between YTHDF3 and c-Jun.In addition,we also found that YTHDF3 could affect the proliferation ability and colony formation ability of A549 cells in low serum culture condition,and also found the effect of YTHDF3 on tumor angiogenesis in vivo.In summary,our study illustrates the interaction between drug efficacy and YTHDF3 expression,which provides a new therapeutic idea to solve the problem of differential drug sensitivity from the perspective of m6A modification.