| Background:N~6-methyladenine(m~6A)modification is the most common epigenetic modification of RNA and is a dynamic process involving the precise regulation of methylases,binding proteins,and demethylases,and its abnormal molecular expression abundance is closely related to tumor development.Insulin-like growth factor II m RNA-binding protein 1(IGF2BP1)is a m6A methylation-modified binding protein that enhances target m RNA stability to regulate transcriptional activity.IGF2BP1 is widely involved in the process of tumor progression by enhancing m RNA stability of a variety of oncogenes.A population of tumor cells with self-renewal and multi-lineage differentiation ability,which can produce heterogeneous cell populations of tumors,called cancer stem cells(CSCs),is an important cause of tumor recurrence,metastasis and drug resistance.IGF2BP1 regulates the biological process of a variety of CSCs,however the mechanism of action of IGF2BP1 in liver cancer stem cells(LCSCs)is not yet clear.Methods:(1)Bioinformatics analysis of changes in the abundance of m~6A methylation modifications in HCC clarified that the m~6A binding protein IGF2BP1 was the most significantly differentially expressed regulator,immunohistochemistry and RT-q PCR detected the expression of IGF2BP1in HCC tissues and cells,combined with bioinformatics database and clinical correlation analysis revealed the relationship between IGF2BP1and HCC prognosis.(2)HCC cells were induced to transform into sphere-forming cells with cancer stem cell properties,and the expression differences of IGF2BP1 in HCC cells and sphere-forming cells were detected by RT-q PCR and Western blotting.Lentiviral transfection of HCC cells was used to interfere with the expression of IGF2BP1,and RT-q PCR and Western blotting were used to verify the knockdown efficiency;the expression abundance of stem cell pluripotent cytokines Nanog,Oct4,and Sox2 was analyzed in HCC cells stably transfected with IGF2BP1,the sphere-forming ability was analyzed by cell sphere-forming assay,the proportion of CD133+cells was analyzed by flow cytometry,the migration and invasion ability was analyzed by cell scratch assay and transwell migration and invasion assay,the tumorigenic ability was analyzed by colony-forming assay and subcutaneous tumor formation assay,and the chemoresistant ability was analyzed by cytotoxic proliferation assay and flow cytometry.(3)Target genes were selected by combining transcriptome sequencing(RNA-seq),RNA immunoprecipitation and high-throughput sequencing(RIP-seq)and methylated RNA immunoprecipitation sequencing(Me RIP-seq)datasets in the GEO database;CD133+/CD44+cells were sorted by flow cytometry to construct CD133+/CD44+cells that stably interfered with IGF2BP1 expression,RT-q PCR and Western blotting were used to verify the target gene transcription and protein expression levels,RIP-q PCR was used to verify that IGF2BP1 directly bound to the m RNA of downstream genes,and Me RIP-q PCR was used to verify that IGF2BP1 bound to m RNA of the target gene in m6A methylation-dependent manner.Transcription efficiency was inhibited using actinomycin D,and the m RNA decay rate of the target gene was analyzed by RT-q PCR.Results:(1)Bioinformatics analysis showed that the expression abundance of m6A methylation regulator was abnormally increased in HCC,of which the expression difference of m~6A binding protein IGF2BP1 was the most significant;immunohistochemistry verified that IGF2BP1 expression was increased in HCC tissues,RT-q PCR verified that IGF2BP1 expression was increased in HCC cells,bioinformatics database and independent clinical center results showed that the high expression level of IGF2BP1 was associated with poor prognosis of HCC,and the above results revealed that IGF2BP1 is a pro-oncogene of HCC and plays an important role in the occurrence and development of HCC.(2)Compared with HCC cells,the expression of IGF2BP1 was significantly increased in LCSCs,interfering with IGF2BP1 expression resulted in decreased expression of Nanog,Oct4 and Sox2,weakened sphere-forming ability,and reduced proportion of CD133+cells in HCC cells,indicating that IGF2BP1 could maintain the stem cell characteristics of HCC cells;wound healing,transwell migration and invasion assays showed that IGF2BP1 promoted HCC cell migration and invasion,colony formation assay and tumor xenograft in vivo assay showed that IGF2BP1promoted HCC cell tumorigenicity,and cell proliferation toxicity assay and flow cytometry indicated that IGF2BP1 promoted HCC cell chemotherapeutic drug resistance.(3)Analyzing the RNA-seq data,RIP-seq data and Me RIP-seq data of IGF2BP1 after interfering with IGF2BP1 in the GEO database,10 target genes were screened,and the candidate target gene MGAT5 was obtained through literature review and bioinformatics analysis.(4)CD133+/CD44+cell subsets were sorted by flow cytometry,and CD133+/CD44+cells with stable knockout of IGF2BP1 were constructed.RT-q PCR and Western blotting showed that the transcription and protein expression levels of MGAT5 decreased after IGF2BP1 knockdown.RIP-q PCR indicated that IGF2BP1 binding to MGAT5 m RNA was significantly reduced after IGF2BP1 knockdown,indicating that IGF2BP1directly bound to MGAT5 m RNA,and Me RIP-q PCR indicated that the abundance of m6A methylation modification with MGAT5 m RNA was significantly reduced after IGF2BP1 knockdown,indicating that IGF2BP1bound to MGAT5 m RNA in a m~6A methylation-dependent manner,and RNA degradation experiments indicated that MGAT5 m RNA stability was significantly decreased after IGF2BP1 knockdown,indicating that IGF2BP1 promoted its expression by enhancing MGAT5 m RNA stability.Conclusion:For the first time,we elucidated that the IGF2BP1regulates MGAT5 m RNA stability through m~6A methylation modification which in turn maintains the stemness characteristics of liver cancer stem cells,and IGF2BP1 may be a new key targeting molecule for liver cancer stem cells... 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