The Functional Study Of M~6A Methylation Recognition Protein YTHDF1 On Breast Cancer Progression

Cancer is the second leading cause of death worldwide.According to the latest statistics of the World Health Organization reported that the number of new cases of breast cancer has surpassed lung cancer,becoming the highest incidence of cancer in the world.Surgery and chemotherapy are now well applied for the early stages of breast cancer,but the early stages of breast cancer are hard for patients to recognize,leading to a gradual progression to the middle and late stages.The environment in which the tumor is located,such as hypoxia,can promote the tumor metastasis through the regulation of abnormal gene expression and abnormal modification.Therefore,early diagnosis and comprehensive treatment of progress is still imminent.Adenosine N-6 methylation(m6A)modification is the most extensive dynamically reversible RNA Post-transcriptional modification in eukaryotes,and many studies have reported abnormal levels of modification during tumorigenesis and progression.It plays an important role in regulating the occurrence and development of cancer.According to the analysis of TCGA database,m6 A recognition protein YTHDF1 was overexpressed in breast tissue,but the mechanism of up-regulation of YTHDF1 was not reported.In this study,YTHDF1,which is highly expressed in breast cancer by TCGA database analysis,was explored the role in the development of breast cancer and its regulatory mechanism.The main research methods and results are as follows:(1)High expression of YTHDF1 was detected in breast cancer tissues by transcriptome and proteomic analysis using TCGA and CPTAC database,respectively.Correlation analysis of patients’ status and YTHDF1 expression in TCGA database showed that high YTHDF1 expression was positively correlated with low prognosis;(2)Selecting clinical samples of breast cancer to detect YTHDF1 expression level by immunohistochemistry and Western Blot,confirming that compared with adjacent tissues,the abundance of YTHDF1 protein in cancer tissues of corresponding patients is significantly increased,and breast cancer cell lines and normal breast cells are also used for the analysis.The comparison also showed that the expression of YTHDF1 in cancer cells increased significantly,which was consistent with the analysis results of the database;(3)To investigate the effect of YTHDF1 on the development and progression of breast cancer in vitro,a specific si RNA targeting YTHDF1 was designed to transfect breast cancer cell line.The results showed that inhibiting the expression of YTHDF1 would inhibit the proliferation,invasion and induce apoptosis of breast cancer cell line.The results showed that YTHDF1 could promote the biological process of breast cancer cells;(4)To explore the mechanism of high expression of YTHDF1 in breast cancer cells in combination with the hypoxia microenvironment,the results showed that hypoxia could significantly induce the expression of YTHDF1.HIF1α knock-down can partly inhibit the expression of YTHDF1 induced by hypoxia,while HIF2α knock-down had no obvious effect on the expression of YTHDF1 induced by hypoxia.It is preliminarily proved that hypoxia partially regulates the expression of YTHDF1 by regulating HIF1α;(5)To further explore the induction of YTHDF1 expression by hypoxia,we used the database to predict the micro RNAs potentially targeting YTHDF1,and verified the differential expression of micro RNAs under hypoxia and the regulation of YTHDF1 expression in cell lines.Subsequently,the interaction between mi R-16-5p and YTHDF1 was further confirmed by dual luciferase assay,which proved that hypoxia could indirectly induce the expression of YTHDF1 by inhibiting the expression of endogenous mi R-16-5p;(6)To explore the regulation of mi R-16-5p targeting YTHDF1 on the development of breast cancer in vitro,we transfected cells with mi R-16-5p.The experimental results showed that cell proliferation and invasion were inhibited and cell apoptosis was significantly induced,confirming that the expression of YTHDF1 was regulated by mi R-16-5p to control the biological behavior of cells;(7)To investigate the regulation mechanism of YTHDF1 in breast cancer,the results showed that YTHDF1 knockdown under hypoxia significantly inhibited glycolysis of the cells,and the results of RIP using YTHDF1 antibody to pull down glycolysis gene showed that PKM2 had strong binding ability to YTHDF1,indicating that PKM2 was the target gene regulated by YTHDF1;(8)By knocking down YTHDF1 while overexpressing PKM2 and overexpressing YTHDF1 while knocking down PKM2,it is further confirmed that YTHDF1 can regulate the expression of PKM2 leading to the change of cell glycolysis and affect the biological process of breast cancer cells.This study further confirmed that m6A-modified related protein(YTHDF1)is involved in the regulation of carcinogenesis and development,and also provides a potential target for the future diagnosis and effective treatment of breast cancer.It also provides the research basis for the development of anti-cancer drugs with small interfering RNA.