The Function And Mechanism Of LncRNA FTO-IT1 Mediated ILF3/FTO Pathway In Hepatocellular Carcinoma

ObjectiveHepatocellular carcinoma(HCC)is a common cancer of the digestive system with limited current treatment effects.Although many studies have reported the specific roles of long non-coding RNA(lncRNA)in HCC,the study of lncRNA still needs to be further strengthened.Therefore,this study aims to explore novel lncRNA involved in the regulation of the occurrence and development of HCC,and clarify its specific regulation mode and provide novel biomarker and therapeutic target for the diagnosis and treatment of HCC.As a result,we can expand the direction and strategy of treatment to achieve the early diagnosis and treatment of HCC,and improve the survival rate of patients.Meanwhile,this study also commits to provide further evidence for the vital function of lncRNA in cancers.MethodsBy analyzing the RNA sequencing data of HCC tissues in TCGA,the lncRNA FTO intron transcript 1(FTO-IT1)that is differentially expressed in HCC and related to the prognosis of HCC was screened.Surgical specimens from patients with HCC in Wuhan Union Hospital were collectted,and expression levels of FTO-IT1 in 32 groups of HCC tissues and paired adjacent tissues(NT)were compared.The patients were divided into high and low groups based on the expression level of FTO-IT1,and the survival time of the two groups was compared.Meanwhile,the expression level of FTO-IT1 in normal liver cells and HCC cells was compare.The subcellular localization of FTO-IT1 in HCC cells was clarify.Small interfering RNAs(siRNAs)were transfected into HCC cells with higher expression level of FTO-IT1 to knockdown FTO-IT1 expression.Then MTT assays,colony forming assays,wound-healing assays and Transwell invasion assays were performed to observe the effect of downregulation of FTO-IT1 on the proliferation,migration and invasion of HCC cells.FTO-IT1 siRNA was packaged using lentivirus and transfected into MHCC97 H cells to construct a cell line that stably knocksdown FTO-IT1.Then the cells used for subcutaneous tumor implantation to construct a xenograft model in nude mice,to evaluate the effect of knockdown of FTO-IT1 on the proliferation of HCC in vivo.The overexpressed plasmid of FTO-IT1 was tranfected into the HCC cells with lower expression lever of FTO-IT1.Then MTT assays,colony-forming assays,wound-healing assays and Transwell invasion assays were performed to observe the effect of upregulation of FTO-IT1 on the proliferation,migration and invasion of HCC cells.Considering that FTO was a potential downstream target of FTO-IT1,the difference expression level in HCC tissue and cells were compared using TCGA data and HCC cell lines.The expression of FTO at protein level were compared using Immunohistochemical staining of HCC tissues.Spearman correlation was used to analyze the expression relationship between FTO-IT1 and FTO in HCC.Hep G2 and MHCC97 H cells were transfected with FTO-IT1 siRNAs,while Huh7 cells were transfected with FTO-IT1 overexpression plasmids.The effects of FTO-IT1 on FTO m RNA and protein levels were observed by q RT-PCR and Western blot.Then,the rescue experiments were performed.FTO-IT1 siRNAs and FTO overexpression plasmid were co-transfected,and FTO-IT1 overexpression plasmid and FTO siRNAs were co-transfected,respectively,and the m RNA and protein levels of FTO were detected.Furthermore,co-transfected cells were used to perform MTT assays,colony-forming assays,wound-healing assays,and Transwell invasion assays to evaluate the effect of FTO-IT1/FTO signal pathway on the proliferation,migration and invasion of HCC cells.The biotin-labeled FTO-IT1 was used to perform RNA pull-down assay combining liquid chromatography-tandem mass spectrometry to identitied proteins interacted with FTO-IT1 in HCC cells.After analyzing the result of mass spectrometry,the RNA binding protein ILF3 was selected as the intermediate protein for FTO-IT1 regulating FTO expression.Western blot was performed on the protein of RNA pull-down to detect the presence of ILF3.Through cat RAPID,the binding ability of FTO-IT1 and ILF3 protein was predicted.Anti-ILF3 antibody was used for RNA immunoprecipitation(RIP)assay,and the expression level of anti-ILF3 antibody-enriched FTO-IT1 was detected by q RT-PCR.The expression level of ILF3 was knockdown in Hep G2 and MHCC97 H cells,while was overexpressed in Huh7 cells,to observe the regulation of FTO expression level by ILF3.The sequence alignment and analysis of data in the AREsite2 were combined to find out the AUrich elements in the 3'UTR of FTO m RNA.RIP was performed to detecte the ILF3 antibodyenriched FTO m RNA.ILF3 was knockdown or overexpressed in HCC cells and then treated with actinomycin D to assess the m RNA stability of FTO.Then,m RNA stability of FTO was tested in FTO-IT1 knockdown or overexpressed cells.Meanwhile,FTO-IT1 siRNAs and ILF3 overexpression plasmids were co-transfected,and FTO-IT1 overexpression plasmids and ILF3 siRNAs were co-transfected,and the m RNA stability,m RNA and protein expression levels of FTO were tested.c-Myc siRNA and overexpression plasmid were transfected into cells,and the expression of FTO-IT1 was tested.Chromatin immunoprecipitation(Ch IP)assay was performed to explore the transcriptional regulation of c-Myc on FTO-IT1.ResultsLncRNA FTO-IT1 is highly expressed in HCC tissues and cells,which was related to the poor prognosis of patients with HCC.Knockdown of FTO-IT1 downregulated the proliferation,migration and invasion of HCC cells,as well as the proliferation of HCC in vivo,while overexpression of FTO-IT1 could promote the proliferation,migration and invasion of HCC cells.FTO was upregulated in HCC tissues and cells,and was positively correlated with the FTO-IT1 in HCC.Knockdown of FTO-IT1 reduced,while overexpression of FTO-IT1 increased the expression levels of FTO m RNA and protein.The rescue experiment verified that FTO-IT1 has specificity in regulating FTO.Overexpression of FTO restored the decrease in proliferation,migration and invasion of HCC cells caused by knockdown of FTO-IT1,while knockdown of FTO reversed the promotion of overexpression of FTO-IT1 on the proliferation,migration and invasion of HCC cells.Results from mass spectrometry and Western blot showed that FTO-IT1 could bind to RNA-binding protein ILF3.Anti-ILF3 antibodies significantly enriched FTO-IT1 transcripts.Meanwhile,anti-ILF3 antibodies could also obviously enrich FTO m RNA.Knockdown of ILF3 downregulated,while overexpression of ILF3 upregulated m RNA stability,m RNA and protein levels of FTO.The rescue experiments showed that overexpression of ILF3 restored the downregulation of FTO m RNA and protein and suppression of m RNA stability by knockdown of FTO-IT1,while knockdown of ILF3 reversed the upregulation of FTO m RNA and protein levels and the promotion of m RNA stability by overexpression of FTO-IT1.Knockdown of c-Myc decreased,while overexpression of c-Myc increased the expression of FTO-IT1.c-Myc activated the expression of FTO-IT1 at the transcriptional level.ConclusionLncRNA FTO-IT1 is highly expressed in HCC tissues and cells,which was related to the poor prognosis of patients with HCC.FTO-IT1 regulated the expression of FTO both at m RNA and protein levels,and promoted progression of HCC by regulating the expression of FTO.Mechanistically,FTO-IT1 bound to ILF3 protein and recruited ILF3 to the 3'UTR of FTO m RNA to facilitate the stability of FTO m RNA and consequently promoted the expression of FTO.In addition,c-Myc ranscriptionally activated the expression of FTO-IT1.In conclusion,this study revealed the regulatory mechanism of lncRNA FTO-IT1 promoting progression of HCC through FTO,providing a limited but reliable theoretical basis for FTOIT1 as a potential diagnostic and therapeutic target for HCC.Meanwhile,this study provided a further evidence for the important role of lncRNA in cancer.