Mechanism Study Of METTL3 Enhancing Homologous Recombination Repair In Breast Cancer Cells Resisting Doxorubicin Chemotherapy

According to the latest global cancer data in 2020,breast cancer has become the highest incidence of cancer in the world,and it is also the leading cancer killer of cancer-related deaths in women.The pathogenesis,detection and treatment of breast cancer have always been the focus and focus of oncology research.Patients with early breast cancer can be treated by surgical resection combined with radiotherapy and chemotherapy,but if the best time for surgical resection is lost,it is mainly treated by chemotherapy and radiotherapy.Platinum,paclitaxel,doxorubicin and 5-fluorouracil are first-line chemotherapeutic drugs for breast cancer.Increasing the sensitivity of tumor cells to chemotherapeutic drugs and reducing the dosage of chemotherapeutic drugs so as to reduce their toxic and side effects is also an important research aspect in the treatment of breast cancer.The N6-methyladenosine modification of RNA exists widely in eukaryotic cells.Firstly,the adenine(A)on RNA is modified by"writer"methyltransferase.After the m~6A modified RNA is recognized by the"reader"protein,it performs a series of downstream functions,including mi RNA processing,m RNA nucleation translation and splicing.In addition,m~6A modification can be demethylated by"eraser"demethylase.m~6A modification can affect many biological processes such as cell proliferation,apoptosis and organ development,and is closely related to tumor.In addition,it has been reported that m~6A modification is involved in the damage repair process of DNA.This paper mainly studies the regulatory effect of METTL3,a key transferase modified by m~6A,on DNA damage and repair of breast cancer cells and its related molecular mechanism,and then studies its effect on the occurrence and development of breast cancer cells and its effect on chemotherapeutic drug sensitivity of breast cancer cells,so as to provide a new target and theoretical basis for the detection and treatment of breast cancer.As the most important methyltransferase modified by m~6A,METTL3 plays an important role in the occurrence,development,migration and invasion of many kinds of malignant tumors.It has been found that METTL3 is highly expressed in breast cancer cells,and the patients with high expression of METTL3 have poor prognosis.We constructed a stable cell line in which MCF-7 knocked down METTL3.The results showed that knockdown METTL3 inhibited the proliferation of breast cancer cells,promoted apoptosis,inhibited their migration and clone formation and so on.Overexpression of METTL3 in breast cancer MCF-7 and MB231 cells showed the opposite result.Subcutaneous tumorigenesis in nude mice also proved that knockdown of METTL3 could inhibit the growth of breast cancer in mice.Our research shows that METTL3 is closely related to the development of breast cancer.In addition,the drug sensitivity test found that knocking down METTL3 can increase the sensitivity of breast cancer cells to the chemotherapeutic drug doxorubicin.Further studies found that knocking down METTL3 could affect the level of DNA damage in breast cancer cells induced by doxorubicin.In order to further explore the relationship between METTL3 and DNA damage repair,we added Doxorubicin into MCF-7 and MB231 cells and recovered for a certain period of time.It was found that knocking down METTL3 could inhibit the repair of intracellular DNA damage,while overexpression of METTL3 could promote its repair.We found and confirmed the target gene EGF of m~6A modification mediated by METTL3 by RNA-seq and Me RIP-qPCR.EGF,a class of polypeptides of 6 k Da with 53 amino acid residues,is a natural ligand of EGFR.EGF can specifically bind to EGFR and regulate the expression of downstream target genes through a series of transcription factors such as NF-?B.Previous studies have shown that EGF can participate in the occurrence and development of a variety of tumors,and may be involved in the process of DNA damage repair.Knockdown of METTL3 could down-regulate the content of EGF in breast cancer cells,while overexpression of METTL3 could up-regulate its expression.In order to verify the related effect,we carried out the recovery experiment.We added Doxorubicin and EGF to the MCF-7 stable transformant cells which knocked down METTL3 and recovered for a certain time.The results showed that the addition of EGF could reverse the increase of intracellular DNA damage caused by knockdown METTL3.The cell lines overexpressing METTL3 were treated with Doxorubicin and the inhibitor of EGFR was added.After a certain period of recovery,it was found that the overexpression of METTL3 could down-regulate the level of intracellular DNA damage caused by Doxorubicin,while the inhibitor of EGFR(Erlotinib or Gefitinib)could inhibit the repair process.We found that EGF can regulate the expression of RAD51,an important protein in the Homologous Recombination repair(HR)pathway by q RT-PCR and Western blot experiments.Through the recovery experiment,we found that the effect of METTL3 on DNA damage and repair induced by Doxorubicin in breast cancer cells was produced by acting on the EGF/RAD51 axis.In addition,in order to determine the"reader"of this process,we constructed a variety of knock-down plasmids related to the reader.Through experiments,we confirmed that YTHDC1 is the m~6A methylated reader of EGF,which can affect the m RNA and protein content of EGF in cells,and affect the expression of downstream RAD51 and the repair of DNA damage.Our studies have shown that in breast cancer cells,METTL3 can methylate EGF,through m~6A methylation and be read by YTHDC1,which promotes the expression of EGF.EGF binds to its receptor EGFR and promotes the expression of RAD51,thus promoting the process of DNA damage repair and affecting the chemosensitivity of breast cancer cells.Our results reveal the relationship between m~6A modification and DNA damage repair and chemotherapy in breast cancer cells,which provides a new scheme and basis for the treatment of breast cancer.