Preliminary Study On The Role And Mechanism Of M6A "reader" Protein YTHDF1 In Promoting The Progression Of HCC

Background and aimsHepatocellular carcinoma(HCC)has caused a severe health burden globally.Therefore,it is an important task to uncover more molecular mechanisms associated with HCC occurrence and progression to develop novel targeted drugs.N6-methyladenosine(m6A)modification,as the most abundant RNA modification,widely participates in the physiological process and inv olves multiple disease progression,especially cancer.Studies showed that YTH N6-methyladenosine RNA binding protein 1(YTHDF1),a pivotal m6 A “reader” protein,was highexpressed in multiple cancers and promoted malignant progression.It was indicated that YTHDF1 played a vital role in the occurrence and development of cancer.However,the function and molecular mechanism of YTHDF1 in HCC are still not fully understood.Consequently,our study aims to explore the expression and role of YTHDF1 in HCC and preliminarily reveal the molecular mechanism of YTHDF1 in HCC.MethodsOur study first used various bioinformatics databases to preliminarily analyze the expression of YTHDF1 in HCC tissues and its correlation with the clinicopathological characteristics and survival of HCC patients;Then,we used western blot,SYBR Green q PCR,and immunofluorescence to examine the expression and subcellular location of YTHDF1 in HCC cells and tissues;To further test its in situ expression,we applied the tissue microarray technology to analyze the expression of YHTDF1 in 90 HCC cases and the relationship between expression and clinicopathological features;We appl ied the loss-of-function method to explore the role of YTHDF1 in HCC involved in Huh7 and MHCC-97 H cells proliferation,cell cycle,migration and invasion by using CCK8,colony-forming assay,Edu assay,flow cytometry,Transwell assay and wound-healing assay;To explore the effect of YTHDF1 on HCC in vivo,we constructed a cell line with stable knockdown of YTHDF1 gene through recombinant lentiviral vector,and structured the subcutaneous implantation model for in vivo experiments;Mechanistically,we performed the gene set enrichment analysis(GSEA)to predict the potential regulatory signaling pathway of YTHDF1 in HCC.Then,we measured the protein expression of key pathway molecules after down-regulated YTHDF1 by transfecting si-YTHDF1;Cells were treated with pathway activators SC79 and TGF-β to detect the response of these key pathway molecules to the YTHDF1 knockdown,so as to confirm whether these signaling pathways mediate the effect of YTHDF1 on HCC.Results(1)Bioinformatic analysis showed that the m RNA level of YTHDF1 was upregulated in HCC tissues and was related to HCC grades and stages,and YTHDF1 high expression indicated poor survival;(2)The m RNA and protein expression level of YTHDF1 in multiple HCC cell lines were higher than the normal liver cell,the protein expression in 12 paired HCC tissues and the in situ expression in 90 paired HCC tissues were higher than adjacent nontumorous tissues,and the expression was associated with HCC grades.The immunofluorescent assay showed that YTHDF1 was located in the cytoplasm and cell nucleus.(3)After knocking down YTHDF1 by transfecting si-YTHDF1,the CCK8 assay showed that HCC cells’ absorbance was significantly reduced;The colony-forming assay showed that the clonal cell clusters were decreased;The Edu assay showed that the proportion of fluorescent fusion cells was decreased;The Transwell assay showed that the number of migratory and invasive cells was significantly reduced;The flow cytometry assay showed that the ratio of HCC cells in the G0/G1 phase was increased.In vivo experiments showed that the size and volume of subcutaneous tumors in nude mice were significantly reduced in the YTHDF1 knockdown group compare with control group;(4)GSEA predicted that the PI3K/AKT/m TOR pathway was significantly enriched in patients with YTHDF1 high-expression.Western blot showed that the expression of AKT,p-AKT,m TOR,and p-m TOR were down-regulated after inhibiting YTHDF1 expression.The suppressed effect of YTHDF1 on HCC cell proliferation was partially rescued after the pathway activator SC79 treatment;(5)Western blot showed that the epithelial cell markers Claudin 1 and ZO-1 were upregulated,and the mesenchymal cell markers MMP2,MMP9,N-cadherin,and Vimentin were down-regulated after inhibiting YTHDF1 expression.The expression changes of MMP9,N-cadherin,and Vimentin and the inhibitory effect of YTHDF1-deficient on cell migration and invasion were partially restored after treating cells with the epithelial-mesenchymal transition(EMT)activator TGF-β.ConclusionsYTHDF1 is significantly highly expressed in HCC and is associated with the grade and stage of HCC.The high-expressed YTHDF1 suggests a poor survival of HCC patients.YTHDF1 is a crucial oncogene.It promotes HCC cell growth by activating the PI3K/AKT/m TOR signaling pathway and promotes HCC cell migration and invasion by inducing EMT.YTHDF1 may be a p otential therapeutic target for HCC.